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WO2001018245A2 - Detection des alterations dans un gene par amplification pcr longue portee a l'aide d'elements mobiles humains - Google Patents

Detection des alterations dans un gene par amplification pcr longue portee a l'aide d'elements mobiles humains Download PDF

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Publication number
WO2001018245A2
WO2001018245A2 PCT/IB2000/001283 IB0001283W WO0118245A2 WO 2001018245 A2 WO2001018245 A2 WO 2001018245A2 IB 0001283 W IB0001283 W IB 0001283W WO 0118245 A2 WO0118245 A2 WO 0118245A2
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gene
interest
alteration
pcr products
test sample
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WO2001018245A3 (fr
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Struan Grant
Thorarinn Blondal
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Decode Genetics ehf
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Decode Genetics ehf
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Priority claimed from US09/470,673 external-priority patent/US6225093B1/en
Priority to EP00954856A priority Critical patent/EP1214449A2/fr
Priority to BR0014017-1A priority patent/BR0014017A/pt
Priority to JP2001521779A priority patent/JP2003508082A/ja
Priority to CA002383857A priority patent/CA2383857A1/fr
Priority to NZ517641A priority patent/NZ517641A/en
Application filed by Decode Genetics ehf filed Critical Decode Genetics ehf
Priority to AU67207/00A priority patent/AU6720700A/en
Priority to MXPA02002507A priority patent/MXPA02002507A/es
Publication of WO2001018245A2 publication Critical patent/WO2001018245A2/fr
Anticipated expiration legal-status Critical
Publication of WO2001018245A3 publication Critical patent/WO2001018245A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • SLE Systemic lupus erythematosus
  • SLE is an autoimmune disease characterized by immune dysregulation resulting in the production of anti -nuclear antibodies, the generation of circulating immune complexes, and the activation of the complement system.
  • SLE leads to inflammation of various parts of the body, especially the skin, joints, blood, kidneys, lungs, heart and nervous system.
  • SLE affects approximately 1 in every 500 Americans, and strikes women 10-15 times more frequently than men. It is more common among Asians, and in China, SLE may be even more common than rheumatoid arthritis.
  • HLA-DR3 and a C4A null allele are frequently co-inherited as the extended haplotype B8,BfS:C2C,C4AQ0,C4Bl;DR3.
  • This is the most common extended haplotype in white SLE patients (Kemp, M.E. et al, Arthritis and Rheumatism 30:1015-22 (1987)).
  • C4AQ0-containing haplotypes have a DNA deletion of approximately 30 kB, extending from the 5' end of the C4A gene to the same position in the C4B gene. This deletion has been classically identified using Southern blotting, and has been found to be a genetic marker for SLE (Kemp, M.E. et al, Arthritis and Rheumatism 30:1015-22 (1987)). Southern blotting, however, is a time consuming and labor intensive process.
  • the invention pertains to methods of identifying an alteration in a gene of interest, particularly a gene of interest in the major histocompatibility region, utilizing long range polymerase chain reaction (LR-PCR) amplification of target DNA that includes all or a portion of a human mobile element.
  • the alteration can be, for example, a deletion, insertion, duplication, or inversion.
  • the human mobile element can be a DNA-based transposable element (e.g., a mariner element), autonomous retrotransposon (e.g., a retrotransposon containing long terminal repeats (LTRs), such as human endogenous retroviruses (HERVs); or lacking LTRs, such as LI elements), or a non-autonomous retrotransposon (e.g., Alu element, pseudogene).
  • a DNA-based transposable element e.g., a mariner element
  • autonomous retrotransposon e.g., a retrotransposon containing long terminal repeats (LTRs), such as human endogenous retroviruses (HERVs); or lacking LTRs, such as LI elements
  • LTRs long terminal repeats
  • HERVs human endogenous retroviruses
  • LI elements such as LI elements
  • HERVs human endogenous retroviruses
  • non-autonomous retrotransposon e.g., Alu element, pseudogene
  • C4AQ0 is an approximately 30 kb deletion that is associated with the extended haplotype B8,BfS:C2C.C4AQ0,C4Bl ;DR3, which is the most common extended haplotype in white SLE patients (Kemp, M.E. et al, Arthritis and Rheumatism 30:1015-22 (1987)).
