WO2002002580A2 - Haplotypes of the etfb gene - Google Patents

Abstract

Novel genetic variants of the Electron-Transfer-Flavoprotein, Beta Polypeptide (ETFB) gene are described. Various genotypes, haplotypes, and haplotype pairs that exist in the general United States population are disclosed for the ETFB gene. Compositions and methods for haplotyping and/or genotyping the ETFB gene in an individual are also disclosed. Polynucleotides defined by the haplotypes disclosed herein are also described.

Description

HAPLOTYPES OF THE ETFB GENE

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/215,984 filed July 5, 2000.

FIELD OF THE INVENTION

This invention relates to variation in genes that encode pharmaceutically-important proteins. In particular, this invention provides genetic variants of the human electron-transfer-flavoprotem, beta polypeptide (ETFB) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.

BACKGROUND OF THE INVENTION

Current methods for identifying pharmaceuticals to treat disease often start by identifying, cloning, and expressing an important target protein related to the disease. A determination of whether an agonist or antagonist is needed to produce an effect that may benefit a patient with the disease is then made. Then, vast numbers of compounds are screened against the target protein to find new potential drugs. The desired outcome of this process is a lead compound that is specific for the target, thereby reducing the incidence of the undesired side effects usually caused by activity at non-intended targets. The lead compound identified in this screening process then undergoes further in vitro and in vivo testing to determine its absorption, disposition, metabolism and toxicological profiles. Typically, this testing involves use of cell lines and animal models with limited, if any, genetic diversity.

What this approach fails to consider, however, is that natural genetic variability exists between individuals in any and every population with respect to pharmaceutically-important proteins, including the protein targets of candidate drugs, the enzymes that metabolize these drugs and the proteins whose activity is modulated by such drug targets. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a pharmaceutically-important protein may be manifested as significant variation in expression, structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in the treatment of individuals with a drug whose design is based upon a single representative example of the target or enzyme(s) involved in metabolizing the drug. For example, it is well-established that some drugs frequently have lower efficacy in some individuals than others, which means such individuals and their physicians must weigh the possible benefit of a larger dosage against a greater risk of side effects. Also, there is significant variation in how well people metabolize drugs and other exogenous chemicals, resulting in substantial mterindividual variation in the toxicity and/or efficacy of such exogenous substances (Evans et al., 1999, Science 286:487-491). This variability in efficacy or toxicity of a drug in genetically-diverse patients makes many drugs ineffective or even dangerous in certain groups of the population, leading to the failure of such drugs in clinical trials or their early withdrawal from the market even though they could be highly beneficial for other groups in the population. This problem significantly increases the time and cost of drug discovery and development, which is a matter of great public concern.

It is well-recognized by pharmaceutical scientists that considering the impact of the genetic variability of pharmaceutically-important proteins in the early phases of drug discovery and development is likely to reduce the failure rate of candidate and approved drugs (Marshall A 1997 Nature Biotech 15: 1249-52; Kleyn PW et al. 1998 Science 281: 1820-21; Kola 1 1999 Curr Opin Biotech 10:589-92; Hill AVS et al. 1999 m. Evolution in Health and Disease Stearns SS (Ed.) Oxford University Press, New York, pp 62-76; Meyer UA. 1999 in Evolution in Health and Disease Stearns SS (Ed.) Oxford University Press, New York, pp 41-49; Kalow W et al. 1999 Clin. Pharm. Therap.

66:445-7; Marshall, E 1999 Science 284:406-7; Judson R et al. 2000 Pharmacogenomics 1:1-12; Roses AD 2000 Nature 405:857-65). However, in practice this has been difficult to do, in large part because of the time and cost required for discovering the amount of genetic variation that exists jn the population (Chakravarti A 1998 Nature Genet 19:216-7; Wang DG et al 1998 Science 280:1077-82; Chakravarti A 1999 Nat Genet 21:56-60 (suppl); Stephens JC 1999 Mol. Diagnosis 4:309-317; Kwok PY and Gu S 1999 Mol. Med. Today 5:538-43; Davidson S 2000 Nature Biotech 18: 1134-5).

The standard for measuring genetic variation among individuals is the haplotype, which is the ordered combination of polymorphisms in the sequence of each form of a gene that exists in the population. Because haplotypes represent the variation across each form of a gene, they provide a more accurate and reliable measurement of genetic variation than individual polymoφhisms. For example, while specific variations in gene sequences have been associated with a particular phenotype such as disease susceptibility (Roses AD supra; Ulbrecht M et al. 2000 Am JRespir Crit Care Med 161: 469- 74) and drug response (Wolfe CR et al. 20005M7320:987-90; Dahl BS 1997 Acta Psychiatr Scand 96 (Suppl 391): 14-21), in many other cases an individual polymorphism may be found in a variety of genomic backgrounds, i.e., different haplotypes, and therefore shows no definitive coupling between the polymorphism and the causative site for the phenotype (Clark AG et al. 1998 Am JHum Genet 63:595- 612; Ulbrecht M et al. 2000 supra; Drysdale et al. 2000 PNAS 97:10483-10488). Thus, there is an unmet need in the pharmaceutical industry for information on what haplotypes exist in the population for pharmaceutically-important genes. Such haplotype information would be useful in improving the efficiency and output of several steps in the drug discovery and development process, including target validation, identifying lead compounds, and early phase clinical trials (Marshall et al., supra).

One pharmaceutically-important gene for the treatment of glutaric acidemia type II is the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene or its encoded product. Electron transfer flavoprotein (ETF) is an obligatory electron acceptor for nine primary flavoprotein dehydrogenases, including the four straight- chain-specific fatty acyl-CoA dehydrogenases, and is located in the mitochondrial matrix (Roberts et al., Proc Natl Acad Sci USA 1996 Dec 10;93(25): 14355-60). ETF is composed of an alpha (ETF A) and a beta (EFTB) suburtit and contains a single equivalent of FAD per dimer (Colombo et al., Hum. Mol. Genet. 1994. 3:429-35). Electrons accepted by ETF are transferred to the mitochondrial respiratory chain by ETF dehydrogenases (ETFDHs). Deficiency of ETF or ETFDH leads to glutaric acidemia type II (GAH), an inherited metabolic disorder that can be fatal in its neonatal form due to the inability to catabolize various fatty acyl-CoAs (Roberts et al., supra). This disease is characterized by severe hypoketotic hypoglycemia and acidosis (White et al., Genomics 1996. 33:131-4). Several polymorphisms that cause amino acid, variations have been identified in the ETFB gene in patients suffering from GAII (Colombo et al., supra). Thus, ETFB is an important target for the treatment of metabolic disorders such as GAII.

The electron-transfer-flavoprotein, beta polypeptide gene is located on chromosome 19ql3.3- ql3.4 and contains 6 exons that encode a 255 amino acid protein. A reference sequence for the ETFB gene is shown in the contiguous lines of Figure 1 (Genaissance Reference No. 767306; SEQ ID NO: 1). Reference sequences for the coding sequence (GenBank Accession No. NM_001985.1) and protein are shown in Figures 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3), respectively.

A polymorphism of cytosine or thymine at a position corresponding to nucleotide 23304 in Figure 1 has been reported in the literature (Colombo et al., supra).

Because of the potential for variation in the ETFB gene to affect the expression and function of the encoded protein, it would be useful to know whether additional polymorphisms exist in the ETFB gene, as well as how such polymorphisms are combined in different copies of the gene. Such information could be applied for studying the biological function of ETFB as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function.

SUMMARY OF THE INVENTION

Accordingly, the inventors herein have discovered 27 noyel polymorphic sites in the ETFB gene. These polymoφhic sites (PS) correspond to the following nucleotide positions in Figure 1 : 3272 (PSl), 3434 (PS2), 3472 (PS3), 3767 (PS4), 3772 (PS5), 3844 (PS6), 3907 (PS7), 3913 (PS8), 3944 (PS9), 3956 (PS10), 16200 (PS11), 16953 (PS12), 16963 (PS13), 16990 (PS14), 17046 (PS15), 19924 (PS16), 20031 (PS17), 23146 (PS18), 23167 (PS19), 23191 (PS20), 23192 (PS21), 23295 (PS22), 23364 (PS24), 23420 (PS25), 23473 (PS26), 25293 (PS27) and 25387 (PS28). The polymoφhisms at ^" these sites are cytosine or thymine at PSl, guanine or adenine at PS2, guanine or adeni e at PS3, cytosine or thymine at PS4, thymine or cytosine at PS5, thymine or cytosine at PS6, guanine or adenine at PS7, cytosine or thymine at PS8, guanine or ademne at PS9, cytosine or thymine at PS 10, guariine or ademne at PSl 1, cytosine or guanine at PS12, cytosine or thymine at PS13, guanine or adenine at PS14, guariine or thymine at PS15, cytosine or thymine at PS16, cytosine or thymine at PS17, cytosine or guanine at PS 18, cytosine or thymine at PS 19, cytosine or thymine at PS20, adenine or guanine at PS21 , cytosine or thymine at PS22, guanine or adenine at PS24, guanine or adenine at PS25, guariine or adenine at PS26, cytosine or thymine at PS27 and guanine or cytosine at PS28. In addition, the inventors have determined the identity of the alleles at these sites, as well as at the previously identified site at nucleotide position 23304 (PS23), in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. From this information, the inventors deduced a set of haplotypes and haplotype pairs for PS1-PS28 in the ETFB gene, which are shown below in Tables 5 and 4, respectively. Each of these ETFB haplotypes defines a natarally-occurring isoform (also referred to herein as an "isogene") of the ETFB gene that exists in the human population. The frequency with which each haplotype and haplotype pair occurs within the total reference population and within each of the four major population groups included in the reference population was also determined.

Thus, in one embodiment, the invention provides a method, composition and kit for genotyping the ETFB gene in an individual. The gehotyping method comprises identifying the nucleotide pair that is present at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28 in both copies of the ETFB gene from the individual. A genotyping composition of the invention comprises an oligoriucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel ETFB polymoφhic sites. A genotyping kit of the invention comprises a set of oligonucleotides designed to genotype each of these novel ETFB polymoφhic sites. In a preferred embodiment, the genotyping kit comprises a set of oligonucleotides designed to genotype each of PS1-PS28. The genotyping method, composition, and kit are useful in determining whether an individual has one of the haplotypes in Table 5 below or has one of the haplotype pairs in Table 4 below.

The invention also provides a method for haplotyping the ETFB gene in an individual. In one embodiment, the haplotyping method comprises determining, for one copy of the ETFB gene, the identity of the nucleotide at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS 19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28. In another embodiment, the haplotyping method comprises determining whether one copy of the. individual's ETFB gene is defined by one of the ETFB haplotypes shown in Table 5, below, or a sub-haplotype thereof. In a preferred embodiment, the haplotyping method comprises determining whether both copies of the individual's ETFB gene are defined by one of the ETFB haplotype pairs shown in Table 4 below, or a sub-haplotype pair thereof. The method for establishing the ETFB haplotype or haplotype pair of an individual is useful for improving the efficiency and reliability of several steps in the discovery and development of drugs for treating diseases associated with ETFB activity, e.g., glutaric acidemia type II.

For example, the haplotyping method can be used by the pharmaceutical research scientist to validate ETFB as a candidate target for treating a specific condition or disease predicted to be associated with ETFB activity. Determining for a particular population the frequency of one or more of the individual ETFB haplotypes or haplotype pairs described herein will facilitate a decision on whether to pursue ETFB as a target for treating the specific disease of interest. In particular, if variable ETFB activity is associated with the disease, then one or more ETFB haplotypes or haplotype pairs will be- found at a higher frequency in disease cohorts than in appropriately genetically matched controls. Conversely, if each of the observed ETFB haplotypes are of similar frequencies in the disease and control groups, then it may be inferred that variable ETFB activity has little, if any, involvement with that disease. In either case, the pharmaceutical research scientist can, without a priori knowledge as to the phenotypic effect of any ETFB haplotype or haplotype pair, apply the information derived from detecting ETFB haplotypes in an individual to decide whether modulating ETFB activity would be useful in treating the disease.

The claimed invention is also useful in screening for compounds targeting ETFB to treat a specific condition or disease predicted to be associated with ETFB activity. For example, detecting which of the ETFB haplotypes or haplotype pairs disclosed herein are present in individual members of a population with the specific disease of interest enables the pharmaceutical scientist to screen for a compound(s) that^'displays the highest desired agonist or antagonist activity for each of the most frequent ETFB isoforms present in the disease population. Thus, without requiring any a priori knowledge of the phenotypic effect of any particular ETFB haplotype or haplotype pair, the claimed haplotyping method provides the scientist with a tool to identify lead compounds that are more likely to show efficacy in clinical trials.

The method for haplotyping the ETFB gene in an individual is also useful in the design of clinical trials of candidate drugs for treating a specific condition or disease predicted to be associated with ETFB activity. For example, instead of randomly assigning patients with the disease of interest to the treatment or control group as is typically done now, determining which of the ETFB haplotype(s) disclosed herein are present in individual patients enables the pharmaceutical scientist to distribute ETFB haplotypes and or haplotype pairs evenly to treatment and control groups, thereby reducing the potential for bias in the results that could be introduced by a larger frequency of an ETFB haplotype or haplotype pair that had a previously unknown association with response to the drug being studied in the trial. Thus, by practicing the claimed invention, the scientist can more confidently rely on the information learned from the trial, without first determining the phenotypic effect of any ETFB haplotype or haplotype pair.

In another embodiment, the invention provides a method for identifying an association between a trait and an ETFB genotype, haplotype, or haplotype pair for one or more of the novel polymoφhic sites described herein. The method comprises comparing the frequency of the ETFB genotype, haplotype, or haplotype pair in a population exhibiting the trait with the frequency of the ETFB genotype or haplotype in a reference population. A higher frequency of the ETFB genotype, haplotype, or haplotype pair in the trait population than in the reference population indicates the trait is associated with the ETFB genotype, haplotype, or haplotype pair. In preferred embodiments, the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug. In a particularly preferred embodiment, the ETFB haplotype is selected from the haplotypes shown in Table 5, or a sub-haplotype thereof. Such methods have applicability in developing diagnostic tests and therapeutic treatments for glutaric acidemia type II.

In yet another embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymoφhic variant of a reference sequence for the ETFB gene or a fragment thereof. The reference sequence comprises the contiguous sequences shown in Figure 1 and the polymoφhic variant comprises at least one polymoφhism selected from the group consisting of thymine at PSl, adenine at PS2, adenine at PS3, thymine at PS4, cytosine at PS5, cytosine at PS6, adenine at PS7, thymine at PS8, adenine at PS9, thymine at PS10, adenine at PS11, guanine at PS12, thymine at PS 13, adenine at PS 1.4, thymine at PS 15, thymine at PS 16, thymine at PS 17, guanine at PS18, thymine at PS19, thymine at PS20, guanine at PS21, thymine at PS22, adenine at PS24, adenine at PS25, adenine at PS26, thymine at PS27 and cytosine at PS28. In a preferred embodiment, the polymoφhic variant comprises an additional polymoφhism of thymine at PS23.

A particularly preferred polymoφhic variant is an isogene of the ETFB gene. An ETFB isogene of the invention comprises cytosine or thymine at PSl, guanine or adenine at PS2, guanine or adenine at PS3, cytosine or thymine at PS4, thymine or cytosine at PS5, thymine or cytosine at PS6, guanine or adenine at PS7, cytosine or thymine at PS8, guanine or adenine at PS9, cytosine or thymine at PS10, guanine or adenine at PSl 1, cytosine or guanine at PS12, cytosine or thymine at PS13, guanine or adenine at PS 14, guanine or thymine at PS 15, cytosine or thymine at PS 16, cytosine or thymine at PS17, cytosine or guanine at PS18, cytosine or thymine at PS19, cytosine or thymine at PS20, adenine or guanine at PS21, cytosine or thymine at PS22, cytosine or thymine at PS23, guanine or adenine at PS24, guanine or adenine at PS25, guanine or adenine at PS26, cytosine or thymine at PS27 and guanine or cytosine at PS28. The invention also provides a collection of ETFB isogenes, referred to herein as an ETFB genome anthology.

In another embodiment, the invention provides a polynucleotide comprising a polymoφhic variant of a reference sequence for an ETFB cDNA or a fragment thereof. The reference sequence comprises SEQ ID NO:2 (Fig.2) and the polymoφhic cDNA comprises at least one polymoφhism selected from the group consisting of thymine at a position corresponding to nucleotide 452, adenine at a position corresponding to nucleotide 521 and adenine at a position corresponding to nucleotide 577. In a preferred embodiment, the polymoφhic variant comprises an additional polymoφhism of thymine at a position corresponding to nucleotide 461. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of an ETFB isogene defined by haplotypes lc, 4c, 7c, 9c, 10c, 12c, 14c, 17c, 18c, 19c, 20c, 24c, 26c, 27c, 29c, 30c, 35c, 36c, 37c, and 39c.