  • the methods can be used to determine whether an individual is at risk for developing systemic lupus erythematosus, as the presence of C4AQ0 correlates with a risk of developing systemic lupus erythematosus.
  • the methods can additionally be used to determine the C4A deletion genotype of an individual (e.g., whether an individual is homozygous for C4AQ0; heterozygous for C4AQ0; or homozygous for the absence of C4AQ0).
  • a test sample of genomic DNA is subjected to long range polymerase chain reaction (LR-PCR) amplification of target DNA that includes a human mobile element (e.g., an endogenous retroviral insert in intron 9 of the C4A gene); the LR-PCR primers are designed such that if the genomic DNA comprises an alteration (e.g., a deletion in the C4A gene), PCR products are formed, and if the genomic DNA does not comprise an alteration (e.g., no deletion in the C4A gene), no PCR products are formed. The presence or absence of PCR products is indicative of the presence or absence of the alteration (e.g., the presence or absence of the deletion in the C4A gene).
  • LR-PCR long range polymerase chain reaction
  • the gene of interest is the C4A gene; the alteration is a deletion; and primers include a forward primer that corresponds to a DNA sequence in the Gl 1 gene (e.g., TCTAGCTTCAGTACTTCCAGCCTGT (SEQ ID NO:l)), and a reverse primer corresponding to a DNA sequence in exon 10 of the C4A gene (e.g., GATGACACAAAATACCAGGATGTGA (SEQ ID NO:2)).
  • TCTAGCTTCAGTACTTCCAGCCTGT SEQ ID NO:l
  • a reverse primer corresponding to a DNA sequence in exon 10 of the C4A gene e.g., GATGACACAAAATACCAGGATGTGA (SEQ ID NO:2)
  • a test sample of genomic DNA is subjected to long range polymerase chain reaction amplification of target DNA that includes a human mobile element (e.g., including the junction between intron 9 and the endogenous retroviral insert in intron 9 of the C4A gene), using primers that are designed such that if the genomic DNA comprises an alteration (e.g., a deletion in the C4A gene), no PCR products are formed, and if the genomic DNA does not comprise an alteration (e.g., no deletion in the C4A gene), PCR products are formed. The presence or absence of PCR products is indicative of the absence or presence of the alteration.
  • a human mobile element e.g., including the junction between intron 9 and the endogenous retroviral insert in intron 9 of the C4A gene
  • the gene of interest is the C4A gene; the alteration is a deletion; and primers include a forward primer that corresponds to a DNA sequence in the Gl 1 gene (e.g., TCTAGCTTCAGTACTTCCAGCCTGT (SEQ ID NO:l)), and a reverse primer corresponding to the junction between intron 9 and the retroviral insert in intron 9 of the C4A gene (e.g.,
  • the primers for the long range polymerase chain reaction amplification are designed such that PCR products having detectably different sizes are produced in the presence and in the absence of the alteration (e.g., in the presence or absence of C4AQ0); an assessment of the size of the PCR products indicates whether the alteration is present or absent.
  • the methods of the invention are simple to perform, provide consistent results, and can be adapted for high-throughput screening of test samples.
  • the methods facilitate genotyping of individuals, thereby affording a quick and reliable means for identification of individuals at risk for inheriting or for developing diseases associated with alterations in a gene of interest, such as systemic lupus erythematosus.
  • Figure 1 is a representation of long range polymerase chain reaction (PCR) detection of the presence of C4AQ0 (presence of the deletion in the C4A gene).
  • PCR polymerase chain reaction
  • Figure 2 is a representation of long range polymerase chain reaction (PCR) detection of the absence of C4AQ0 (absence of the deletion in the C4A gene).
  • a gene "of interest,” as used herein, refers to a gene (e.g., a nucleic acid encoding a polypeptide) which is to be assessed for the presence or absence of an alteration.