Polynucleotides complementary to these ETFB genomic and cDNA variants are also provided by the invention. It is believed that polymoφhic variants of the ETFB gene will be useful in studying the expression and function of ETFB, and in expressing ETFB protein for use in screening for candidate drugs to treat diseases related to ETFB activity. In other embodiments, the invention provides a recombinant expression vector comprising one of the polymoφhic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector. The recombinant vector and host cell may be used to express ETFB for protein structure analysis and drug binding studies. In yet another embodiment, the invention provides a polypeptide comprising a polymoφhic variant of a reference amino acid sequence for the ETFB protein. The reference amino acid sequence comprises SEQ ID NO:3 (Fig.3) and the polymoφhic variant comprises at least one variant amino acid selected from the group consisting of phenylalanine at a position corresponding to amino acid position 151, histidine at a position corresponding to amino acid position 174 and threonine at a position • corresponding to amino acid position 193. In some embodiments, the polymoφhic variant also comprises methionine at a position corresponding to amino acid position 154. A polymoφhic variant of ETFB is useful in studying the effect of the variation on the biological activity of ETFB as well as on the binding affinity of candidate drugs targeting ETFB for the treatment of glutaric acidemia type II. The present invention also provides antibodies that recognize and bind to the above polymoφhic ETFB protein variant. Such antibodies can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods.

The present invention also provides nonhuman transgenic. animals comprising one of the ETFB polymoφhic genomic variants described herein and methods for producing such animals. The transgenic animals are useful for studying expression of the ETFB isogenes in vivo, for in vivo screening and testing of drugs targeted against ETFB protein, and for testing the efficacy of therapeutic agents and compounds for glutaric acidemia type II in a biological system.

The present invention also provides a computer system for storing and displaying polymoφhism data determined for the ETFB gene. The computer system comprises a computer processing unit; a display; and a database containing the polymoφhism data. The polymoφhism data includes the polymoφhisms, the genotypes and the haplotypes identified for the ETFB gene in a reference population. In a preferred embodiment, the computer system is capable of producing a display showing ETFB haplotypes organized according to their evolutionary relationships.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a reference sequence for the ETFB gene (Genaissance Reference No.

767306; contiguous lines), with the start and stop positions of each region of coding sequence indicated with a bracket ([ or ]) and the numerical position below the sequence and the polymoφhic site(s) and polymoφhism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymoφhic site in the sequence. SEQ ID NO: 1 is equivalent to Figure 1,. with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WIPO standard ST.25). SEQ ID NO: 141 is a modified version of SEQ ID NO: 1 that shows the context sequence of each polymoφhic site, PS1-PS28, in a uniform format to facilitate electronic searching. For each polymoφhic site, SEQ ID NO: 141 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymoφhic site at the 30 position, followed by 60 bases of unspecified sequence to represent that each PS is separated by genomic sequence whose composition is defined elsewhere herein.

Figure 2 illustrates a reference sequence for the ETFB coding sequence (contiguous lines; SEQ ID NO:2), with the polymoφhic site(s) and polymoφhism(s) identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymoφhic site in the sequence. Figure 3 illustrates a reference sequence for the ETFB protein (contiguous lines; SEQ ID NO:3), with the variant amino acid(s) caused by the polymoφhism(s) of Figure 2 positioned below the polymoφhic site in the sequence.

DESCRIPTION OF THE PREFERRED EMBODTJVIENTS

The present invention is based on the discovery of novel variants of the ETFB gene. As described in more detail below, the inventors herein discovered 41 isogenes of the ETFB gene by characterizing the ETFB gene found in genomic DNAs isolated from an Index Repository that contains immortalized cell lines from one chimpanzee and 93 human individuals. The human individuals included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (21 individuals), African descent (20 individuals), Asian (20 individuals), or Hispanic/Latino (18 individuals). To the extent possible, the members of this reference population were organized into population subgroups by their self-identified ethnogeographic origin as shown in Table 1 below.

In addition, the Index Repository contains three unrelated indigenous American Indians (one from each of North, Central and South America), one three-generation Caucasian family (from the CEPH Utah cohort) and one two-generation African- American family.

The ETFB isogenes present in the human reference population are defined by haplotypes for 28 polymoφhic sites in the ETFB gene, 27 of which are believed to be novel. The ETFB polymoφhic sites identified by the inventors are referred to as PS1-PS28 to designate the order in which they are located in the gene (see Table 3 below), with the novel polymoφhic sites referred to as PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28. Using the genotypes identified in the Index Repository for PS1-PS28 and the methodology described in the Examples below, the inventors herein also determined the pair of haplotypes for the ETFB gene present in individual human members of this repository. The human genotypes and haplotypes found in the repository for the ETFB gene include those shown in Tables 4 and 5, respectively. The polymoφhism and haplotype data disclosed herein are useful for validating whether ETFB is a suitable target for drugs to treat glutaric acidemia type II, screening for such drugs and reducing bias in clinical trials of such drugs.

In the context of this disclosure, the following terms shall be defined as follows unless otherwise indicated: Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.

Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these. Gene - A segment of DNA that contains all the information for the regulated biosynthesis of an

RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

Genotype - An unphased 5' to 3' sequence of nucleotide pair(s) found at one or more polymoφhic sites in a locus on a pair of homologous chromosomes in an individual. As used herein, genotype includes a full-genotype and/or a sub-genotype as described below.

Full-genotype - The unphased 5 ' to 3 ' sequence of nucleotide pairs found at all polymoφhic sites examined herein in a locus on a pair of homologous chromosomes in a single individual. Sub-genotype - The unphased 5 ' to 3 ' sequence of nucleotides seen at a subset of the polymoφhic sites examined herein in a locus on a pair of homologous chromosomes in a single individual.

Genotyping - A process for determining a genotype of an individual. Haplotype - A 5 ' to 3 ' sequence of nucleotides found at one or more polymoφhic sites in a locus on a single chromosome from a single individual. As used herein, haplotype includes a full- haplotype and/or a sub-haplotype as described below. Full-haplotype - The 5 ' to 3 ' sequence of nucleotides found at all polymoφhic sites examined herein in a locus on a single chromosome from a single individual.

Sub-haplotype - The 5 ' to 3 ' sequence of nucleotides seen at a subset of the polymoφhic sites examined herein in a locus on a single chromosome from a single individual.

Haplotype pair - The two haplotypes found for a locus in a single individual. Haplotyping - A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and or statistical inference.

Haplotype data - Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.

Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.

Isogene - One of the isoforms of a gene found in a population. An isogene contains all of the polymoφhisms present in the particular isoform of the gene. Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term "isolated" is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.

Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.

Naturally-occurring - A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.

Nucleotide pair - The nucleotides found at a polymoφhic site on the two copies of a chromosome from an individual.

Phased - As applied to a sequence of nucleotide pairs for two or more polymoφhic sites in a locus, phased means the combination of nucleotides present at those polymoφhic sites on a single copy of the locus is known.

Polymorphic site (PS) - A position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.

Polymorphic variant - A gene, mRNA, cDNA, polypeptide or peptide whose nucleotide or amino acid sequence varies from a reference sequence due to the presence of a polymoφhism in the gene.

Polymorphism - The sequence variation observed in an individual at a polymoφhic site. Polymoφhisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.

Polymorphism data - Information concerning one or more of the following for a specific gene: location of polymoφhic sites; sequence variation at those sites; frequency of polymoφhisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.

Polymorphism Database - A collection of polymoφhism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.

Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.

Population Group - A group of individuals sharing a common ethnogeographic origin. Reference Population - A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.

Single Nucleotide Polymorphism (SNP) - Typically, the specific pair of nucleotides observed at a single polymoφhic site. In rare cases, three or four nucleotides may be found. Subject - A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.

Treatment - A stimulus administered internally or externally to a subject. Unphased - As applied to a sequence of nucleotide pairs for two or more polymoφhic sites in a locus, unphased means the combination of nucleotides present at those polymoφhic sites on a single copy of the locus is not known.

As discussed above, information on the identity of genotypes and haplotypes for the ETFB gene of any particular-individual as well as the frequency of such genotypes and haplotypes in any particular population of individuals is expected to be useful for a variety of drug discovery and development applications. Thus, the invention also provides compositions and methods for detecting the novel ETFB polymoφhisms and haplotypes identified herein.

^■ The compositions comprise at least one ETFB genotyping oligonucleotide. In one embodiment, an ETFB genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymoφhic sites described herein. As used herein, the term "oligonucleotide" refers to a polynucleotide molecule having less than about 100 nucleotides. A preferred oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan. The oligonucleotide maybe comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives. Alternatively, oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc. (1995), pages 617-620). Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion. The oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like. Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of an ETFB polynucleotide, i.e., an ETFB isogene. As used herein, specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-ETFB polynucleotide under the same hybridizing conditions. Preferably, the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions. The skilled artisan can readily design and test oligonucleotide probes and primers suitable for detecting polymoφhisms in the ETFB gene using the polymoφhism information provided herein in conjunction with the known sequence information for the ETFB gene and routine techniques.

A nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect" or "complete" complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule. A nucleic acid molecule is "substantially complementary" to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described,. for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D. et al. in Nucleic Acid Hybridization, A Practical Approach, IRL Press,

Washington, D.C. (1985). While perfectly complementary oligonucleotides are preferred for detecting polymoφhisms, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region. For example, an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region. Alternatively, non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.

Preferred genotyping oligonucleotides of the invention are allele-specific oligonucleotides. As used herein, the term allele-specific .oligonucleotide (ASO) means an oligonucleotide that is able, under sufficiently stringent conditions, to hybridize specifically to one allele of a gene, or other locus, at a target region containing a polymoφhic site while not hybridizing to the corresponding region in another allele(s). As understood by the skilled artisan, allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps. Examples of hybridization and washing conditions typically used for ASO probes are found in Kogan et al., "Genetic Prediction of Hemophilia A" in PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990 and Ruaiio et al., 87 Proc. Natl. Acad. Sci. USA 6296-6300, 1990. Typically, an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele.

Allele-specific, oligonucleotides of the invention include ASO probes and ASO primers. ASO probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymoφhic site in the target region (e.g., approximately the 7^th or 8^th position in a 15mer, the 8^th or 9^th position in a 16mer, and the 10^th or 11^th position in a 20mer). An ASO primer of the invention has a 3 ' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present. ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention. ASO probes and primers listed below use the appropriate nucleotide symbol (R= G or A, Y= T or C, M= A or C, K= G or T, S= G or C, and W= A or T; WEPO standard ST.25) at the position of the polymoφhic site to represent the two alternative allelic variants observed at that polymoφhic site.

A preferred ASO probe for detecting ETFB gene polymoφhisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:

TGCGGGTYGTGGGGA (SEQ ID NO 4) and its complement,

AGGGGACRTGGGCCT (SEQ ID NO 5) and its complement,

TGGGGACRACAΪGGA (SEQ ID NO 6) and its complement,

GGCCTTTYCTTCTCT (SEQ ID NO: 7) and its complement,

TTCCTTCYCTCTCCA (SEQ ID NO. 8) and its complement,

CCCTTCCΫTCCGGCC (SEQ ID NO 9) and its complement,

GCTGACCRTGCTGCG (SEQ ID NO 10 and its complement,

CGTGCTGYGCGCAGG (SEQ ID NO 11 and its complement,

GGGGGCGRGGGTGGT (SEQ ID NO 12 and its complement,

GGTGGGGYTTCTGGA (SEQ ID NO 13 and its complement,

GATGAAGRTCTTGGG (SEQ ID NO 14 and its complement,

GATGGGGSCCTGGAC (SEQ ID NO 15 and its complement,

TGGACTCYTGGGTCT (SEQ ID NO- 16 and its complement,

GCTGCGARCCAGACT (SEQ ID NO 17 and its complement,

GTCCTTGKCAGGAGA (SEQ ID NO 18 and its complement,

TGTCATAYAGTCTTC (SEQ ID NO 19 and its complement,

TGGGGAGYACCTGCT (SEQ ID NO 20 ) and its complement,

TCTAAACSTCATGGG (SEQ ID NO 21 and its -complement,

CATTGTTYGCTCCCT (SEQ ID NO 22 ) and its complement,

ACTAGTCYATGCGTG (SEQ ID NO 23 and its complement,

CTAGTCCRTGCGTGT (SEQ ID NO 24 and its complement,

TTCGCCTYCCAGGTG (SEQ ID NO 25 and its complement,

ACCCTGCRCCTGAAG (SEQ ID NO 26 ) and its complement,

CCGCTACRCCACGCT (SEQ ID NO 27 and its complement,

AGGGCCTRGGGGTGG (SEQ ID NO 28 and its complement,

AGAGACCYATTATTT (SEQ ID NO 29 ) and its complement, and

TGATGCGSGGGACAA (SEQ ID NO 30 ) and its complement.

A preferred ASO primer for detecting ETFB gene polymoφhisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:

CTTTTCTGCGGGTYG (SEQ^' ID NO: 31) CCTGCGTCCCCACRA SEQ ID NO: 32)

GCCAGGAGGGGACRT (SEQ ID NO: 33) CTCCAAAGGCCCAYG SEQ ID NO 34)

AGGCTGTGGGGACRA (SEQ ID NO: 35)_^ TTCACCTCCATGTYG SEQ ID NO 36) CCGCCAGGCCTTTYC (SEQ ID NO: 37) CTGGAGAGAGAAGRA SEQ ID NO 38)

AGGCCTTTCCTTCYC (SEQ ID NO: 39) GGGACCTGGAGAGRG SEQ ID NO 40)

ACACCTCCCTTCCYT (SEQ ID NO; 1) TCGAGAGGCCGGARG SEQ ID NO. 42)

TGACTGGCTGACCRT (SEQ ID NO: 3) CCTGCGCGCAGCAYG SEQ ID NO 44)

GCTGACCGTGCTGYG (SEQ ID NO: 5) TCTGCGCCTGCGCRC SEQ ID NO 46) GGCGCGGGGGGCGRG (SEQ^' ID NO: 47) AGCCCCACCACCCYC SEQ ID NO 48)

GGGGGTGGTGGGGYT (SEQ ID NO: 49) GCTCAGTCCAGAARC SEQ^' ID NO- 50)

CCAGGTGATGAAGRT (SEQ ID NO: 51) CCCCTCCCCAAGAYC SEQ ID NO 52)

GGAGGGGATGGGGSC (SEQ ID NO: 53) CCAGGAGTCCAGGSC SEQ ID NO 54)

GGGCCCTGGACTCYT (SEQ ID NO: 55) TTCCTCAGACCCARG SEQ ID NO 56) GGAGGGGCTGCGARC (SEQ ID NO: 57) CCTAGGAGTCTGGYT SEQ ID NO 58)

CCCTCTGTCCTTGKC (SEQ ID NO: 59) GAATCGTCTCCTGMC SEQ ID NO 60)

ACTGATTGTCATAYA (SEQ ID NO: 61) GAGACAGAAGACTRT SEQ ID NO 62) CTGGAGTGGGGAGYA (SEQ ID NO 63); GCCCTCAGCAGGTRC (SEQ ID NO: 64) ;

CTCGATTCTAAACST (SEQ ID NO 65) ; CAGGGTCCCATGASG (SEQ ID NO: 66) ;

ACCCTGCATTGTTYG (SEQ ID NO 67) ; TGAATGAGGGAGCRA (SEQ ID NO: 68) ;

TCATTCACTAGTCYA (SEQ ID NO 69) ; TGAGTACACGCATRG (SEQ ID NO: 70) ;

CATTCACTAGTCCRT (SEQ ID NO 71) ; CTGAGTACACGCAYG (SEQ ID NO:72

GGCACATTCGCCTYC (SEQ ID NO 73) ; CAGCGTCACCTGGRA (SEQ ID NO: 74) ;

CTGGAGACCCTGCRC (SEQ ID_. NO 75); TGGCAGCTTCAGGYG (SEQ ID NO: 76

CGAGCCCCGCTACRC (SEQ ID NO 77) ; TTGGGCAGCGTGGYG (SEQ ID, NO: 78

GCACTGAGGGCCTRG (SEQ ID NO 79) ; GCTTGCCACCCCYA (SEQ .ID NO: 80) ;

TTTCCTAGAGACCYA (SEQ ID NO 81) ; TGAACCAAATAATRG (SEQ ID NO: 82)

CTGTATTGATGCGSG (SEQ ^■ID NO 83) ; and TCCTCATTGTCCCSC (SEQ ID NO 84)

Other genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymoφhic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymoφhisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides". In a preferred embodiment, the 3 '-terminus of a primer- extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymoφhic site.