  • the gene is a gene in the major histocompatibility region (MHC).
  • the methods can be employed, for example, in assessment of alterations associated with complement-based diseases, such as rheumatoid arthritis, ankylosing spondylitis, scleroderma, subacute sclerosing panencephalitis, systemic lupus erythematosus, or renal disease such as nephropathy (e.g., IgA nephropathy).
  • An "alteration" in the gene is a change in the nucleic acid sequence of the gene (e.g., in a test sample) as compared to a known or expected nucleic acid sequence of the gene.
  • the alteration can be, for example, a deletion (e.g., of one or more nucleotides), insertion (e.g., of one or more nucleotides), duplication (e.g., of all or a portion of the gene), or inversion.
  • PCR polymerase chain reaction
  • long range PCR long range PCR
  • LR PCR polymerase chain reaction
  • the human mobile element can be a DNA-based transposable element (e.g., a mariner element).
  • the human mobile element can be an autonomous retrotransposon, for example, a retrotransposon containing long terminal repeats (LTRs), such as short or long interspersed elements (SINEs or LINEs), or human endogenous retroviruses
  • HERVs HERVs
  • the human mobile element can also be a non-autonomous retrotransposon (e.g., Alu element, pseudogene).
  • the human mobile element of interest is a human endogenous retrovirus (HERV).
  • HERVs are particularly useful for analysis of genes of interest of the major histocompatibility complex (MHC), because of the prevalence of HERVs within the MHC, as well as the possible relationship between HERVs and complement-based or other MHC-related diseases (see, e.g., Andersson, G. et al, Trends Genet. 14(3):109-114 (1998); Kulski, J.K. et al, J. Mol.
  • MHC major histocompatibility complex
  • the human mobile element used in the methods is referred to as the "human mobile element of interest.”
  • the primers used in the LR PCR reaction are designed such that in the presence of an alteration in the gene of interest, a PCR product is produced which can be detected, and in the absence of the alteration, no PCR product is produced which can be detected, or a PCR product of a detectably different size can be detected.
  • the absence of the alteration can be confirmed by LR PCR using primers designed such that in the absence of the alteration, a PCR product is produced which can be detected, and in the presence of the alteration, no PCR product is produced which can be detected (or PCR products of a detectably different size can be detected).
  • a deletion in the C4A gene is detected.
  • the deletion C4AQ0 which extends from the 5' end of the C4A gene to the same position in the C4B gene, is detected.
  • This deletion serves as a marker for systemic lupus erythematosus (SLE) (see, e.g., Kemp, M.E. et al, Arthritis and Rheumatism 30:1015-22 (1987); Arnett, F.C., Clin. Immunol.
  • the primers used in the LR PCR reaction are designed such that in the presence of a deletion in the C4A gene (e.g., in the presence of C4AQ0), a PCR product is produced which can be detected, and in the absence of the deletion (e.g., in the absence of C4AQ0), no PCR product is produced which can be detected, or a PCR product of a detectably different size can be detected.
  • the absence of the deletion can be confirmed by LR PCR using primers designed such that in the absence of the deletion, a PCR product is produced which can be detected, and in the presence of the deletion, no PCR product is produced which can be detected (or PCR products of a detectably different size can be detected).
  • the methods can be used to identify individuals at risk for developing SLE or for inheriting SLE, or to confirm a diagnosis of SLE in an individual suspected of having SLE.
  • POLYMERASE CHAIN REACTION AMPLIFICATION Polymerase chain reaction (PCR) amplification is a well-known tool for amplification of nucleotide sequences.
  • PCR Polymerase chain reaction
  • a double-stranded target DNA sequence is denatured; primers are annealed to each strand of the denatured target; and the primers are extended by a DNA polymerase. This cycle is repeated, generally between 25 and 40 times, in order to concentrate the number of copies of a target DNA sequence in a sample.
  • the primers used in PCR are designed to anneal to the denatured target DNA sequence strands in a position and orientation such that the extended primers are complementary copies of the target DNA sequences. On subsequent amplification cycles, the extended primers can also serve as targets for amplification.
  • PCR is described in detail in U.S. Patent No.s 4,683,202; 4,683,195; 4,800,159; and 4,965,188; the entire teachings of these patents are incorporated herein by reference.
  • Long range PCR utilizes amplification conditions which improve target strand denaturation (e.g., higher denaturation temperatures, addition of cosolvents), and which protect DNA from degradation; utilizes longer extension times; and minimize incorporation of erroneous nucleotides by utilizing polymerases having exonuclease activity to reduce mismatches, thereby enabling amplification of extended strands of DNA.
  • Long range PCR is described in detail, for example, in U.S. Patent 5,512,462; in Burland, V. and Kusukawa, N., Biotechniques 23:1070-1072, 1074-1075 (1997)); and Cheng, S. et al, Proc. Natl. Acad. Sci. USA 91:5695-5699 (1994)); the entire teachings of which are incorporated by reference herein.
  • a test sample comprising genomic DNA is used.
  • the test sample is obtained from an individual suspected of having (or of carrying a defect associated with) an alteration in a gene of interest (e.g., an individual suspected of having or of carrying a defect associated with systemic lupus erythematosus (SLE)) (the "test individual").
  • the individual can be an adult, child or fetus.
  • the test sample can be from any source which contains genomic DNA, such as a blood or tissue sample (e.g., from skin or other organs).
  • the test sample is obtained from a blood sample, a fibroblast skin sample, from hair roots, or from cells obtained from the oral cavity (e.g., via mouthwash).
  • test sample is obtained from fetal cells or tissue by appropriate methods, such as by amniocentesis or chorionic villus sampling.
  • the test sample is subjected to LR PCR amplification, and the presence or absence of an alteration (e.g., the presence or absence of a deletion in the C4A gene) is then detected.
  • primers are designed to amplify DNA adjacent to, and/or including at least part of, a gene of interest.
  • the DNA that is amplified contains all or a portion of a human mobile element of interest (e.g., the retroviral insert in the C4A gene), as well as the part of the gene (or DNA adjacent to the gene) which may contain the alteration of interest.
  • the human mobile element of interest can be a part of the gene of interest (e.g., not solely adjacent to the gene of interest), and can contain the alteration of interest.
  • the DNA that is targeted for amplification, which contains the human mobile element of interest is referred to herein as the "target" DNA.
  • primer refers to an oligonucleotide that is capable of serving as an initiation point for nucleic acid synthesis during PCR, under appropriate conditions as described below.
  • the primer typically ranges from 15 to 50 nucleotides , and/or has a T m of approximately 50-
  • the primer is approximately 25-30 nucleotides in length, and/or has a T m of approximately 60-65 °C.
  • Primers can be prepared by a variety of methods, including chemical synthesis (see, e.g., Narang et al, Meth. Enzymol 68:90-99 (1979); Brown et al, Meth. Enzymol. (55:109-151 (1979); Beucage et al, Tetrahedron Lett. 22:1859-1862 (1981); U.S.patent 4,458,066; the entire teachings of these references are incorporated herein in their entirety).
  • the primers are designed such that the PCR products (as described below) obtained from the primers will differ in size, depending on the presence or absence of alteration (e.g., the presence or absence of a deletion in the C4A gene, such as C4AQ0). That is, in the presence of the alteration, the primers will yield PCR products of a certain (first) size, and in the absence of the alteration, the same primers will yield PCR products of a (second) size that is detectably different from the size of the PCR products in the presence of the deletion (the first size).
  • a "detectably different" size indicates that the differences in the sizes of the products can be identified, using standard techniques, such as described below.
  • the primers are designed such that no PCR products are produced either in the presence or in the absence of the alteration.
  • the primers will yield PCR products of a certain size in the presence of the alteration, and will yield no PCR products in the absence of the deletion.
  • the primers will yield PCR products of a certain size in the absence of the alteration, and will yield no PCR products in the presence of the alteration.
  • the nucleotide sequences of the primers correspond to DNA sequences adjacent to or present in the human mobile element of interest (e.g., adjacent to the C4A gene or the retroviral insert).