A particularly preferred oligonucleotide primer for detecting ETFB gene polymoφhisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:

TTCTGCGGGT (SEQ ID NO. 85); GCGTCCCCAC (SEQ ID NO 86) ;

AGGAGGGGAC (SEQ ID NO 87); CAAAGGCCCA (SEQ ID NO 88) ;

CTGTGGGGAC (SEQ ID NO 89); ACCTCCATGT (SEQ ID -NO 90) ;

CCAGGCCTTT (SEQ ID NO 91); GAGAGAGAAG (SEQ ID NO 92);

CCTTTCCTTC (SEQ ID NO 93); ACCTGGAGAG (SEQ ID NO 94) ;

CCTCCCTTCC (SEQ ID NO 95); AGAGGCCGGA (SEQ ID NO 96) ;

CTGGCTGACC (SEQ ID NO 97); GCGCGCAGCA (SEQ ID NO 98);

GACCGTGCTG (SEQ ID NO_. 99); GCGCCTGCGC (SEQ ID NO 100)

GCGGGGGGCG (SEQ ID NO: 101) , CCCACCACCC (SEQ ID NO 102)

GGTGGTGGGG (SEQ ID NO 103) , CAGTCCAGAA (SEQ ID NO 104)

GGTGATGAAG (SEQ ID NO .105) , CTCCCCAAGA (SEQ ID NO 106)

GGGGATGGGG (SEQ ID NO 107) GGAGTCCAGG (SEQ ID NO 108)

CCCTGGACTC (SEQ ID NO 109) ; CTCAGACCCA (SEQ ID NO 110)

GGGGCTGCGA (SEQ ID NO 111) AGGAGTCTGG (SEQ ID NO 112)

TCTGTCCTTG (SEQ ID NO 113). TCGTCTCCTG (SEQ ID NO 114)

GATTGTCATA (SEQ ID NO 115) ACAGAAGACT (SEQ ID NO 116)

GAGTGGGGAG (SEQ ID NO 117) CTCAGCAGGT (SEQ ID NO 118)

GATTCTAAAC (SEQ ID NO- 119) ; GGTCCCATGA (SEQ ID NO 120)

CTGCATTGTT (SEQ ID NO 121) ATGAGGGAGC (SEQ ID NO 122)

TTCACTAGTC (SEQ ID NO 123) GTACACGCAT . (SEQ ID NO 124)

TCACTAGTCC (SEQ ID NO 125) : AGTACACGCA (SEQ ID NO 126)

ACATTCGCCT (SEQ ID NO 127) ; CGTCACCTGG (SEQ ID NO 128)

GAGACCCTGC (SEQ ID NO 129) ^• CAGCTTCAGG (SEQ ID NO 130)

GCCCCGCTAC (SEQ ID NO 131) ; GGCAGCGTGG (SEQ ID NO 132)

CTGAGGGCCT (SEQ ID^' NO 133) TTGCCACCCC (SEQ ID NO 134)

CCTAGAGACC (SEQ ID NO 135) ; ACCAAATAAT (SEQ ID NO 136)

TATTGATGCG (SEQ ID NO 137) ; and TCATTGTCCC (SEQ ID NO 138) In some embodiments, a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymoφhic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymoφhic site.

ETFB genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Such immobilized genotyping oligonucleotides may be used in a variety of polymoφhism detection assays, including but not limited to probe hybridization and polymerase extension assays. Immobilized ETFB genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymoφhisms in multiple genes at the same time. In another embodiment, the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers. The kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container. Alternatively, where the oligonucleotides are to be used to amplify a target region, the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.

The above described oligonucleotide compositions and kits are useful in methods for genotyping'and/or haplotyping the ETFB gene in an individual. As used herein, the terms "ETFB genotype" and "ETFB haplotype" mean the genotype or haplotype contains the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymoφhic sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymoφhic sites in the ETFB gene. The additional polymoφhic sites may be currently known polymoφhic sites or sites that are subsequently discovered.

One embodiment of the genotyping method involves isolating from the individual a nucleic acid sample comprising the two copies of the ETFB gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3,PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS2^*5, PS26, PS27 an PS28 in the two copies to assign an ETFB genotype to the individual. As will be readily understood by the skilled artisan, the two "copies" of a gene in an individual may be the same allele or may be different alleles. In a preferred embodiment of the genotyping method, the identity_, of the nucleotide pair at PS23 is also determined. In a particularly preferred embodiment, the genotyping method comprises determining the identity of the nucleotide pair at each of PS1-PS28.

Typically, the nucleic acid sample is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. The nucleic acid sample may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from a tissue in which the ETFB gene is expressed. Furthermore it will be understood by the skilled artisan that RNA or cDNA preparations would not be used to detect polymoφhisms located in introns or in 5 ' and- 3 ' untranslated regions. If an ETFB gene fragment is isolated, it must contain the polymoφhic site(s) to be genotyped.

One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid sample contai ng only one of the two copies of the ETFB gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15,PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28 in that copy to assign an ETFB haplotype to the individual. The nucleic acid may be isolated using any method capable of separating the two copies of the ETFB gene or fragment such as one of the methods described above for preparing ETFB isogenes, with targeted in vivo cloning being the preferred approach. As will be readily appreciated by those skilled in the art, any individual clone will only provide haplotype information on one of the two ETFB gene copies present in an individual. If haplotype information is desired for the individual's other copy, additional ETFB clones will need to be examined. Typically, at least five clones should be examined to have more than a 90% probability of haplotyping both copies of the ETFB gene in an individual. In some embodiments, the haplotyping. method also comprises identifying the nucleotide at PS23. In a particularly preferred embodiment, the nucleotide at each of PS 1 -PS28 is identified.

In another embodiment, the haplotyping method comprises determining whether an individual has one or more of the ETFB haplotypes shown in Table 5. This can be accomplished by identifying, for one or both copies of the individual's ETFB gene, the phased sequence of nucleotides present at each of PS1-PS28. The present invention also contemplates that typically only a subset of PS1-PS28 will need to be directly examined to assign to an individual one or more of the haplotypes shown in Table 5. This is because at least one polymoφhic site in a gene is frequently in strong linkage disequilibrium with one or more other polymoφhic sites in that gene (Drysdale, CM et al. 2000 PNAS 97:10483-10488; Rieder MJ et al. 1999 Nature Genetics 22:59-62). Two sites are said to be in linkage disequilibrium if the presence of a particular variant at one site enhances the predictability of another variant at the second site (Stephens, JC 1999, Mol. Diag. 4:309-317). Techniques for determining whether any two polymoφhic sites are in linkage disequilibrium are well-known in the art (Weir B„S. 1996 Genetic Data Analysis II, Sinauer Associates, Inc. Publishers, Sunderland, MA).

In a preferred embodiment, an ETFB haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more polymoφhic sites selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28 in each copy of the ETFB gene that is present in the individual. In a particularly preferred embodiment, the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS1-PS28 in each copy of the ETFB gene. When haplotyping both copies of the gene, the identifying step is preferably performed with each copy of the gene being placed in separate containers. However, it is also envisioned that if the two copies are labeled with different tags, or are otherwise separately distinguishable or identifiable, it could be possible in some cases to perform the method in the same container. For example, if first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymoφhic site(s), then detecting a combination of the first and third dyes would identify the polymoφhism in the first gene copy while detecting a combination of the second and third dyes would identify the polymoφhism in the second gene copy.

In both the genotyping and haplotyping methods, the identity of a nucleotide (or nucleotide pair) at a polymoφhic site(s) may be determined by amplifying a target region(s) containing the polymoφhic- site(s) directly from one or both copies of the ETFB gene, or a fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymoφhic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site. The polymoφhism may be identified directly, known as positive type identification, or by inference, referred to as negative-type identification. For example, where a SNP is known to be guanine and cytosine in a reference population, a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site. Alternatively, the site.may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanme/guanine).

The target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988).

Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al, Proc. Natl. Acad. Sci. USA 89:392-396, 1992).

A polymoφhism_. in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art. Typically, allele-specific oligonucleotides are utilized in performing such methods. The allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant. In some embodiments, more than one polymoφhic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs. Preferably, the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymoφhic sites being detected.

Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid. The genotype or haplotype for the ETFB gene of an individual may also be determined by hybridization of a nucleic acid sample containing one or both copies of the gene, or fragment(s) thereof, to nucleic acid arrays and subarrays such as described in WO 95/11995. The arrays would contain a battery of allele-specific oligonucleotides representing each of the polymoφhic sites to be included in the genotype or haplotype. The identity of polymoφhisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230: 1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variant alleles can be identified by single strand conformation polymoφhism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al, Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used to identify the polymoφhism(s). Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis" method (W092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524. Related methods are disclosed in WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283. Extended primers containing a polymoφhism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798. Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17:8392, 1989; Ruano et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al, J. Clin. Invest. 95: 1635-1641, 1995). In addition, multiple polymoφhic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).

In addition, the identity of the allele(s) present at any of the novel polymoφhic sites described herein may be indirectly determined by genotyping another polymoφhic site that is in linkage disequilibrium with the polymoφhic site that is of interest. Polymoφhic sites in linkage disequilibrium with the presently disclosed polymoφhic sites may be located in regions of the gene or in other genomic regions not examined herein. Genotyping of a polymoφhic site in linkage disequilibrium with the novel polymoφhic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymoφhic site. In another aspect of the invention, an individual's ETFB haplotype pair is predicted from its

ETFB genotype using information on haplotype pairs known to exist in a reference population. In its broadest embodiment, the haplotyping prediction method comprises identifying an ETFB genotype for the individual at two or more ETFB polymoφhic sites described herein, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing ETFB haplotype pairs identified in a reference ^"population, and assigning a haplotype pair to the individual that is consistent with the data. In one embodiment, the reference haplotype pairs include the ETFB haplotype pairs shown in Table 4.

Generally, the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world. A preferred reference population for use in the methods of the present invention comprises an approximately equal number of individuals from Caucasian, African-descent, Asian and Hispanic-Latino population groups with the minimum number of each group being chosen based on how rare a haplotype one wants to be guaranteed to see. For example, if one wants to have a q% chance of not missing a haplotype that exists in the population at a p% frequency of occurring in the reference population, the number of individuals (n) who must be sampled is given by 2n=log(l-q)/log(l-p) where p and q are expressed as fractions. A preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each of the four population groups named above. A particularly preferred reference population includes a 3 -generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.

In a preferred embodiment, the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L. Hartl et al., Principles of Population Genomics, Sinauer Associates (Sunderland, MA), 3^rd Ed., 1997) postulates that the frequency of finding the haplotype pair H_x /H₂is equal to p_H__w(H_x IH₂) = 2p(H_λ)p⁽H₂) if H ≠ H₂ and p_H__w(H_λ I H₂) = p(H_x)p(H₂) if H, = H₂ . A statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may ^■ wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System^™ technology (U.S. Patent No. 5,866,404), single molecule dilution, or allele-specific long-range PCR (Michalotos-Beloin et al., Nucleic Acids Res. 24:4841-4843, 1996).

In one embodiment of this method for predicting an ETFB haplotype pair for an individual, the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual. Occasionally, only one haplotype represented in the reference haplotype pairs is consistent with a possible haplotype pair for an individual, and in such cases the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair. Alternatively, the haplotype pair in an individual may be predicted from the individual's genotype for that gene using reported methods (e.g., Clark et al. 1990 Mol Bio Evol 7: 111-22) or through a commercial haplotyping service such as offered by Genaissance Pharmaceuticals, Inc. (New Haven, CT). In rare cases, either no haplotypes in the reference population are consistent with the possible haplotype pairs, or alternatively, multiple reference haplotype pairs are consistent with the possible haplotype pairs. In such cases, the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System^™ technology (U.S. Patent No. 5,866,404), SMD, or allele-specific long-range PCR (Michalotos-Beloin et al., supra). A preferred process for predicting ETFB haplotype pairs from ETFB genotypes is described in U.S. Provisional Application Serial No. 60/198,340 and the corresponding International Application, PCT/USO 1/12831.

The invention also provides a method for determining the frequency of an ETFB genotype, haplotype, or haplotype pair in a population. The method comprises, for each member of the population, determining the genotype or the haplotype pair for the novel ETFB polymoφhic sites described herein, and calculating the frequency any particular genotype, haplotype, or haplotype pair is found in the population. The population may be a reference population, a family population, a same sex population, a population group, or a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).

In another aspect of the invention, frequency data for ETFB genotypes, haplotypes, and/or haplotype pairs are determined in a reference population and used in a method for identifying an association between a trait and an ETFB genotype, haplotype, or haplotype pair. The trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment. The method involves obtaining data on the frequency of the genotype(s), haplotype(s), or haplotype pair(s) of interest in a reference population as well as in a population exhibiting the trait. Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above. The haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above. In another embodiment, the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic form. For example, the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s), haplotype(s), or haplotype pair(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes, haplotypes, and/or haplotype pairs observed in the populations are compared. If a particular ETFB genotype, haplotype, or haplotype pair is more frequent in the trait population than in the reference population at a statistically significant amount, then the trait is predicted to be associated with that ETFB genotype, haplotype or haplotype pair. Preferably, the ETFB genotype, haplotype, or haplotype pair being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables^' 4 and 5, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.

In a preferred embodiment of the method, the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting ETFB or response to a therapeutic treatment for a medical condition. As used herein, "medical condition" includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders. As used herein the term "clinical response" means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects). In order to deduce a correlation between clinical response to a treatment and an ETFB genotype, haplotype, or haplotype pair, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population". This clinical data may be obtained by analyzing the results of a clinical trial that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials. As used herein, the term "clinical trial" means any research study designed to collect clinical data on responses to a particular treatment, and includes but is hot limited to phase I, phase II and phase III clinical trials. Standard methods are used to define the patient population and to enroll subjects.

It is preferred that the individuals included in the clinical population have been graded for the existence of the medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome. This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.

The therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses. In addition, the ETFB gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.

After both the clinical and polymoφhism data have been obtained, correlations between individual response and ETFB genotype or haplotype content are created. Correlations may be produced in several ways. In one method,, individuals are grouped by their ETFB genotype or haplotype (or haplotype pair) (also referred to as a polymoφhism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymoφhism group are calculated.

These results are then analyzed to determine if any observed variation in clinical response between polymoφhism groups is statistically significant. Statistical analysis methods which may be used are described in L.D. Fisher and G. vanBelle, "Biostatistics: A Methodology for the Health Sciences", Wiley-Interscience (New York) 1993. This analysis may also include a regression calculation of which polymoφhic sites in the ETFB gene give the most significant contribution to the differences in phenotype. One regression model useful in the invention is described in PCT Application Serial No. PCT/US00/17540, entitled "Methods for Obtaining and Using Haplotype Data". A second method for finding correlations between ETFB haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms. One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry" in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997). Simulated annealing (Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K. Knight, "Artificial Intelligence", 2^nd Edition (McGraw-Hill, New York, 1991, Ch. 18), standard gradient descent methods (Press et al., supra, Ch. 10), or other global or local optimization approaches (see discussion in Judson, supra) could also be used. Preferably, the correlation is found using a genetic algorithm approach as described in PCT Application Serial No. PCT/US00/17540.

Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymoφhic sites in the ETFB gene. As described in PCT Application Serial No. PCT/USOO/17540, ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10).

From the analyses described above, a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of ETFB genotype or haplotype content. Preferably, the model is validated in one or more follow-up clinical trials designed to test the model.

The identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the ETFB gene may be the basis for designing a diagnostic method to determine those individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug. The diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymoφhic sites in the ETFB gene), a serological test, or a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying ETFB genotype or haplotype that is in turn correlated with the clinical response. In a preferred embodiment, this diagnostic method uses the predictive haplotyping method described above. In another embodiment, the invention provides an isolated polynucleotide comprising a polymoφhic variant of the ETFB gene or a fragment of the gene which contains at least one of the novel polymoφhic sites described herein. The nucleotide sequence of a variant ETFB gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymoφhic sites PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28, and may also comprise an additional polymoφhism of thymine at PS23. Similarly, the nucleotide sequence of a variant fragment of the ETFB gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymoφhic sites described herein. Thus, the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence of the ETFB gene, which is defined by haplotype 25, (or other reported ETFB sequences) or to portions of the reference sequence (or other reported ETFB sequences), except for genotyping oligonucleotides as described above. The location of a polymoφhism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO:l. The polymoφhism is selected from the group consisting of thymine at PSl, adenine at PS2, adenine at PS3, thymine at PS4, cytosine at PS5, cytosine at PS6, adenine at PS7, thymine at PS8, adenine at PS9, thymine at PS10, adenine at PSl 1, guanine at PS12, thymine at PS13, adenine at PS14, thymine at PS15, thymine at PS16, thymine at PS17, guanine at PS18, thymine at PS19, thymine at PS20, guanine at PS21, thymine at PS22, adenine at PS24, adenine at PS25, adenine at PS26, thymine at PS27 and cytosine at PS28. In a preferred embodiment, the polymoφhic variant comprises a naturally-occurring isogene of the ETFB gene which is defined by any one of haplotypes 1- 24 and 26 - 41 shown in Table 5 below.

Polymoφbic variants of the invention may be prepared by isolating a clone containing the ETFB gene from a human genomic library. The clone may be sequenced to determine the identity of the nucleotides at the novel polymoφhic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.

ETFB isogenes may be isolated using any method that allows separation of the two "copies" of the ETFB gene present in an individual, which, as readily understood by the skilled artisan, may be the . same allele or different alleles. Separation methods include targeted in vivo cloning (TIVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614. Another method, which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets. Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al., 1989, supra; Ruano et al., 1991, supra; Michalatos-Beloin et al., supra).

The invention also provides ETFB genome anthologies, which are collections of ETFB isogenes found in a given population. The population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population. An ETFB genome anthology may comprise individual ETFB isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the ETFB isogenes in the anthology may be stored in separate containers. Individual isogenes or groups of isogenes in a genome anthology may be stored in any convenient and stable form, including but not limited to in buffered solutions, as DNA precipitates, freeze-dried preparations and the like. A preferred ETFB genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 5 below.

An isolated polynucleotide containing a polymoφhic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded ETFB protein in a prokaryotic or a eukaryotic host cell. Examples of expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40. Other regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host cell. Of course, the correct combinations of expression regulatory elements will depend on the host system used. In addition, it is understood that the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers. Such expression vectors are commercially available or are readily . constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York). Host cells which may be used to express the variant ETFB sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art. The recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In a preferred aspect, eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used. Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, heφes virus vectors, and baculovirus transfer vectors. Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282:1145-1147). Particularly preferred host cells are mammalian cells.

As will be readily recognized by the skilled artisan, expression of polymoφhic variants of the ETFB gene will produce ETFB mRNAs varying from each other at any polymoφhic site retained in the spliced and processed mRNA molecules. These mRNAs can be used for the preparation of an ETFB cDNA comprising a nucleotide sequence which is a polymoφhic variant of the ETFB reference coding sequence shown in Figure 2. Thus, the invention also provides ETFB mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig. 2), or its corresponding RNA sequence, except for having one or more polymoφhisms selected from the group consisting of thymine at a position corresponding to nucleotide 452, adenine at a position corresponding to nucleotide 521 and adenine at a position corresponding to nucleotide 577, and may also comprise an additional polymoφhism of thymine at a position corresponding to nucleotide 461. A particularly preferred polymoφhic cDNA variant comprises the coding sequence of an ETFB isogene defined by haplotypes lc, 4c, 7c, 9c, 10c, 12c, 14c, 17c, 18c, 19c, 20c, 24c, 26c, 27c, 29c, 30c, 35c, 36c, 37c, and 39c. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymoφhisms described herein. The invention specifically excludes polynucleotides identical to previously identified and characterized ETFB cDNAs and fragments thereof. Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized. As used herein, a polymoφhic variant of an ETFB gene fragment comprises at least one novel polymoφhism identified herein and has a length of at least 10 nucleotides and may range up to the full length of the gene. Preferably, such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.

In describing the ETFB polymoφhic sites identified herein, reference is made to the sense strand of the gene for convenience. However, as recognized by the skilled artisan, nucleic acid molecules containing the ETFB. gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand. Thus, reference may be made to the same polymoφhic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymoφhic site. Thus, the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the ETFB genomic variants described herein.

Polynucleotides comprising a polymoφhic gene variant or fragment may be useful for therapeutic puφoses. For example, where a patient could benefit from expression, or increased expression, of a particular ETFB protein isoform, an expression vector encoding the isoform may be administered to the patient. The patient may be one who lacks the ETFB isogene encoding that isoform or may already have at least one copy of that isogene.

In other situations, it may be desirable to decrease or block expression of a particular ETFB isogene. Expression of an ETFB isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene. Alternatively, oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3 ' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred. Similarly, inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). Antisense oligonucleotides may also be designed to block translation of ETFB mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of ETFB mRNA transcribed from a particular isogene.

The oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo. Alternatively, the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient. Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life. Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guariine, thymine, and uracil which are not as easily recognized by endogenous nucleases. The invention also provides an isolated polypeptide comprising a polymoφhic variant of the reference ETFB amino acid sequence shown in Figure 3. The location of a variant amino acid in an ETFB polypeptide or fragment of the invention is identified by aligning its sequence against SEQ ID NO: 3 (Fig. 3). An ETFB protein variant of the invention comprises an amino acid sequence identical to SEQ ID NO:3 except for having one or more variant amino acids selected from the group consisting of phenylalariine at a position corresponding to amino acid position 151, histidine at a position corresponding to amino acid position 174 and threonine at a position corresponding to amino acid position 193, and may also comprise an additional variant amino acid of metMonine at a position corresponding to amino acid position 154. The invention specifically excludes amino acid sequences identical to those previously identified for ETFB, including SEQ ID NO:3, and previously described fragments thereof. ETFB protein variants included within the invention comprise all amino acid sequences based on SEQ ID NO: 3 and having the combination of amino acid variations described in Table 2 below. In preferred embodiments, an ETFB protein variant of the invention is encoded by an isogene defined by one of the observed haplotypes shown in Table 5. Table 2. Novel Polymoφhic Variants of ETFB

Polymoφhic Amino Acid I Position and Ic

Variant

Number 151 154 174 193

1 S T R T

2 S T H A

3 S T H T

4 S M R T

5 S M H A

6 S M H T

7 F T R A

8 F T R T

9 F T H A

10 F T H T

11 F M R A

12 F M R T

13 F M H A

14 F M H T The invention also includes ETFB peptide variants, which are any fragments of an ETFB protein variant that contain one or more of the amino acid variations shown in Table 2. An ETFB peptide variant is at least 6 amino acids in length and is preferably any number between 6 and 30 amino acids long, more preferably between 10 and 25, and most preferably between 15 and 20 amino acids long. Such ETFB peptide variants may be useful as antigens to generate antibodies specific for one of the above ETFB isoforms. In addition, the ETFB peptide variants may be useful in drug screening assays.

An ETFB variant protein or peptide of the invention may be prepared by chemical synthesis or by expressing one of the variant ETFB genomic and cDNA sequences as described above.

Alternatively, the ETFB protein variant may be isolated from a biological sample of an individual having an ETFB isogene which encodes the variant protein. Where the sample contains two different ETFB isoforms (i.e., the individual has different ETFB isogenes), a particular ETFB isoform of the invention can be isolated by immunoaffinity chromatography using an antibody which specifically binds to that particular ETFB isoform but does not bind to the other ETFB isoform. The expressed or isolated ETFB protein may be detected by methods known in the art, including Coomassie blue staining, silver staining, and Western blot analysis using antibodies specific for the isoform of the ETFB protein as discussed further below. ETFB variant proteins can be purified by standard protein purification procedures known in the art, including differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography and the like. (Ausubel et. al., 1987, In Current Protocols in Molecular Biology John Wiley and Sons, New York, New York). In the case of immunoaffinity chromatography, antibodies specific for a particular polymoφhic variant may be used.

A polymoφhic variant ETFB gene of the invention may also be fused in frame with a heterologous sequence to encode a chimeric ETFB protein. The non-ETFB portion of the chimeric protein may be recognized by a commercially available antibody. In addition, the chimeric protein may also be engineered to contain a cleavage site located between the ETFB and non-ETFB portions so that the ETFB protein may be cleaved and purified away from the non-ETFB portion.

An additional embodiment of the invention relates to using a novel ETFB protein isoform in any of a variety of drug screening assays. Such screening assays may be performed to identify agents that bind specifically to all known ETFB protein isoforms or to only a subset of one or more of these isoforms. The agents may be from chemical compound libraries, peptide libraries and the like. The ETFB protein or peptide variant may be free in solution or affixed to a solid support. In one embodiment, high throughput screening of compounds for binding to an ETFB variant may be accomplished using the method described in PCT application WO84/03565, in which large numbers of test compounds are synthesized on a solid substrate, such as plastic pins or some other surface, contacted with the ETFB protein(s) of interest and then washed. Bound ETFB protein(s) are then detected using methods well-known in the art.

In another embodiment, a novel ETFB protein isoform may be used in assays to measure the binding affinities of one or more candidate drugs targeting the ETFB protein. In yet another embodiment, when a particular ETFB haplotype or group of ETFB haplotypes encodes an ETFB protein variant with an amino acid sequence distinct from that of ETFB protein isoforms encoded by other ETFB haplotypes, then detection of that particular ETFB haplotype or group of ETFB haplotypes may be accomplished by detecting expression of the encoded ETFB protein variant using any of the methods described herein or otherwise commonly known to the skilled artisan. In another embodiment, the invention provides antibodies specific for and immunoreactive with one or more of the novel ETFB variant proteins described herein. The antibodies may be either monoclonal or polyclonal in origin. The ETFB protein or peptide variant used to generate the antibodies may be from natural or recombinant sources or produced by chemical synthesis using synthesis techniques known in the art. If the ETFB protein variant is of insufficient size to be antigenic, it may be conjugated, complexed, or otherwise covalently linked to a carrier molecule to enhance the antigenicity of the peptide. Examples of carrier molecules, include, but are not limited to, albumins (e.g., human, bovine, fish, ovine), and keyhole limpet hemocyanin (Basic and Clinical Immunology, 1991, Eds. D.P. Stites, and A.I. Terr, Appleton and Lange, Norwalk Connecticut, San Mateo, California).

In one embodiment, an antibody specifically immunoreactive with one of the novel protein isoforms described herein is administered to an individual to neutralize activity of the ETFB isoform expressed by that individual. The antibody may be formulated as a pharmaceutical composition which includes a pharmaceutically acceptable carrier.

Antibodies specific for and immunoreactive with one of the novel protein isoforms described herein may be used to immunoprecipitate the ETFB protein variant from solution as well as react with ETFB protein isoforms on Western or immunoblots of polyacrylamide gels on membrane supports or substrates. In another preferred embodiment,.the antibodies will detect ETFB protein isoforms in paraffin or frozen tissue sections, or in cells which have been fixed or unfixed and prepared on slides, coverslips, or the like, for use in immunocytochemical, immunohistochemical, and immunofluorescence techniques.

In another embodiment, an antibody specifically immunoreactive with one of the novel ETFB protein variants described herein is used in immunoassays to detect this variant in biological samples. In this method, an antibody of the present invention is contacted with a biological sample and the formation of a complex between the ETFB protein variant and the antibody is detected. As described, suitable immunoassays include radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme linked immunoassay (ELISA), chemiluminescent assay, immunohistochemical assay, immunocytochemical assay, and the like (see, e.g., Principles and Practice of Immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Press, New York, New York; Current Protocols in Molecular Biology, 1987, Eds. Ausubel et al., John Wiley and Sons, New York, New York). Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Ed., Eds. Rose and Bigazzi, John Wiley and Sons, New York 1980; and Campbell et al., 1984, Methods in Immunology, W.A. Benjamin, Inc.). Such assays may be direct, indirect, competitive, or noncompetitive as described in the art (see, e.g., Principles and Practice of immunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Pres, NY, NY; and Oellirich, M., 1984, J. Clin. Chem. Clin. Biochem., 22:895-904). Proteins may be isolated from test specimens and biological samples by conventional methods, as described in Current Protocols in Molecular Biology, supra. Exemplary antibody molecules for use in the detection and therapy methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, or those portions of immunoglobulin molecules that contain the antigen binding site. Polyclonal or monoclonal antibodies may be produced by methods conventionally known in the art (e.g., Kohler and Milstein, 1975, Nature, 256:495-497; Campbell Monoclonal Antibody Technology, the Production and Characterization of Rodent and Human Hybridomas, 1985, In: Laboratory Techniques in Biochemistry and Molecular Biology, Eds. Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam). The antibodies or antigen binding fragments thereof may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject of PCT patent applications, publication number WO 901443, WO 901443 and WO 9014424 and in Huse et al., 1989, Science, 246:1275-1281. The antibodies may also be humanized (e.g., Queen, C. et al. 1989 Proc. Natl. Acad. Sci/USA 86; 10029).

Effect(s) of the polymoφhisms identified herein on expression of ETFB may be investigated by preparing recombinant cells and/or nonhuman recombinant organisms, preferably recombinant animals, containing a polymoφhic variant of the ETFB gene. As used herein, "expression" includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into ETFB protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function. To prepare a recombinant cell of the invention, the desired ETFB isogene may be introduced • into the cell in a vector such that the isogene remains extrachromosomal. ^' In such a situation, the gene will be expressed by the cell from the extrachromosomal location. In a preferred embodiment, the ETFB isogene is introduced into a cell in such a way that it recombines with the endogenous ETFB gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired ETFB gene polymoφhism. Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner. Examples of cells into which the ETFB isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the ETFB isogene. Such recombinant cells can be used to compare the biological activities of the different protein variants. Recombinant nonhuman organisms, i.e., transgenic animals, expressing a variant ETFB gene are prepared using standard procedures known in the art. Preferably, a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one-cell stage, or generally not later than about the eight-cell stage. Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art. One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053. Another method involves directly injecting a transgene into the embryo. A third method involves the use of embryonic stem cells. Examples of animals into which the ETFB isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see "The Introduction of Foreign Genes into Mice" and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. ZoUer; W.H. Freeman and Company, New York, pages 254-272). Transgenic animals stably expressing a human ETFB isogene and producing human ETFB protein can be used as biological models for studying diseases related to abnormal ETFB expression and/or .activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases. An additional embodiment of the invention relates to pharmaceutical compositions for treating disorders affected by expression or function of a novel ETFB isogene described herein. The pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel ETFB isogenes; an antisense oligonucleotide directed against one of the novel ETFB isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel ETFB isogene described herein. Preferably, the composition contains the active ingredient in a therapeutically effective amount. By therapeutically effective amount is meant that one or more of the symptoms relating to disorders affected by expression or function of a novel ETFB isogene is reduced and/or eliminated. The composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose; and water. Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist. The pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound. Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intrayentricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

For any composition, determination of the therapeutically effective dose of active ingredient and/or the appropriate route of administration is well within the capability of those skilled in the art. For example, the dose can be estimated initially either in cell culture assays or in animal models. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment. Any or all analytical and mathematical operations involved in practicing the methods of the. present invention may be implemented by a computer. In addition, the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the ETFB gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymoφhism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations). The ETFB polymoφhism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymoφhism data may be stored on the computer's hard drive or may* for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.

Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

EXAMPLES The Examples herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the scope of the invention in any way. The Examples do not include detailed descriptions for conventional methods employed, such as in the performance of genomic DNA isolation, PCR and sequencing procedures; Such methods are well-known to those skilled in the art and are described in numerous publications, for example, Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2^nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989).

EXAMPLE 1 This example illustrates examination of various regions of the ETFB gene for polymoφhic sites.

Amplification of Target Regions

The following target regions were amplified using either the PCR primers represented below or 'tailed' PCR primers, each of which includes a universal sequence forming a noncomplementary 'tail' attached to the 5 ' end of each unique sequence in the PCR primer pairs. The universal 'tail' sequence for the forward PCR primers comprises the sequence 5 '-TGTAAAACGACGGCCAGT-3 ' (SEQ ID NO: 139) and the universal 'tail' sequence for the reverse PCR primers comprises the sequence 5 '- AGGAAACAGCTATGACCAT-3' (SEQ ID NO: 140). The nucleotide positions of the first and last nucleotide of the forward and reverse primers for each region amplified are presented below and correspond to positions in Figure 1.

PCR Primer Pairs

Fragment No. Forward Primer Reverse Primer PCR Product Fragment 1 3209-3230 complement of 3844-3824 635 nt Fragment 2 3530-3559 complement of 4169-4149 640 nt Fragment 3 3757-3778 complement of 4212-4192 456 nt Fragment 4 15854-15876 complement of 16424-16402 571 nt Fragment 5 16857-16876 complement of 17358-17336 502 nt Fragment 6 19785-19807 complement of 20161-20139 377 nt Fragment 7 23098-23120 complement of 23633-23611 536 nt Fragment 8 24901-24922 complement of 25420-25398 520 nt

These primer pairs were used in PCR reactions containing genomic DNA isolated from immortalized cell lines for each member of the Index Repository. The PCR reactions were carried out under the following conditions:

Reaction volume = 10 μl

10 x Advantage 2 Polymerase reaction buffer (Clontech) = l μl

100 ng of human genomic DNA = l μl

10 mM dNTP = 0.4 μl

Advantage 2 Polymerase enzyme mix (Clontech) = 0.2 μl

Forward Primer (10 μM) = 0.4 μl

Reverse Primer (10 μM) = 0.4 μl

Water = 6.6μl

Amplification profile: 97°C - 2 min. 1 cycle

97°C - 15 sec. 70°C - 45 sec. 10 cycles 72°C - 45 sec.

97°C - 15 sec. 64°C - 45 sec. 35 cycles 72°C - 45 sec.

Sequencing of PCR Products

The PCR products were purified using a Whatman/Polyfiltronics 100 μl 384 well unifϊlter plate essentially according to the manufacturers protocol. The purified DNA was eluted in 50 μl of distilled water. Sequencing reactions were set up using Applied Biosystems Big Dye Terminator chemistry essentially according to the manufacturers protocol. The purified PCR products were sequenced in both directions using either the primer sets represented below with the positions of their first and last nucleotide corresponding to positions in Figure 1, or the appropriate universal 'tail' sequence as a primer. Reaction products were purified by isopropanol precipitation, and run on an Applied Biosystems 3700 DNA Analyzer. Sequencing Primer Pairs

Fragment No. Forward Primer Reverse Primer Fragment 1 3285-3303 complement of 3816-3797 Fragment 2 Tailed Sequence Fragment 3 3825-3844 complement of 4188-4170 Fragment 4 15908-15925 complement of 16322-16303 Fragment 5 16919-16939 complement of 17329-17310 Fragment 6 19809-19828 complement of 20137-20118 Fragment 7 23158-23177 complement of 23562-23543 Fragment 8 24932-24951 complement of 25396-25377

Analysis of Sequences for Polymoφhic Sites

Sequence information for a minimum of 80 humans was analyzed for the presence of polymoφhisms using the Polyphred program (Nickerson et al., Nucleic Acids Res. 14:2745-2751, 1997). The presence of a polymoφhism was confirmed on both strands. The polymoφhisms and their locations in the ETFB gene are listed in Table 3 below.

Table 3. Polymoφhic Sites Identified in the ETFB Gene

Polymoφhic Nucleotide Reference Variant CDS Variant AA

Site Number Polyld³ Position Allele Allele Position Variant

PSl 767426' 3272 C T

PS2 767422 3434 G A

PS3 767420 3472 G A

PS4 8889542 3767 C T

PS5 767413 3772 T C

PS6 767411 3844 T C

PS7 767409 3907 G A

PS8 767407 3913 C T

PS9 8890111 3944. G A

PS10 767405 3956 C T

PS11 767397 16200 G A

PS12 767395 16953 C G

PS13 767393 16963 C T

PS14 767385 16990 G A

PS15 767383 17046 G T

PS16 767381 19924 C T

PS17 767377 20031 C T

PS18 - 767369 23146 C G

PS19 767367 23167 C T

PS20 767365 23191 C T

PS21 767363 23192 A G

PS22 767361 23295 C T 452 S151F

PS23^R 767359 23304 C T 461 T154M

PS24 767357 23364 G A 521 R174H

PS25 767355 23420 G A 577 A193T

PS26 767353 23473 G A

PS27 767349 25293 C T

PS28 767347 25387 ^: G C ^aPolyId is a unique identifier assigned to each PS by Genaissance Pharmaceuticals, Inc. ^RReported in the literature. EXAMPLE 2 This example illustrates analysis of the ETFB polymoφhisms identified in the Index Repository for human genotypes and haplotypes. The different genotypes containing these polymoφhisms that were observed in the reference population are shown in Table 4 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below. In Table 4, homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides. Missing nucleotides in any given genotype in Table 4 were inferred based on linkage disequilibrium and/or Mendelian inheritance.