  • a primer that "corresponds to" a DNA sequence is a primer that has the same nucleotide sequence as the DNA sequence, or that is sufficiently complementary to the DNA sequence that it hybridizes under PCR conditions to the DNA sequence.
  • a DNA sequence that is "adjacent to" the a human mobile element of interest is DNA sequence that is in physical proximity to the mobile element of interest.
  • a DNA sequence that is "adjacent to" the C4A gene is a DNA sequence that is in physical proximity to the C4A gene on chromosome 6, such as a DNA sequence in a gene next to the C4A gene (e.g., the Gi l, C2, TEN, Bf, 21-OH or RD genes, preferably the Gl 1 gene).
  • An "upstream” or “forward” primer a primer that hybridizes to the non-coding strand of the target DNA and forms the 5' end of the amplified product of the coding strand
  • a primer that hybridizes to the non-coding strand of the target DNA and forms the 5' end of the amplified product of the coding strand
  • downstream or reverse primer a primer that hybridizes to the coding strand of the target DNA and forms the 5' end of the amplified product of the non-coding strand
  • the forward primer corresponds to a unique DNA sequence upstream of the part of the gene of interest that may contain the alteration
  • the reverse primer corresponds to a unique DNA sequence downstream of the part of the gene of interest that may contain the alteration; either the forward primer, or the reverse primer, comprises all or a portion of the human mobile element.
  • the forward primer corresponds to a unique DNA sequence upstream of the target DNA (e.g., a forward primer corresponding to a DNA sequence in the Gl 1 gene), and the reverse primer corresponds to a DNA sequence in an exon of the C4A gene that is after (downstream of) the location of the target DNA which is a retroviral insert (e.g., the second half of intron 9, exon 10, or beyond).
  • the same forward primer as described above is used; the reverse primer corresponds to a DNA sequence within the target DNA which is a retroviral sequence (e.g., the DNA sequence in the junction between the retroviral sequence and intron 9).
  • the forward primer TCTAGCTTCAGTACTTCCAGCCTGT (SEQ ID NO:l), which corresponds to a DNA sequence in the Gl 1 gene, is used, and the reverse primer GATGACACAAAATACCAGGATGTGA (SEQ ID NO:2), which corresponds to a DNA sequence in exon 10 of the C4A gene, is used.
  • the same forward primer TCTAGCTTCAGTACTTCCAGCCTGT (SEQ ID NO: 1) is used, and the reverse primer TGGTCCCCAACATGTCTGTGCATGCTG (SEQ ID NO:3), which corresponds to a DNA sequence of the junction between the retroviral sequence and intron 9 of the C4A gene, is used.
  • test sample of genomic DNA and the primers are used in LR PCR amplification. Long range PCR amplification is performed, for example, as described in U.S. Patent 5,512,462. Briefly, the test sample of genomic DNA and the primers are mixed in an amplification reaction mixture.
  • An "amplification reaction mixture” contains reagents necessary for amplification of the target DNA sequence (e.g., nucleotides, enzymes, buffers, etc.).
  • Representative amplification reaction mixtures include commercial kits, such as the rTth DNA POLYMERASE XL kit (Perkin Elmer, catalog number N808-0187).
  • the amplification reaction mixture, including the test sample and primers, is then subjected to cycles of varying temperature. If a commercial kit is used, the manufacturer's suggested protocol can be used for amplifying the target DNA.
  • a 20 ⁇ l reaction mixture can be used, including the following components:
  • Template DNA (genomic) 1 ⁇ l (approximately 100 ng) 3.3 x Buffer 6 ⁇ l (e.g., rTth Polymerase XL kit buffer, Perkin-Elmer)
  • Reverse primer (5 ⁇ M) 2 ⁇ l (Final: 0.5 ⁇ M) rTth polymerase 0.4 ⁇ l**
  • ** rTth pol has exonuclease activity; PCR is started immediately after addition of rTth pol, which is added last to the mixture.
  • the reaction mixture is then subjected to cycling conditions, such as the following:
  • PCR products refers to copies of the target DNA sequence that are produced during PCR amplification (i.e., DNA which has been amplified during the PCR process). If no DNA has been amplified during PCR, no PCR products will be generated.
  • Analysis of the PCR products includes detecting the presence (or absence) of detectable PCR products; in a preferred embodiment, analysis of the PCR products includes determining the size of any detectable PCR products.
  • the PCR products can be detected by a variety of methods; in a preferred embodiment, methods which separate the DNA by size, such as gel electrophoresis (e.g., agarose or acrylamide gel electrophoresis), or HPLC, are used to separate PCR products, and are followed by detection of the size fractionated DNA by methods such as staining (e.g., with ethidium bromide), or hybridization of labeled probes. Representative methods are described in Current Protocols in Molecular Biology, Ausubel, F. et al, eds., John Wiley & Sons, including all supplements through 1999. In a preferred embodiment, ethidium bromide agarose gel electrophoresis is used, and the presence or absence of PCR products is then detected.
  • gel electrophoresis e.g., agarose or acrylamide gel electrophoresis
  • HPLC e.g., agarose or acrylamide gel electrophoresis
  • staining e.g.,
  • the presence of absence of an alteration in a gene of interest can be determined based on the presence or absence, or the size, of the PCR products. For example, as shown in Figure 1, if the test sample comprises genomic DNA containing C4AQ0, then a forward primer in the Gi l gene and a reverse primer in exon 10 (e.g., SEQ ID NO:l and SEQ ID NO:2) will yield a PCR product of approximately 5.4 kb.
  • test sample comprises genomic DNA that does not contain C4AQ0
  • the DNA between these primers will be approximately 11.8 kb, which is too big a product to be generated (that is, no PCR products will be generated).
  • detection of a PCR product, and particularly of a PCR product of approximately 5.4 kb is indicative of the presence of a deletion, and particularly of the presence of C4AQ0 which is associated with SLE.
  • Lack of a PCR product is indicative of the absence of C4AQ0.
  • a PCR product is detected (i.e., if the test sample contains genomic DNA that comprises a deletion, such as C4AQ0), further experiments can also be performed to determine whether the test sample is homozygous or heterozygous for the deletion (e.g., heterozygous or homozygous for C4AQ0).
  • a forward primer in the Gl 1 gene e.g., SEQ ID NO:l
  • a reverse primer which corresponds to a DNA sequence of the junction between the retroviral sequence and intron 9 of the C4A gene e.g., SEQ ID NO:3
  • test sample comprises genomic DNA that does not contain the deletion (i.e., is heterozygous for C4AQ0)
  • these primers will yield a PCR product of approximately 5.2 kb.
  • genomic DNA that does contain the deletion i.e., is homozygous for C4AQ0
  • no PCR products will be generated.
  • detection of a PCR product, and particularly of a PCR product of approximately 5.2 kb is indicative of the absence of the deletion, and particularly the absence of C4AQ0.
  • Lack of a PCR product is indicative of the presence of the deletion, particularly the presence of C4AQ0 which is associated with SLE.
  • a test sample of genomic DNA from an individual such as an individual suspected of being at risk for developing or inheriting a disease or condition associated with an alteration in a gene of interest (e.g., SLE) is analyzed for the presence or absence of the alteration in the gene of interest (e.g., the deletion in the C4A gene, such as an analysis for the presence or absence of C4AQ0). If a test sample from the individual comprises genomic DNA that contains the alteration (e.g., a deletion in the C4A gene, such as the presence of C4AQ0), the presence of the deletion therefore indicates that the individual is at risk for developing or inheriting the disease or condition associated with the alteration in the gene of interest.
  • a gene of interest e.g., SLE
  • a test sample of genomic DNA from an individual can be analyzed homozygosity or heterozygosity of an alteration of interest.
  • a test sample of genomic DNA can be analyzed for the presence or absence of deletions in the C4A gene (e.g., for the presence or absence of C4AQ0), and the C4A deletion genotype (for example, whether the individual is homozygous for (the presence of) C4AQ0; heterozygous for C4AQ0; or homozygous for the absence of C4AQ0) can be determined.