Table 4 (Parti). Genotypes and Haplotype Pairs Observed for ETFB Gene Genotype Polymorphic Sites

Number PSl PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 HAP Pai:

1 C G .G C T T G C G C 33 33

2 C G G C T T ^'G c- G C 35 35

3 C G G C T T G C G C 26 26

4 C G G C T T G C G C 25 25

5 C G G C ^' T c G C G C 15 11

6 C G G C T T ^• G/A C G C 30 18

7 . C G G C T T/C G C G C . 30 14

^'8 T/C G G C T c G C G C 40 16

9 C G- G C T/C T G C G C 33 6

. 10 C G G C - T T G C G C 33 ^• 24

11 C G G C T T G C G - C 33 22

12 C G G C T T G C G/A C 33 21

13 C G G C T/C T G C G C 33 7

14 C G G C T T G C . G C 33 32

15 C G G C T/C T G/A C/T G C 31 5

16 C G G C T T/C G C G C 35 17

17 C G G/A C T T G C G C 33 3

18 C G G C T T G . C - G C 25 28

19 C G G C T T G C G C 35 26

20 C G G C T T G/A C G C 35 20

21 C G G C/T T T G C G C 27 39

22^' C G G C T T/C G C G C 35 12

23 c G G/A C T T G C G c 33 2

24 c G G C T/C T G/A C G c 28 4

25 c G G C T T/C G/A c G c 31 8

26 c G G c T C/T G c G c 15 31

27 c G G c T T G/A ^' c G c 35 19

28 c G G c - T T G c G c 33 •36

29 c G G c T T G c G c 35 37

30 c G G c T T/C G . c G c 33 10

31 c G G c T T/C G c G . c 33 13

32 c G/A G c T T G ' c G c 35 1

33 c G G c T T G c G c 35 27

34 c G G c T T G c G c 35 29

35 C/T G ^•G c T T/C G c G c 33 40

36 c G G c T T G c G C/T 33 38

37 c G G c T T G c G c 33 28

38 c G G c T T G c G c 33 23

39 c G ^' G c T T/C G c G c^* 35 ^• 9

40 c G G c T T G c G c 33 25

41 C/T G G c T T/C G c G c 33 41

42 c G G c T C/T G c G c 16 34 Table 4(Part2). Genotypes and Haplotype Pairs Observed for ETFB Gene Genotype Polymorphic Sites

Number PS11 PS12 PS13 PS14 PS15 PS16 PS17 PS18 PS19 PS20 HAP Pair

1 G G T G G C c c c c 33 33

2 G G T G G c c c c c 35 35

3 G C C G G C c c c c 26 26

4 G C C G G^' C c c c c 25 25

5 G G T G/A G C c c c c 15 11

^' 6 G G/C T/C G G C c c c c 30 18

7 G G T G ^•G C ^"c c c c 30 14

8 G G T A/G G C ^■ c c c c 40 16

9 G G/C T/C G G C c c c c 33 6

^• 10 G G/C T/C G G C c ^' c c c 33 ^{' 1} 24

11 G/A G T G G C c c c c 33 22

12 G G T G G C c c c c 33 21

13 G G T G G . C c c c c 33 7

14 G G T G G C c c c c 33 32

15 .- G G T G/A G . C c c c c 31 5

^' 16 G G T G G C/T c c c c 35 17

17 G G T G G c c c c c 33 3

18 G C/G C/T G/A G c . c c c c 25 28

19 ^• G G/C T/C G G ' c c c c c 35 ^' 26

20 G G T G G c c c c c 35 20

21 G C c G G c ^■ c c T c 27 ^•39

22 G G T G/A G c c c c c 35 12

23 G G/C T/C G G c c c c c 33 2

24 G G T A G c C/T c c c 28 4

25 G G T G/A G c C/T c c c 31 8

26 G G T G G c c c c c 15 31

27 G G T G/A G c c^* C/G c c 35 19

28 G G T G G - c c c c c 33 36

29 G G T G G c c c c c 35 37

30 G G T G/A - G c c c c c 33 10

31 G ^" G T G/A G/T c C/T c c c 33 13

32 G G/C T/C G/A G c c c c c 35^' 1

33 G . G/C T/C G G c c c C/T c 35 27

34 G G T G/A G c c C/G c c 35 29

35 G G T G/A G c c c c c 33 40

36 G G T G G c c c c c 33 38

37 G G T G/A G c c c c c ^'33 ■ 28

38 G G/C T/C G/A G c c c c C/T 33 23-

39 G G/C T/C G G c c c - c c 35 9

40 G G/C T/C G G c c c c c 33 25

41 G G - T - G G c c c c ^• c 33 41

42 G G T G G c c c c c 16 34 A(Part3) . Genotypes and Haplotype Pairs Observed for ETFB Gene Genotype Polymorphic Sites

Number PS21 PS22 PS23 PS24 PS25 PS26 PS27 PS28 HAP Pair -

I A C C G G G C G 33 33 2 A C T G G G C G 35 35

3- G C T G G G C G 26 26

4 A ^'C C G G G C G 25 25

5 A C C G G A C G 15_, 11 ^'

6 A C C A G A C G 30 18 7 A C C A G A C G 30 14

8 A C C G G G C G 40 16

9 A C C G G G C G 33 6

10 A C C G G/A G C G 33 24

II ^' A C C - G G G C G 33 22 12 A C C G G G C G^' 33 21

13 A ^' _.C C/T G G G C/T G 33 ^' 7

14 A . C C G G G C G/C 33 32

15 A C C G G A C G 31 5

16 A/G C- T G G G C G 35 17 17 A C C G G G C ^' G 33 3

18 A C C G G G C G 25^' 28

19 A/G C T G G G C G 35 26^'

20 A C . T G ^' G G C G 35 - 20

21 G C T G G G C ^' G 27 39 22 A C T G G G C G 35 12

23 ^' A C C G G G C G 33 2

24 C C/T G G G C G 28 4

25 A C C G G A C G 31 8

26 A C C G G A C G 15 31 27^' A C T G G G C G 35 19

28 A C/T C G G G C G 33 36

29 A/G C T G G G C G 35 37

30 A C C G/A G G/A C G 33 10

31 A C C G G G C G 33 13 32 A C T G G G C G 35 1

33 ^' A/G^' C T G G G C G 35 27

34 A C T G G G C G 35 29

35 A C C G G G C G 33 40

36 A C C G G G C G 33 38 37 A C C G G G C G 33 28

38 A C C G G G C G 33 23

39 A/G C T G G G C G 35 ^' 9

40 A C C G G G C G 33 25

41 .. _. A C G G G G C G 33 41 42 ^' A C C G G G C/T G 16 34

The haplotype pairs shown in Table 4 were estimated from the unphased genotypes using a computer-implemented extension of Clark's algorithm (Clark, A.G.1990 Mol Bio Evol 7, 111-122) for assigning haplotypes to unrelated individuals in a population sample, as described in U.S. Provisional ^' Application Serial No.60/198,340 entitled "A Method and System for Determining Haplotypes from a

Collection of Polymoφhisms" and the corresponding International Application, PCT/US01/12831. In this method, haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites. This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals. In our analysis, the list of haplotypes was augmented with haplotypes obtained from two families (one three- generation Caucasian family and one two-generation African-American family).

By following this protocol, it was determined that the Index Repository examined herein and, by extension, the general population contains the 41 human ETFB haplotypes shown in Table 5 below.

An ETFB isogene defined by a full-haplotype shown in Table 5 below comprises the regions of the SEQ ID NOS indicated in Table 5, with their corresponding set of polymoφhic locations and identities, which are also set forth in Table 5'.

^aAUeles for ETFB haplotypes are presented 5 ' to 3 ' in each column ^bPS = polymoφhic site; ^cPosition of PS within the indicated SEQ ID NO, with the Imposition number referring to the first SEQ ID NO and the 2^nd position number referring to the 2^nd SEQ ID NO; ^dl^st SEQ ID NO refers to Figure 1, with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol; 2^nd SEQ ID NO is a modified version of the

1^st SEQ ID NO that comprises the context sequence of each polymoφhic site, PS1-PS28, to facilitate electronic searching of the haplotypes; ^eRegion examined represents the nucleotide positions defining the start and stop positions within the 1^st SEQ ID NO of the sequenced region.

SEQ ID NO: 1 refers to Figure 1 , with the two alternative allelic variants of each polymoφhic site indicated by the appropriate nucleotide symbol. SEQ ID NO: 141 is a modified version of SEQ JD NO:l that shows the context sequence of each of PS1-PS28 in a uniform format to facilitate electronic searching of the ETFB haplotypes. For each polymoφhic s.ite, SEQ ID NO: 141 contains a block of 60 bases of the nucleotide sequence encompassing the centrally-located polymoφhic site at the 30^th position, followed by 60 bases of unspecified sequence to represent that each polymoφhic site is separated by genomic sequence whose composition is defined elsewhere herein. 42". 42. OJ OJ t t o © o o

n 42- 42- OJ OJ to to

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ethnogeographic background. The number of unrelated individuals in the Index Repository provides a sample size that is sufficient to detect SNPs and haplotypes that occur in the general population with high statistical certainty. For instance, a haplotype that occurs with a frequency of 5% in the general population has a probability higher than 99.9% of being observed in a sample of 80 individuals from the general population. Similarly, a haplotype that occurs with a frequency of 10% in a specific population group has a 99% probability of being observed in a sample of 20 individuals from that population group. In addition, the size and composition of the Index Repository means that the relative frequencies determined therein for the haplotypes and haplotype pairs of the ETFB gene are likely to be similar to the relative frequencies of these ETFB haplotypes and haplotype pairs in the general U.S. population and in the four population groups represented in the Index Repository. The genetic diversity observed for the three Native Americans is presented because it is of scientific interest, but due to the small sample size it lacks statistical significance.

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained. As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be inteφreted as illustrative and not in a limiting sense.

All references cited in this specification, including patents and patent applications, are hereby incoφorated in their entirety by reference. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

Claims

What is Claimed is:

1. A method for haplotyping the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene of an individual, which comprises determining which of the ETFB haplotypes shown in the table immediately below defines one copy of the individual's ETFB gene, wherein each of the ETFB haplotypes comprises a set of polymoφhisms whose locations and identities are set forth in the table immediately below:

Haplotype Number³ PS^b PS

1 2 3 4 5 6 7 8 9 ^" 10 Number Position' c C . C C C C C C C C 1 3272

A G G G G G G G G G 2 ^' 3434

G A A^' G ^' G G G G G G 3 3472

C C C C C C C C C C 4 3767

T T T C C C C T T T 5 3772

T T T T T T T C c C 6 3844

G G G A A G G A G G 7 3907

C C C C T C C C C C 8. 3913

G G G G G G G G G G 9 3944

C C C C C C C C C C ^■ 10 3956

G G G G G G G G G G 11 16200

C C G G G C G G C G 12 16953

C C T T T C T T C T 13 16963

A G G A A G G A G A 14 16990

G G G G G G G G G G 15 17046

C C C C C C C C C C 16 19924

C C C T C C C T C C 17 20031 c C C C C C C c C C 18 23146 c C C C C C C c C C 19 23167. c c C C .C C C c C C . 20 23191

A A A- A A A A A G A 21 23192

C C C C C C C C C C 22 23295

T C C T C C T C T C 23 23304

G G G G G G G . G G A 24 23364

G G G G G G G G G G 25 23420

G G G G A G. G A G A 26 23473

C C C C C C T C C C 27 25293

G G G G G G G G G G 28 25387

Haplotype Number³ PS^b PS

11 12 13 14 15 16 17 18 19 ^'20 Number Position' c C C C C C C C C C l^' 3272

G G G G G G G G G G 2 3434

G G G G G G G G G G 3 3472

C C C C C C C C C C 4 3767

T T T T T T T T. T ^'T 5 3772 c C c C c c c T T T 6 3844

G G G G G G G A A A . 7 3907

C C C C C C C C C C 8 3913

G G G G G G G G G G 9 3944

C C C C C C C C C C 10 3956

G G G G G G ^' G G G G 11 16200

G G G G G G G C G G 12 16953

T T T T T T T C T T 13 16963

A A A G G G G G A G 14 16990

G G T G G G G G G G 15 17046

C C c C C C T C C C 16 19924

C C T C C C C C C C 17 20031

C C c C C C C C G C 18 23146

C C c C C C C C C C 19 23167

C C c C C C C C C C 20 23191

A A A A . A A G A A A 21 23192

C C C C C ^' ' C C C C C 22 23295

C T C C c C T C T T 23 23304

G G G A^' G G G A G G 24 23364

G G G G G G G G G G 25 23420

A G G A A G G A G G 26 23473

C C C C C C C C C C 27 25293

G G G G G G ^' G G G G 28 25387

Haploitype Number³ PS^b PS

21 22 23 24 25 26 27 28 29 30 Number Position'

C C C C C C C C C C 1 3272

G G G G G G G G G G 2 3434

G G G G G G G G G G 3 3472

C C C C C C C 'C C C 4 3767

T T T T T T • T T T T 5 3772

T T T T T T T T T T 6 3844

G G G G G G G G G G 7 3907

C C C C C C C C C C 8 3913

A G G G G G G G G ^'G 9 3944

C C C C C C C C C C 10 3956

G A G G G G , G G G G 11 16200

G G C C C C C G G G 12 .16953

T T C C C C C T T T 13 16963

G G A G G G G A A G 14 16990

G G G G G G G G G G 15 1-7046

C C C ■ C C C C C C C 16 19924

C C C C C C C C C C 17 20031 c C C C C C C c G C 18 23146 c C C C c C T c C C 19 23167 c C T C c C c c C C 20 23191

A A A - A A G G A A A 21 23192

C C C C C C C C C C 22 23295 c C C C C T T C T C 23 23304

G G G G G G G G G A 24 23364

G G G A G G G G G G 25 23420

G G G G G G G G ^•G A 26 23473

C C C C C C C C C C 27 25293

G G G G G G G G G G 28 25387 Haplotype Number^a PS^b PS

31 32 33 34 35 36 37 38 39 40 41 Number Position⁰

C C C C C C C C C T T 1 3272

G G G G G G G G G G G 2 3434

G G G G G G G G G G G 3 3472

C C C C C C C C ^'T C C 4 3767

T T T T T T T T T T T 5 3772

T T T T T T T T T C C 6 3844

G G G G G G G G G G G 7 • 3907

C C C C C C C C C C C 8- ^' 3913

G ^■ G G G G G G ^■ G G G G 9 3944

C C C C C C C T C C C 10 3956

G G G G G G G G G G G 11 16200

G G G G G G G G C G G 12 • 16953

T T T T T T T T C T T 13 16963

G G G G G G G G G A G 14, 16990

G G G G G G G G G G G 15 17046

C C C C C C C C C C C 16 19924

C C C C C C C ^■ C C C C 17 20031 c C C C C C C C C C C 18 23146 c C c C C C C C T C C 19 23167 c C c C C C C C C C C 20 23191

A A A A A A G A G A A 21 23192

C C C C C T C C C C C 22 23295

C C C C ■ T C T C T C C 23 23304

G G G G G G G G G G G 24 23364

G G G G G G G G G G G 25 23420

A G G G G G G G G G G 26 23473

C C C T C C ' ^■c C C C C 27 25293

G G CC G G G G G G G G G 28 25387 ^aAlleles for haplotypes are presented 5 ' to 3 ' in each column ^bPS = polymoφhic site;

Position of PS within SEQ ID NO:l.

2. The method of claim 1, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PS1-PS28 on the one copy of the individual's ETFB gene.

3. A method for haplotyping the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene of an individual, which comprises determining which of the ETFB haplotype pairs shown in the table immediately below defines both copies of the individual's ETFB gene, wherein each of the ETFB haplotype pairs consists of first and second haplotypes which comprise first and second sets of polymoφhisms whose locations and identities are set forth in the table immediately below: Haplotype Paira PS^b PS

33/33 35/35 26/26 25/25 15/11 30/18 30/14 40/16 Number Position'

C/C C/C C/C C/C C/C C/C C/C T/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G . 2 3434

G/G G/G G/G G/G G/G G/G G/G . G/G 3 3472.

C/C C/C C/C C/C C/C • C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/T T/T T/T T/T C/C . T/T T/C C/C 6 3844

G/G G/G G/G G/G G/G G/A G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G G/G C/C C/C G/G ■ G/C G/G G/G 12 16953

T/T T/T C/C C/C T/T.- T/C T/T T/T 13 16963

G/G G/G G/G G/G G/A G/G G/G A/G 14 ^'16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A G/G A/A A/A A/A A/A A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C T/T T/T C/C C/C C/C C/C C/C 23 23304

G/G G/G G/G G/G G/G A/A A/A G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G A/A A/A A/A G/G 26 23473.

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS 3/6 33/24 33/22 ! 33/21 33/7 33/32 . 31/5 35/17 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C - C/C C/C C/C C/C 4 3767

T/C- T/T T/T T/T T/C ^■ T/T T/C T/T 5 3772 -

T/T T/T T/T T/T T/T T/T T/T T/C 6 3844

G/G G/G G/G G/G G/G G/G G/A G/G 7 ^' 3907

-C/C C/C C/C C/C C/C C/C C/T C/C 8 3913

G/G G/G G/G G/A G/G G/G G/G G/G . 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/A G/G G/G G/G G/G G/G 11 16200

G/C G/C G/G G/G G/G G/G G/G G/G 12 16953

T/C T/C ,T/T T/T T/T T/T T/T T/T 13 16963

G/G G/G G/G G/G G/G G/G G/A G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/T 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A -A/A A/A A/A A/G 21 ^' 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C C/C C/C C/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/A G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G A/A G/G 26 23473

C/C C/C C/C C/C C/T C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/C G/G G/G 28 25387 Haplotype Pair³ PS^b PS

33/3 25/28 35/26 35/20 27/39 35/12 33/2 28/4 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434 ^' G/A G/G G/G G/G G/G G/G G/A G/G 3 3472

C/C . C/C C/C C/C C/T C/C C/C C/C 4 -3767

T/T T/T .T/T T/T T/T T/T T/T T/C 5 3772

T/T T/T T/T T/T , T/T T/C T/T T/T 6 3844

G/G^' G/G G/G G/A G/G G/G G/G G/A 7 - 3907 C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G. G/G G/G G/G G/G 11 16200

G/G C/G G/C G/G C/C G/G G/C G/G 12 16953 T/T C/T T/C T/T C/C T/T T/C T/T 13 16963

G/G G/A G/G G/G G/G G/A G/G A/A 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/T 17 20031 C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C T/T C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/G A/A G/G A/A A/A A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295 C/C C/C T/T T/T T/T T/T C/C C/T 23 23304

G/G G/G G/G G/G G/G G/G- G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293 G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ ' PS^b PS

31/8 15/31 35/1933/36 35/37 33/10 33/13 35/1 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C . 1 3272 G/G G/G G/G G/G G/G G/G G/G G/A ^' 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/C C/T T/T T/T T/T T/C T/C T/T 6 3844 G/A G/G G/A G/G G/G G/G G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C .8 3913

G/G G/G G/G G/G . G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200 G/G G/G G/G G/G G/G G/G G/G G/C 12 16953

T/T T/T T/T T/T T/T T/T T/T T/C 13 16963

G/A G/G G/A G/G G/G G/A G/A G/A 14 16990

G/G G/G G/G G/G G/G G/G G/T G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924 C/T C/C C/C C/C C/C C/C C/T C/C 17 20031 ^'

C/C C/C C/G C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/G A/A A/A A/A 21 23192 C/C C/C C/C C/T C/C C/C C/C C/C 22 23295

C/C C/C T/T C/C T/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/A G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

A/A A/A G/G G/G G/G G/A G/G G/G 26 23473 C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387 Haplotype Pair³ PS^b PS

35/27 35/29 33/40 33/3833/28 33/23 35/9 33/25 Number Position⁰

C/C C/C C/T C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434 G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T -T/T T-/T T/T T/T T/T T/T 5 3772

T/T T/T T/C T/T T/T T/T T/C T/T 6 3844

G/G G/G G/G G/G G/G G/G G/G G/G 7 3907 C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/T C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/C G/G G/G G/G G/G G/C G/C G/C 12 16953 T/C T/T T/T T/T T/T T/C T/C T/C 13 16963

G/G G/A G/A G/G G/A G/A G/G G/G 14 16990

G/G G/G G/G • G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C ^' 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031 C/C C/G C/C C/C C/C C/C C/C C/C 18 23146

C/T C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/T C/C^" C/C 20 23191

A/G A/A A/A A/A A/A A/A A/G A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295 T/T T/T C/C C/C C/C C/C T/T C/C 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G, G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293 G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS

33/41 16/34 Number Position

C/T C/C 1 3272 G/G G/G 2 3434

G/G G/G 3 3472

C/C C/C 4 3767

T/T T/T 5 3772

T/C C/T 6 3844 G/G G/G 7 3907

C/C C/C 8 3913

G/G G/G 9 3944

C/C C/C 10 3956

G/G G/G 11 16200 G/G G/G 12 16953

T/T T/T 13 16963

G/G G/G 14 16990

G/G G/G 15 17046

C/C C/C 16 19924 C/C C/C 17 20031

C/C C/C 18 23146

C/C C/C 19 23167

C/C C/C 20 23191

A/A A/A 21 23192 C/C C/C 22 23295

C/C C/C 23 23304

G/G G/G 24 23364

G/G G/G 25 23420

G/G G/G 26 23473 C/C C/T 27 25293

G/G G/G 28 25387 ^aHaplotype pairs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column; ^bPS = polymoφhic site; "Position of PS in SEQ ID NO: 1.

4. The method of claim 3, wherein the determining step comprises identifying the phased sequence of nucleotides present at each of PS1-PS28 on both copies of the individual's ETFB gene.

5. A method for genotyping the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene of an individual, comprising determining for the two copies of the ETFB gene present in the individual the identity of the nucleotide pair at one or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and

PS28, wherein the one or more PS have the location and alternative alleles shown in SEQ ID NO:l.

6. The method of claim 5, wherein the determining step comprises:

(a) isolating from the individual a nucleic acid mixture comprising both copies of the ETFB gene, or a fragment thereof, that are present in the individual;

(b) amplifying from the nucleic acid mixture a target region containing the selected polymoφhic site;

(c) hybridizing a primer extension oligonucleotide to one allele of the amplified target region;

(d) performing a nucleic acid template-dependent, primer extension reaction on the hybridized genotyping oligonucleotide in the presence of at least two different terminators of the reaction, wherein said terminators are complementary to the alternative nucleotides present at the selected polymoφhic site; and

(e) detecting the presence and identity of the terminator in the extended genotyping oligonucleotide.

7. The method of claim 5, which comprises determining for the two copies of the ETFB gene present in the individual the identity of the nucleotide pair at each of PS1-PS28.

8. A method for haplotyping the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene of an individual which comprises determining, for one copy of the ETFB gene present in the individual, the identity of the nucleotide at two or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28, wherein the selected PS have the location and alternative alleles shown in SEQ ID NO: 1.

9. The method of claim 8, further comprising determining the identity of the nucleotide at PS23, which has the location and alternative alleles shown in SEQ ID NO:l.

10. The method of claim 8, wherein the determining step comprises: (a) isolating from the individual a nucleic acid sample containing only one of the two copies of the ETFB gene, or a fragment thereof, that is present in the individual;

(b) amplifying from the nucleic acid sample a target region containing the selected polymoφhic site;

(c) hybridizing a primer extension oligonucleotide to one allele of the amplified target region;

(d) performing a nucleic acid template-dependent, primer extension reaction on the hybridized genotyping oligonucleotide in the presence of at least two different terminators of the reaction, wherein said terminators are complementary to the alternative nucleotides present at the selected polymoφhic site; and

(e) detecting the presence and identity of the terminator in the extended genotyping oligonucleotide.

11. A method for predicting a haplotype pair for the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene of an individual comprising:

(a) identifying an ETFB genotype for the individual, wherein the genotype comprises the nucleotide pair at two or more polymoφhic sites (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PSl 1, PS12, PS13, PS14, PS15,

PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28, wherein the selected PS have the location and alternative alleles shown in SEQ ID NO: 1 ;

(b) enumerating all possible haplotype pairs which are consistent with the genotype;

(c) comparing the possible haplotype pairs to the haplotype pair data set forth in the table immediately below; and

(d) assigning a haplotype pair to the individual that is consistent with the data

Haplotypes Pair^;a PS^b PS

33/33 35/35 26/26 25/25 15/11 30/18 30/14 40/16 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C T/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G - 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 • 3772

T/T T/T T/T T/T C/C T/T T/C C/C 6 3844

G/G G/G G/G G/G G/G G/A G/G G/G - 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C ^' C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G G/G C/C ^' C/C G/G G/C G/G G/G 12 16953

T/T ^• _τ/_τ C/C C/C T/T T/C T/T T/T 13 16963

G/G G/G G/G G/G G/A G/G G/G A/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C ^' C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/G C/C C/C C/C C/C .20 23191

A/A A/A G/G A/A A/A A/A A/A A/A 21 231.92

C/C C/C C/C C/C C/C C/C- C/C C/C 22 23295

C/C T/T T/T C/C C/C C/G C/C C/C 23 23304

G/G G/G G/G G/G G/G A/A A/A G/G 24 2.3364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G A/A A/A A/A G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS 3/6 33/24 33/22 : 33/21 33/7 33/32 31/5 35/17 Number Position⁰

C/C C/C C/C c/c C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G -G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/C T/T T/T T/T T/C T/T T/C T/T 5 3772

T/T T/T T/T T/T T/T T/T T/T T/C 6 3844

G/G G/G G/G G/G G/G G/G G/A G/G 7 3907

C/C C/C C/C C/C C/C C/C C/T C/C 8 3913

G/G G/G G/G G/A G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/A G/G G/G G/G G/G G/G 11 16200

G/C G/C G/G G/G G/G G/G G/G G/G 12 16953

T/C T/C T/T T/T T/T T/T T/T T/T 13 16963

G/G G/G G/G G/G G/G G/G G/A G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C. C/C C/C C/T 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167.

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/A A/A A/A A/G 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C C/C C/C C/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/A G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G A/A G/G 26 23473

C/C C/C C/C C/C C/T C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/C G/G G/G 28 25387 Haplotype Pair³ PS^b PS

33/3 25/28 35/26 35/20 27/39 35/12 33/2 28/4 Number. Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/A G/G G/G G/G G/G G/G G/A G/G 3 3472

C/C C/C C/C C/C C/T C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/C 5 3772

T/T T/T T/T T/T T/T T/C T/T T/T 6 3844

G/G G/G G/G G/A G/G G/G G/G G/A 7 3907

C/C C/C C/C C/C ^'C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G. G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G C/G G/C G/G C/C G/G G/C G/G 12 16953

T/T C/T T/C T/T C/C T/T T/C T/T 13 16963

G/G G/A G/G G/G G/G G/A G/G A/A 14 16990

G/G G/G G/G G/G^' G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/T 17 20031

C/C C/C C/C C/C C/C C/C C/c C/C 18 23146

C/C C/C C/C C/C T/T C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A Α/A A/G A/A G/G A/A A/A A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C T/T T/T T/T T/T C/C C/T 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G. G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS

31/8 15/31 35/19 33/36 35/37 33/10 33/13 35/1 Number Position'

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/A 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/C C/T T/T T/T T/T T/C T/C T/T 6 ' 3844

G/A G/G G/A G/G G/G G/G G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C. C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G G/G G/G G/G G/G G/G G/G G/C 12 16953

T/T T/T T/T T/T T/T T/T T/T T/C 13 16963

G/A G/G G/A G/G G/G G/A G/A G/A 14 16990

G/G G/G G/G G/G G/G G/G G/T G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/T C/C C/C C/C C/C C/C C/T C/C 17 20031

C/C C/C C/G C/C C/C C/C C/C C/C 1^'8 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/G A/A A/A A/A 21 23192

C/C C/C C/C C/T C/C C/C C/C C/C 22 23295

C/C C/C T/T C/C T/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/A G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

A/A A/A G/G G/G G/G G/A G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387 Haplotype Pair³ PS^b PS

35/27 35/29 33/40 33/38 33/28 33/23 35/9 33/25 Number Position⁰

C/C C/C C/T C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/T T/T T/C T/T T/T T/T T/C τ/τ^' 6 3844

G/G G/G G/G G/G G/G G/G G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/T C/C C/C C/C C/C 10 3956-

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/C ^■ G/G G/G G/G G/G G/C G/C G/C 12 16953

T/C T/T T/T T/T T/T T/C T/C T/C 13 16963

G/G G/A G/A G/G G/A G/A G/G G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/G C/C C/C C/C C/C C/C C/C 18 23146

C/T C/C. C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/T C/C C/C 20 23191

A/G A/A A/A A/A A/A A/A A/G A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

T/T T/T C/C C/C C/C C/C T/T C/C 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387 laplotype Pair³ PS PS

33/41 16/34 Number^b Position⁰

C/T C/C 1 3272

G/G ^■ G/G 2 3434

G/G G/G 3 3472

C/C C/C 4 3767

T/T T/T 5 3772

T/C C/T 6 3844

G/G G/G 7 3907

C/C C/C 8 3913

G/G G/G 9 3944

C/C C/C 10 3956

G/G G/G 11 16200

G/G G/G 12 16953

T/T T/T 13 16963

G/G G/G 14 • 16990

G/G G/G 15. 17046

C/C C/C 16 19924

C/C C/C 17 20031

C/C C/C 18 . 23146

C/C C/C 19 23167

C/C C/C 20 23191

A/A A/A 21 23192

C/C C/C 22 23295

C/C C/C 23 23304

G/G G/G 24 23364

G/G G/G 25 23420

G/G G/G 26 23473

C/C C/T 27 25293

G/G G/G 28 25387 Ηaplotype pairs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column; ^bPS = polymoφhic site;

"Position of PS in SEQ ID NO: 1.

12. The method of claim 11, wherein the identified genotype of the individual comprises the nucleotide pair at each of PS1-PS28, which have the location and alternative alleles shown in SEQ ID NO:l.

13. A method for identifying an association between a trait and at least one haplotype or haplotype pair of the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene which comprises comparing the frequency of the haplotype or haplotype pair in a population exhibiting the trait with the frequency of the haplotype or haplotype pair in a reference population, wherein the haplotype is selected from haplotypes 1-41 shown in the table presented immediately below, wherein each of the haplotypes comprises a set of polymoφhisms whose locations and identities are set forth in the table immediately below:

Haplotype Number³ PS^b PS

1 2 3 4 5 6 7 8 9 10 Number Position' c C C C C C C C C C 1 3272

A G G G G G G G G G 2 3434

G A A G G G G G G G 3 3472

C C C C C C C C C C 4 3767

T T T C C C c T T T 5 3772

T T T T T T T c c c 6 3844

G G G A A G G A G G 7 3907

C C C C T C C C C C 8 ^• 3913

G G G G G G G G G G 9 3944

C C C C C C C C C C 10 3956

G G G G G G G G G G 11 16200

C C G G G C G G C G 12 ^' 16953

C C T T T C T T C T 13 16963

A G G A A G G A G A 14 16990

G G G G G G G G G G 15 17046

C C C C C . C C C C C 16 19924

C C C T C C C T C c 17 20031

C C C c C C C c c c 18 ^' 23146 c C C c C C C c c c ^' 19 23167 c C C c C C C c c c 20 23191

A A A A A A A A G A 21 23192

C C C C C C C C C C 22 23295

T C C T C C T C T C 23 23304

G - G G G G G G G G A 24 23364

G G G G G G G G G G 25 23420

G G G G A G G A G A 26 23473

C C C C C C T C C C 27 25293

G G G G G G G G G G 28 25387 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι^ Ω Ω Ω ' Ω Ω ι-3 H Ω Ω Ω to Ω ^ Ω Ω Ω Ω ^ Ω Ω Ω Ω Ω &a H Ω Ω Ω Ω Ω a Ω Ω Ω l-¹

H> l-> Ω Ω Ω Ω Ω Ω Ω ^I Ω Ω Ω Ω Ω Ω Ω ^ Ω DΪ' Ω Ω Ω Ω I-3 I-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ^ H Ω Ω Ω Ω Ω ^ Ω Ω Ω Ω Ω Ω Ω H i-a Ω Ω Ω Ω Ω Ω Ω ^! Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω I-3 I-3 Ω Ω Ω Ω

-3

ΩΩΏΩΩΩO ΩΩΩΩΩΩΩΩΩΩΩΩOΩH ΩΩΩΩ ro c- Ω Ω ^ Ω Ω Ω Ω ^ Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Ω Ω H Ω Ω O Ω cn Cπ

CD ΩΩΏΩΩ ΩΏΩΩΩΩΩΩΩΩΩΩΩΩΩΩ^HΩΩΩΩ ro H -Ω Ω O O Ω Ω Ω >' Ω Ω Ω Ω Ω Q Ω >-3 0 0 Ω Ω Ω Ω Ω >-3 Ω Ω O Ω

CΛ CΛ

Ω Ω Ω Ω Ω ι-3 Ω Ω Ω β Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-a ι-3 Ω Ω Ω Ω t Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω ι-3 Ω Ω ι-3 Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω μ-'

-J

O Ω Ω Ω O Ω Ω Ω Ω Ω Ω Ω O ⁾ I-E Ω Ώ Ω O Ω Ώ >-- H Ω O O Ω to Ω ^ O ^ Ω Ω ^ Ω Ω Ω Ω Ω Ω Ω O Ω Ω Ω iP i-a a Ω Ω Ω I-¹

00 co Ώ Ω Ω Ω Ώ H Ω |> Ω Ω Ω Ω Ω Ώ Ω Ώ Ω Ώ Ω Ω H I-- Ω Ω Ώ Ω ro Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Ω Ω I-- Ω Ω Ω Ω Ω ^, I-3 H Ω Ω Ω Ω

CO id

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^ Ω Ω Ώ Ω Ω ^I Ω Ω Ω Ω Ω Ώ Ω H Ω Ώ Ω Ώ Ω Ω I-- Ω Ω Ω Ω ω Ω Ω Ω Ω Ω H Ω < Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω H H Ω Ω Ω Ω ro o O

N M M M N M M M t H H H P H H H H P H ID C -J-CΛ Ul *. U) M H O ro M to r M M M rθ M ^J l-^Λ VO CO -J CΛ (-rι . Cθ rθ h-' g oo -J n ui ^ U M H O D ∞ o σi Cn Λ ω t H O g 3 TJ o- co ^ oi ui i^ ω M μ o iD ∞ J oi ui iMij M μ o 3

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H ro r M M tO t M r r M M r I-^> W CO UJ- LO G U LO OJ Gu J TJ T) W M M M M M M N W M t M μ μ μ μ μ μ u u u u u i ω u ω ω TJ ui ϋι ω ω ω u ω w iλ) U ) o iD j (?ι oι σι σι iD nfl iD c ^ j ι2. *. κι o cn uι uι ω ω ω ω ω ω w ω ω o iD -J fi cfι σι n ti) iD iD U3 ω i ^ι *. ^ N O ω ω *. Λ Ui ω M H μ μ H θ ιo o i£HD U) M Ui fc μ o ιC i (n i u ^ι ω ro ι^ α^ ω ω t > ^j θ Cβ o ι^ ^ U3 M αi ιi^ μ^j θ ιc. ~j cΛ --J U) -j cn

CO (D l M t!1 0 UHD (£) (31 *. W N ^ l!) m Ul O n *' U -J t. W l Kl fc [ P- CO U) -J M σi O U) D lD n ι^ U M ι2. U fl Ul θ σi Jⁱ ω -J *' tVI ^ M ^ M H-

-J io u o *. ^ tti w j (j\ μ t- fι o ω u o rt •J θ) ω o *. *> uι ic μ j ή μ ιt σι o ω ω o rr

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0 3

Haplotype Number³ PS^b PS

31 32 33 34 35 36 37 38 39 40 41 Number Position'

C C C C C C C C C T T 1 3272

G G G G G G G G G G G 2 3434

G G G G G G G G G G G 3 3472

C C C C C C C C T C C 4 3767

T T f T T T T T T T T.. 5 3772

T T T T T T T T T C C 6 3844

G G G G G G G G G G G 7 3907

C C C C C C C C^' C C C 8 3913

G G G G G G G G G G G 9 3944

C C C C C C C T C C C 10 3956

G G G G G^' G G G G G G 11 16200

G G G G G G G G C G G 12 16953

T T T T T T T T C T T 13 16963

G G G G G G G G G A G 14 16990

G G G G G G G G G G G 15 17046

C . C C C C C C C C C C 16 19924

C C ■ C C C C C C C C C 17 20031

C C C C C . C C C C C C 18 23146

C C C C C c C C T C C 19 23167

C C C C C c C C - C C C 20 23191

A A A A A - A G A G A A 21 23192

C C C C C T - C C C C C 22 23295

C C C C T c T C T C C 23 23304

G G G G G G G G G G G 24 23364

G. G G G G G G G G G G 25 23420

A G G G G G G G G G G 26 23473

C C C T C C C C C C C 27 25293

G C G G G G G G G G G 28 25387

Alleles for haplotypes are presented 5 ' to 3 ' in each column 'PS = polymoφhic site; Position of PS in SEQ ID NO:l; and wherein the haplotype pair is selected from the haplotype pairs shown in the table immediately below, wherein each of the ETFB haplotype pairs consists of first and second haplotypes which comprise first and second sets of polymoφhisms whose locations and identities are set forth in the table immediately below:

Haplotype Pair³ PS^b PS

33/33 35/35 26/26 25/25 15/11 30/18 30/14 40/16 Number Position'

C/C C/C C/C C/C C/C C/C C/C T/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/T T/T T/T T/T C/C T/T T/C C/C 6 3844

G/G G/G G/G G/G G/G G/A G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G G/G C/C C/C G/G G/C G/G G/G 12 16953

T/T T/T C/C C/C T/T T/C T/T T/T 13 16963

G/G G/G G/G G/G G/A G/G G/G A/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C ■C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A G/G A/A A/A A/A - A/A A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C T/T T/T C/C C/C C/C C/C C/C 23 23304

G/G G/G G/G G/G G/G A/A A/A G/G 24 23364 -

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G A/A A/A A/A G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS

33/6 33/24 33/22 '. 33/21 33/7 33/32 : 31/5 35/17 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/C T/T T/T T/T T/C T/T T/C T/T 5 3772

T/T T/T T/T T/T T/T T/T T/T T/C 6 3844

G/G G/G G/G G/G G/G G/G G/A G/G 7 3907

C/C C/C C/C C/C C/C C/C C/T C/C 8 3913

G/G G/G G/G G/A G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/A G/G G/G G/G G/G G/G 11 16200

G/C G/C G/G G/G G/G G/G G/G G/G 12 16953

T/C T/C T/T T/T T/T T/T T/T T/T 13 16963

G/G G/G G/G G/G G/G G/G G/A G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/T 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/A A/A A/A A/G 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C C/C C/C C/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/A G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G A/A G/G 26 23473

C/C C/C C/C C/C C/T C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/C G/G G/G ^' 28 25387 Haplotype Pair³ PS^b PS

33/3 25/28 35/26 35/20 27/39 35/12 33/2 28/4 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/A G/G G/G G/G G/G G/G G/A G/G 3 3472

C/C C/C C/C C/C C/T C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/C 5 3772

T/T T/T T/T T/T T/T T/C T/T T/T 6 3844

G/G G/G G/G G/A G/G G/G G/G G/A 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G C/G G/C G/G C/C G/G G/C G/G 12 16953

T/T C/T T/C T/T C/C T/T T/C T/T 13 16963

G/G G/A G/G G/G G/G G/A G/G A/A 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924 -

C/C C/C C/C C/C C/C C/C C/C C/T 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C T/T C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/G A/A G/G A/A A/A A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C T/T T/T T/T T/T C/C C/T 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS

31/8 15/31 35/1933/36 35/37 33/10 33/13 35/1 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/A 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C . C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/C C/T T/T T/T T/T T/C T/C T/T 6 3844

G/A G/G G/A G/G G/G G/G G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G G/G G/G G/G G/G G/G G/G G/C 12 16953

T/T T/T T/T T/T T/T T/T T/T T/C 13 16963

G/A G/G G/A G/G G/G G/A G/A G/A 14 . 16990

G/G G/G G/G G/G G/G G/G G/T G/G 15 ^' 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/T C/C C/C C/C C/C C/C C/T C/C 17 20031

C/C C/C C/G C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/G A/A A/A A/A 21 23192

C/C C/C C/C C/T C/C C/C C/C C/C 22 23295

C/C C/C T/T C/C T/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/A G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

A/A .A/A G/G G/G G/G G/A G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G. 28 25387 Haplotype Pair³ PS^b PS

35/27 35/29 33/40 33/38 33/28 33/23 35/9 33/25 Number Position⁰

C/C C/C C/T C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 ^• 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/T T/T T/C T/T T/T T/T T/C T/T 6 3844

G/G G/G G/G G/G G/G G/G G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/T C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/C G/G G/G G/G G/G G/C G/C G/C 12 16953

T/C T/T T/T T/T T/T T/C T/C T/C 13 16963

G/G G/A ^'G/A G/G G/A G/A G/G G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/G C/C C/C C/C C/C C/C C/C 18 23146 ^'

C/T C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/T C/C C/C 20 23191

A/G A/A A/A A/A A/A A/A A/G A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 2^'3295

T/T T/T C/C C/C C/C C/C T/T C/C 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387 Haplotype Pair³ PS^b PS

33/41 16/34 Number Position⁰

C/T C/C 1 3272

G/G G/G 2 3434

G/G G/G 3 3472

C/C C/C 4 3767

T/T T/T 5 3772

T/C C/T 6 3844

G/G G/G 7 3907

C/C ^' C/C ^' 8 3913

G/G G/G^' 9 3944

C/C C/C 10 3956

G/G G/G 11 16200

G/G G/G 12 16953

T/T T/T 13 16963

G/G G/G 14 16990

G/G G/G 15 17046

C/C C/C 16 19924

C/C C/C 17 20031

C/C C/C 18 23146

C/C C/C 19 23167

C/C C/C 20 23191

A/A A/A 21 23192

C/C C/C 22 23295

C/C C/C 23 23304

G/G G/G 24 23364

G/G G/G 25 23420

G/G G/G 26 23473

C/C C/T 27 25293

G/G ^' G/G 28 25387 Ηaplotype pairs are represented as 1^st haplotype/2^nd haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column; ^bPS = polymoφhic site; "Position of PS in SEQ ID NO: 1;

wherein a higher frequency of the haplotype or haplotype pair in the trait population than in the reference population indicates the trait is associated with the haplotype or haplotype pair.

14. The method of claim 13, wherein the trait is a clinical response to a drug targeting ETFB.

15. An isolated genotyping oligonucleotide for detecting a polymoφhism in the electron-transfer- flavoprotein, beta polypeptide (ETFB) gene at a polymoφhic site (PS) selected from the group consisting of PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 and PS28, wherein the selected PS have the location and alternative alleles shown in SEQ ID NO:l.

16. The isolated genotyping oligonucleotide of claim 15, which is an allele-specific oligonucleotide that specifically hybridizes to an allele of the ETFB gene at a region containing the polymoφhic site.

17. The allele-specific oligonucleotide of claim 16, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:4-30, the complements of SEQ ID NOS:4-30, and SEQ ID NOS:31-84.'

18. The isolated genotyping oligonucleotide of claim 15, which is a primer-extension oligonucleotide.

19. The primer-extension oligonucleotide of claim 18, which comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:85-138.

20. A kit for genotyping the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene of an individual, which comprises a set of oligonucleotides designed to genotype each of polymoφhic sites (PS) PSl, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9, PS10, PS11, PS12, PS13, PS14, PS15, PS16, PS17, PS18, PS19, PS20, PS21, PS22, PS24, PS25, PS26, PS27 andPS28, wherein the selected PS have the location and alternative alleles shown in SEQ ID NO:l.

21. The kit of claim 20, which further comprises oligonucleotides designed to genotype PS23, having the location and alternative alleles shown in SEQ ID NO:l.

22. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) a first nucleotide sequence which comprises a electron-transfer-flavoprotein, beta polypeptide (ETFB) isogene, wherein the ETFB isogene is selected from the group consisting of isogenes 1- 24 and 26 - 41 shown in the table immediately below and wherein each of the isogenes comprises the regions of SEQ ID NO: 1 shown in the table immediately below and wherein each of the isogenes 1- 24 and 26 - 41 is further defined by the corresponding set of polymoφhisms whose locations and identities are set forth in the table immediately below

Alleles for isogenes are presented 5' to 3' in each column ^bPS = polymoφhic site; "Position of PS in SEQ ID NO:l;

Region examined represents the nucleotide positions defining the start and stop positions within the 1^st SEQ ID NO of the sequenced region.

(b) a second nucleotide sequence which comprises a fragment of the first nucleotide sequence, wherein the fragment comprises one or more polymoφhisms selected from the group consisting of thymine at PSl, adenine at PS2, adenine at PS3, thymine at PS4, cytosine at PS5, cytosine at PS6, adenine at PS7, thymine at PS8, adenine at PS9, thymine at PS10, adenine at PSl 1, guanine at PS12, thymine at PS13, adenine at PS14, thymine at PS15, thymine at PS16, thymine at PS17, guanine at PS18, thymine at PS19, thymine at PS20, guanine at PS21, thymine at PS22, adenine at PS24, adenine at PS25, adenine at PS26, thymine at PS27 and cytosine at PS28, wherein the selected polymoφhism has the location set forth in the table immediately above; and

(c) a third nucleotide sequence which is complementary to the first or second nucleotide sequence.

23. The isolated polynucleotide of claim 22, which is a DNA molecule and comprises both the first and third nucleotide sequences and further comprises expression regulatory elements operably linked to the first nucleotide sequence.

24. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 22, wherein the organism expresses an ETFB protein encoded by the first nucleotide sequence.

25. The recombinant nonhuman organism of claim 24, which is a transgenic animal.

26. The isolated polynucleotide of claim 22 which consists of the second nucleotide sequence.

27. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) a coding sequence for a electron-transfer-flavoprotein, beta polypeptide (ETFB) isogene wherein the coding sequence is defined by a haplotype selected from the group consisting of lc, 4c, 7c, 9c, 10c, 12c, 14c, 17c, 18c, 19c, 20c, 24c, 26c, 27c, 29c, 30c, 35c, 36c, 37c, and 39c shown in the table immediately below and wherein the coding sequence comprises SEQ ID NO:2 except at each of the polymoφhic sites which have the locations and polymoφhisms set forth in the table immediately below:

Coding Sequence Haplotype Number³ PS PS lc 4c 7c 9c 10c 12c 14c 17c 18c 19c Number^b Position⁰ c C C C • C C C C C C 22 452

T T T T C T C T C T 23 461

G G G G A G A G A G 24 521

G G G G G G G G G G 25 577 lodiirig Sequence Haplotype Number³ - PS PS

20c 24c 26c 27c 29c 30c 35c 36c 37c 39c Number^b Position'

C C C C C C C T C C 22 452

T C T T T C - T C T T 23 461

G G G G G A G G G G 24 521

G A G G G G G G G G 25 577 ^aAlleles for coding sequence haplotypes are presented 5' to 3' in each column; the numerical portion of the coding sequence haplotype number represents the number of the parent full ETFB haplotype; ^bPS = polymoφhic site; "Position of PS in SEQ ID NO:2; and (b) a fragment of the coding sequence, wherein the fragment comprises at least one polymoφhism selected from the group consisting of thymine at a position corresponding to nucleotide 452, adenine at a position corresponding to nucleotide 521 and adenine at a position corresponding to nucleotide 577, wherein said positions in the coding sequence and the fragment refer to SEQ ID NO:2.

28. A recombinant nonhuman organism transformed or transfected with the isolated polynucleotide of claim 27, wherein the organism expresses a electron-transfer-flavoprotein, beta polypeptide (ETFB) protein encoded by the polymoφhic variant sequence.

29. The recombinant nonhuman organism of claim 28, which is a transgenic animal.

30. An isolated polypeptide comprising an amino acid sequence which is a polymoφhic variant of a reference sequence for the electron-transfer-flavoprotein, beta polypeptide (ETFB) protein or a fragment thereof, wherein the reference sequence comprises SEQ ID NO:3 and the polymoφhic variant comprises one or more variant amino acids selected from the group consisting of phenylalanine at a position corresponding to amino acid position 151, histidine at a position corresponding to amino acid position 174 and threonine at a position corresponding to amino acid position 193.

31. An isolated monoclonal antibody specific for and immunoreactive with the isolated polypeptide of claim 30.

32. A method for screening for drugs targeting the isolated polypeptide of claim 30 which comprises contacting the ETFB polymoφhic variant with a candidate agent and assaying for binding activity.

33. A computer system for storing and analyzing polymoφhism data for the electron-transfer- flavoprotein, beta polypeptide gene, comprising: -

(a) a central processing unit (CPU);

(b) a communication interface;

(c) a display device;

(d) an input device; and

(e) a database containing the polymoφhism data; wherein the polymoφhism data comprises the haplotypes set forth in the table immediately below:

Ω Ω Ω Ω Ω Ω ;s^, Ω Ω Ω Ω Ω Ω U2^| >-3 Ω Ω Ω Ω Ω Ω Ω ι-- Ω Ω Ω Ω H^J Ω Ω Ω Ω H Ω | Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 3 ^μ Ω Ω > Ω

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-3 ι-3 Ω fi Ω Ω t

Ω Ω Ω Ω Ω μ3 Ω Ω Ω Ω Ω Ω Ω >-3 0 Ω Ω Ω Ω Ω Ω μ3 Ω Ω Ω Ω I-' > Ω Ω Ω Ω Ω Ω ro K

E" Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω a ^μ3 Ω Ω Ω

TJ

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω >-3 Ω μ3 ι-3 Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω μ-> μ-'

CO O Ω Ω Ω Ω Ω H Ω Ω Ω Ω ι-3 Ω Ω »-3 Ω Ω Ω Ω Ω 3 Ω Ω Ω Ω Ω j2- rt

Ω Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ώ Ώ Ω Ώ Ω Ώ Ω Ώ Ω Ω Ώ Ω Ω I-' 'O< Ω Ω Ω Ω Ω Ω

>t- CD > Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω ι-3 'p n Ω Ω Ω Ω Ω π

3 Ω Ω Ω Ω Ω Ω Ω

Ω Ω tf Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω >-3 Ω Ω Ω Ω Ω Ω Ω jf Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω I-' d

Cπ g

Ω Ω Ω Ω H Ω > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω h^J h σ> <* Ω Ω Ω Ω Ω Ω > Ω Ω Ω ι-3 Ω Ω > Ω Ω Ω Ω Ω Ω 3 Ω Ω Ω Ω

Ω Ω Ω Ω Ω t-3 Ω Ω Ω Ω Ω Ω H Ω Ω ι-3 Ω^"Ω Ω Ω Ω Ω Ω - Ω Ω Ω Ω t-¹ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ^ Ω Ω Ω Ω

-J

^■Ω Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω >-3 μ3 Ω Ω Ω Ω μ-> > Ω Ω Ω Ω Ω Ω Ω 3 Ω Ω Ω Ω co a

Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Da μ3 Ω Ω Ω Ω cz 5 w ro to ro t to to t ro μ-¹ μ-> 00 -J t μ->

Ω Ω Ω Ω Ω 3 Ω Ω Ω Ω Ω Ω Ω H H Ω Ω Ω Ω r ro l-^» Cπ ro μ-> o H o ro ro ro ro ro M N N M H H H H H H H M H H lo ro vl cri tJl ib I H M M M W N M M M M M t M μ μ μ μ μ H ω ω ω ω ω ω ω ω ω co ι-rj co -J C cπ it- i M H o ω co ^i fi Lii Λ ω t μ o TJ n n ω ω ω ω co ώ ω i i w o co ^i σi oi m σi ω co cD o co -j j ^ ib cn ro o σ ι t ιb *. ω ω M μ μ o «) o iD U) ^ M ϋi «> μ o *> ^ σi i ω -J ω co u) ~j M n o o co CD σι J^!' Cι) M ι& o σι n o σι ^ ω ^i ιfc N) ^i M ^ ro p- ~J Co co θ ιJ2. ιi-. Cπ ro μ-' -J C μ-' *- CΛ θ Co co o rt

H- 'fl 0 cn P

Ω Ω ^ Ω Ω Ω Ω &a Ω Ω Ω Ω Ω Ω Ω i-S Ω Ω Ω Ω Ω Ω H H Ω Ω Ω Ω CO Ω Ω Ω Ω Ω Ω Ω tW Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω > Ω Ω Ω Ω Ω Ω ro

Ω Ω Ώ Ώ Ω Ω Ω ^ Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ώ Ω Ω I^ ^ Ω Ώ Ώ Ω CO Ω ro a ΏΩΏΏΏΩΩ^ΩΩΩΩΩΏ Ω > Ω Ω Ω Ω Ω Ω Ω Ω ro to

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ^μ3 Ω Ω Ω Ω Ω Ω 3 3 Ω Ω Ω Ω Co -^J Ω Ω Ω Ω Ω Ω Ω ia ^ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ro

CO O rt Ω H Ω Ω Ω Ω Ω ΪC Ω Ω Ω Ω Ω Ω S Ω Ω Ω Ω Ω H S Ω Ω CoO Ω Ω Ω ^ Ω Ω Ω iWΩ ΩΩ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω ro

^ - ^' 2

Ω Ω Ω Ω Ω ^μS Ω ^ Ω Ω Ω Ω Ω Ω Ω i-S Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Co C Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ro π g ΩΩΏΏΏΩΩ^ΩΩΩΩΩΏ cπ

CD Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω Ω >-3 Ω Ω Ω Ω Ω Ω ι-3 !-3 Ω Ω Ω Ω Co μj Ω Ω Ω Ω Ω I-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ro

Ω Ω Ω Ω Ω ^μS Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 3 ^' H3 Ω Ω Ω Ω CO Ώ Ω Ώ Ώ Ώ ^ Ω Ώ. H Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω ro

-J

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω μ3 Ω Ω H Ω Ω Ω μ3 H Ω Ω Ω Ω C0 ΩΩ Ω Ω Ω Ω Ω lP Ω ΩΩ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ro co

Ω Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω μ3 H H Ω Ω Ω C0 Ω Ω Ω Ω Ω H Ω ^ Ω Ω Ω ΩΩΩ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ro to Ώ Ω Ώ Ώ Ώ Ω Ω Ω Ω Ω Ω Ω Ω ' H Ω Ώ Ω Ω Ω Ώ Ω H Ω Ώ Ώ I^ ^ Ω Ω Ω Ω Ω ^ Ω ΩΩ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω

O o

Ω Ω Ώ Ώ Ω Ω Ω D=^, Ω Ω Ω Ω Ω Ώ Ώ « Ω Ώ Ω Ω Ω Ώ Ω H Ω Ώ Ω J²'

M M M M M M M M M μ μ μ μ μ -o ro a cj co ^ι σι Ln *. ω κ) μ o ifl co -J σι cιι ro

ro ro ro ro ro ro ro ro ro to to ro μ^j μ^j

Cπ Cπ C CO CO CO C C C CO CO O CO -J σ> ro ro to ro ro.ro w w M M μ μ μ μ μ μ μ μ cπ ^ ro a L W Λ ιt. to u t μ μ μ μ o iD θ ro cπ o ω co i n cn ib ω M o co co ^i u n it- ω M o n co co -j to cΛ θ co co co crι *. co ro ιi-. o -o ro ro i ω ω o *. ι-- uι (o μ i σι μ *> cn o o rt H- O 3

M M M M M M M M M M M μ μ μ μ μ μ i ω u u ω ω u ω u ω il 'fl n ϋi ω ω ω u ω c u ω ω o ω J n iJi σi m iD iD ifl iD CO i i ^ fc t o Cn U M it j- u u N μ o uj o io io ϊi M ui fc μ o fc ^ ή i U m

OO CO ^J N3 CTl O lD CO U) 01 i£. ) M ib CO Cn ( 10 cri *> U) ^l it- N ^ M i-- M H-

-j ω ω o fc ^ uι N μ J m μ *> σι θ Cι) LJ o rt

H- O 0

-J

OJ

TJ cn

M M W M M t M M μ μ μ μ μ μ μ μ μ μ «) co J tι Ui *. ω — J cΛ Cπ >e- co ro μ-' o co co ι cΛ Cπ --. co to μ-' o (D ri w t M M M M W M M M M M μ μ μ μ μ u ω u ui ω ω o ui i ui f ifl w w ω ω ω ω ω ω ω ω ω o o _^i cn σi σi ri Co o o o ra _^i ^ ^ ^ M O n

C_ΛJ M ιi2. ^ C CΛ) M μ-¹ O CO O CO CD Cθ ro Cπ ιt- μ-' O ιt2. --J CΛ --J CO ~J ω

CO CO J M m θ CO ) lO Λ ^ M *' CD <Jl Cπ θ [!_l *. ) ^l * M ^ M *> r μ-

-J Co co o iti. ic. cji ro μ-' - cΛ μ-' it. cΛ O Co co o rt

H- o

Haplotype Pair³ PS° - PS 3/6 33/24 33/22 33/21 33/7 33/32 31/5 35/17- Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/C - T/T T/T T/T T/C T/T T/C T/T^' 5 3772

T/T T/T T/T T/T T/T T/T T/T T/C 6 3844

G/G G/G G/G G/G G/G G/G G/A G/G 7 3907

C/C C/C C/C C/C C/C C/C C/T C/C 8 3913

G/G G/G G/G G/A G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/A G/G G/G G/G G/G G/G 11 16200

G/C G/C G/G G/G G/G G/G G/G G/G 12 16953

T/C T/C T/T T/T T/T T/T T/T T/T 13 16963

G/G G/G G/G G/G G/G G/G G/A G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/T 16 19924

C/C. C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/C C/C C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/A A/A A/A A/G 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C C/C C/C C/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/G G/G ^•G/G 24 23364

G/G G/A G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G A/A G/G 26 23473

C/C C/C C/C C/C C/T C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/C G/G G/G 28 25387

Haplotype Pair³ PS^b PS

33/3 25/28 35/26 35/20 27/39 35/12 33/2 28/4 Number Position⁰

C/C C/C C/C C/C C/C C/C ^■ C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/A G/G G/G G/G G/G G/G G/A G/G 3 3472

C/C C/C C/C C/C C/T C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/C 5 3772

T/T T/T T/T T/T T/T T/C T/T T/T 6 3844

G/G G/G G/G G/A G/G G/G G/G G/A 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G C/G G/C G/G C/C G/G- G/C G/G 12 16953

T/T C/T T/C T/T C/C T/T T/C T/T 13 16963

G/G G/A G/G G/G G/G G/A G/G A/A 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/T 17 20031

C/C C/C C/C C/C C/C C/C C/C- C/C 18 23146

C/C C/C C/C C/C T/T C/C C/C C/C 19 23167

C/C • C/C C/C C/C C/C C/C C/C C/C 20 - 23191

A/A A/A A/G A/A G/G A/A A/A A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

C/C C/C T/T T/T T/T T/T C/C C/T 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420-

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387 Haplotype Pair³ PS^b PS

31/8 15/31 35/19 33/36 35/37 33/10 33/13 35/1 Number Position⁰

C/C C/C C/C C/C C/C C/C C/C C/C 1 - 3272

G/G G/G G/G G/G G/G G/G G/G G/A 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C ■ C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T ^• T/T T/T T/T T/T 5 3772

T/C C/T T/T T/T T/T T/C T/C T/T 6 3844

G/A G/G G/A G/G G/G G/G G/G G/G 7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/C C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/G G/G G/G G/G G/G G/G G/G G/C 12 16953

T/T T/T T/T T/T T/T T/T T/T T/C . 13 16963

G/A G/G G/A G/G G/G G/A G/A .G/A 14 16990

G/G G/G G/G G/G G/G G/G G/T G/G 15 17046

C/C C/C C/C C/C C/C C/C C/C C/C 16 19924

C/T C/C C/C C/C C/C C/C C/T C/C 17 20031

C/C C/C C/G C/C C/C C/C C/C C/C 18 23146

C/C C/C C/C C/C C/C C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/C C/C C/C 20 23191

A/A A/A A/A A/A A/G A/A A/A A/A 21 23192

C/C C/C C/C C/T .C/C C/C C/C C/C 22. 23295

C/C C/C T/T C/C T/T C/C C/C T/T 23 23304

G/G G/G G/G G/G G/G G/A G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

A/A A/A G/G G/G G/G G/A G/G G/G 26 - 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387

Haplotype Pair³ PS^b PS

35/27 35/29 33/40 33/38 33/28 33/23 35/9 33/25 Number Position⁰

C/C C/C C/T C/C C/C C/C C/C C/C 1 3272

G/G G/G G/G G/G G/G G/G G/G G/G 2 3434

G/G G/G G/G G/G G/G G/G G/G G/G 3 3472

C/C C/C C/C C/C C/C C/C C/C C/C 4 3767

T/T T/T T/T T/T T/T T/T T/T T/T 5 3772

T/T T/T T/C T/T T/T T/T T/C T/T 6 3844

G/G G/G G/G G/G G/G G/G G/G G/G ^•7 3907

C/C C/C C/C C/C C/C C/C C/C C/C 8 3913

G/G G/G G/G G/G G/G G/G G/G G/G 9 3944

C/C C/C C/C C/T C/C C/C C/C C/C 10 3956

G/G G/G G/G G/G G/G G/G G/G G/G 11 16200

G/C G/G G/G G/G G/G G/C G/C G/C 12 16953

T/C T/T T/T T/T T/T T/C T/C T/C 13 16963

G/G G/A G/A G/G G/A G/A G/G G/G 14 16990

G/G G/G G/G G/G G/G G/G G/G G/G 15 17046

C/C C/C C/C C/C C/C ^' C/C C/C C/C 16 19924

C/C C/C C/C C/C C/C C/C C/C C/C 17 20031

C/C C/G C/C C/C C/C C/C C/C C/C 18 23146

C/T C/C C/C C/C C/C ^' _, C/C C/C C/C 19 23167

C/C C/C C/C C/C C/C C/T C/C C/C 20 23191

A/G A/A A/A A/A A/A A/A A/G A/A 21 23192

C/C C/C C/C C/C C/C C/C C/C C/C 22 23295

T/T T/T C/C C/C C/C C/C T/T C/C 23 23304

G/G G/G G/G G/G G/G G/G G/G G/G 24 23364

G/G G/G G/G G/G G/G G/G G/G G/G 25 23420

G/G G/G G/G G/G G/G G/G G/G G/G 26 23473

C/C C/C C/C C/C C/C C/C C/C C/C 27 25293

G/G G/G G/G G/G G/G G/G G/G G/G 28 25387 Haplotype Pair³ PS^b PS

33/41 16/34 Number Position⁰

C/T C/C 1 3272

G/G G/G 2 3434

G/G G/G 3 3472

C/C C/C 4 3767

T/T T/T 5 3772

T/C C/T 6 3844

G/G G/G 7 3907

C/C C/C 8 3913

G/G G/G 9 3944

C/C C/C 10 3956

G/G G/G 11 16200

G/G G/G 12 16953

T/T T/T 13 16963

G/G G/G 14 16990

G/G G/G 15 -17046

C/C C/C 16 19924

C/C C/C 17 20031

C/C C/C 18 23146

C/C C/C 19 23167

C/C C/C 20 23191

A/A A/A 21 23192

C/C C/C 22 23295

C/C^' C/C 23 23304

G/G G/G 24 23364

G/G G/G 25 23420

G/G G/G 26 23473

C/C C/T 27 25293

G/G G/G 28 25387

^aHaplotype pairs are represented as 1^st Haplotype/2^nd Haplotype; with alleles of each haplotype shown 5 ' to 3 ' as 1^st polymoφhism/2^nd polymoφhism in each column;

"Location of PS in SEQ ID NO:l.

34. A genome anthology for the electron-transfer-flavoprotein, beta polypeptide (ETFB) gene which comprises ETFB isogenes defined by any one of haplotypes 1-41 set forth in the table shown below:

Ω Ω !» Ω Ω Ω Ω > Ω Ω Ω Ω Ω Ω > Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω > Ω

Ω Ω Ω Ω Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω Ω > Ω Ω Ω ι-3 Ω H > Ω Ω Ω Ω Ω Ω Ω i-S Ω Ω Ω Ω

Ω Ω Ω . Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω μS Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω

-Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Ω Ω Ω Ω H Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H μ3 Ω Ω Ω Ω a

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω > H Ω Ω Ω Ω Ω > H ι-3 Ω Ω Ω Ω g ro ro ro ro ro to ro ro to μ-> μ-¹ μ-¹ cπ co ro

-J Ω co ^i cn n it- co ro μ-> o co cπ

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω μ 3 Ω Ω Ω Ω ω ro O

2 g TJ t ro ro t ro ro ro ro ro cπ it- co ro μ-¹ B CT. n σ W M t to M t w M M t M M μ μ μ μ u io L ω ω ω ω ω ω u ij π ro o cn ro o (D Ln n ω ω ω ω ω ω ω ω ω o o -J cn m σi m o cD co ci αi ^i ^i iΛ ifc w o l-f C M ιb ιfc. ω ω t0 μ^J O D O C0 0 CΩ W Un ιt- O ιJ^ ^ C --J 00 -J 0 co o J w σι θ co u) o σι Λ ω t ιfr D σι n o m ι W ^i Λ M ^i M ιt. M H- ro ro ro ro ro ro ro ro ro ro ro ro ω o co co co o o co TJ Tj -J C0 C0 O it. iC. Cn r0 -J C l it. (Tl O C0 C0 O rt π cπ o co co its. it. ro 0 n H- ro ro o o to cπ o *■ σi ω ω o to o ro o *. -j it. ro .ro ιt> ro H- 3

-o ro o o rt H- O P

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ^μ3 Ω Ω Ω Ω Ω ι-3 ι-3 Ω Ω Ω Ω t μ-¹

-

Ω Ω Ω Ω Ω Ω Ω ^ Ω Ω Ω Ω Ω Ω Ω ι-3 Ω Ω Ω Ω Ω ι-3 3 Ω Ω Ω Ω to ro

Ω Ω Ω Ω Ω Ω Ω |> f-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 3 3 Ω Ω Ω Ω ro

Ω Ω Ω > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 1-3 ^μa Ω Ω Ω Ω ro

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω a ^μ3 Ω Ω Ω Ω ro cπ

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-3 H Ω Ω Ω Ω ro

Ω Ω Ω Ω Ω Ω Ω Ω 1-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω ι-3 3 Ω Ω Ω Ω ro

Ω Ω Ω Ω Ω Ω Ω |f Ω Ω Ω Ω Ω Ω- a Ω Ω Ω Ω Ω Ω 3 ι-3 Ω Ω Ω Ω ro

Ω Ω Ω Ω Ω >-3 Ω Ω Ω Ω Ω Ω Ω a Ω Ω Ω Ω Ω Ω i-3 ι-3 Ω Ω Ω Ω ro oo Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω a Ω Ω Ω Ω Ω Ω ^μa 1-3 Ω Ω Ω Ω o

t to ro ro ro ro ro ro ro π ro cπ ro o cπ ro o &

CD

H ro ro o ro ro ro ro ro ro to ro to ω TJ cπ o ro 0 to ro o o ro Cπ o ω ro o ro cπ o ro ro ro H- o cπ ro \-> o rt H- O

VD

ro to t ro ro o ro M M H P H H H H H H P CO CO ^l CTl Ul it t N H Z 'fl

00 CΛ n J-. CO ro μ-' O CD Oo -o cΛ Cπ ij-. co ro μ-' o d en

CD Hi ro to ro ro ro ro ro ro ro M tO M H CO CO CO CO CO CO CO CO CO CO TJ TJ cπ cπ co co co co co CO CO CO CO O CD -O CΛ CΛ CΛ CΛ CO CD CD CO CO -J ^l ib ib rO O n co ro >i2- it- co co ro μ u_ι μ 1— i μ 1— i i—i ro~ι i(Dn rθ~ιi isn ttfon ion M K-i U tπi .*t.. μ 1— l o r-i .*t.. _^1i n(ji -s.1i fω.l - —j 1 r ωn

CO CO I ro CΛ O CD CO CO CΛ ib CO rO ib cO CΛ Cπ O CΛ ib CO -O ib rO -J rO ib tO μ-

-J co Co o i it. cπ to μ-' -J cΛ μ-' ib cΛ o co co o rt μ- O