  • a test sample comprising genomic DNA from the individual can be subjected to long range polymerase chain reaction amplification of target DNA comprising a retroviral insert in intron 9 of the C4A gene, as described above. If the test sample comprises genomic DNA comprising a deletion in the C4A gene, PCR products are formed, and if the test sample does not comprise genomic DNA comprising a deletion in the C4A gene, no PCR products are formed; thus, the presence of PCR products indicates that the individual is either homozygous or heterozygous for a deletion in the C4A gene (e.g., C4AQ0), and the absence of PCR products indicates that the individual is homozygous for the absence of a deletion in the C4A gene (e.g., C4AQ0).
  • C4AQ0 homozygous or heterozygous for a deletion in the C4A gene
  • Homozygosity or heterozygosity for the deletion can be determined by subjecting a test sample comprising genomic DNA from the individual to long range polymerase chain reaction amplification of target DNA comprising a junction between intron 9 and retroviral insert in intron 9 of the C4A gene, as described above.
  • test sample comprises genomic DNA comprising a deletion in the C4A gene (e.g., C4AQ0)
  • no PCR products are formed, and if the test sample does not comprise genomic DNA comprising a deletion in the C4A gene, PCR products are formed; the absence of PCR products indicates that the individual is homozygous for a deletion in the C4A gene (e.g., C4AQ0), and the presence of PCR products indicates that the individual is heterozygous for a deletion in the C4A gene (e.g., C4AQ0).
  • This method provides an advantage over protein-based methods of analysis, as the C4A protein is produced in very low amounts, rendering it difficult to determine genotype by analysis of the amount of C4A protein.
  • Genotyping allows an assessment of an individual's risk for developing (or inheriting) SLE. Individuals with no deletion are at a reduced risk of developing SLE, compared with individuals with one or more deletions. At least one C4AQ0 (i.e., a heterozygous or homozygous genotype) has been identified in up to 50% or more patients with SLE, and homozygosity for C4 AQO is reported in 11 - 13 % of SLE patients, compared to 2-3% of controls (Schur, P.H., Dubois' Lupus Erythematosus 5 th ed:254-261 (1977); Arnett, F.C., Moulds, J.M., Clin.
  • C4AQ0 i.e., a heterozygous or homozygous genotype
  • the methods of the invention which have been exemplified by an analysis of the C4A gene for the presence of absence of a deletion (e.g., for the presence or absence of C4AQ0), are equally applicable to analysis of other genes for the presence of absence of other alterations (e.g., deletions, duplications, insertions, and inversions) in a gene of interest.
  • the methods are particularly useful for the identification of alterations (e.g., deletions) within the genes of the major histocompatibility complex (MHC) region, as they enable analysis of sizable regions of DNA such as that of the MHC.
  • MHC major histocompatibility complex
  • methods can be employed, for example, in assessment of alterations associated with complement- based diseases, such as rheumatoid arthritis, ankylosing spondylitis, scleroderma, subacute sclerosing panencephalitis, and renal disease such as nephropathy (e.g., IgA nephropathy).
  • complement- based diseases such as rheumatoid arthritis, ankylosing spondylitis, scleroderma, subacute sclerosing panencephalitis, and renal disease such as nephropathy (e.g., IgA nephropathy).
  • primers are designed to amplify regions of DNA including and/or surrounding the alteration.
  • the annealing temperature (T m ) of the primers should be designed to ensure success of the polymerase chain reaction under the conditions necessary for long range PCR amplification.
  • the T m of the primers should be approximately 50-75°C, preferably approximately 60-65°C.
  • the primers are designed such that the PCR products, as described above, that are obtained from the primers will differ in size, depending on the presence or absence of the alteration in the gene of interest.
  • the primers in the presence of the alteration, will yield PCR products of a certain (first) size, and in the absence of the alteration, the same primers will yield PCR products of a (second) size that is detectably different from the size of the PCR products in the presence of the alteration (the first size).
  • the primers are designed such that no PCR products are produced either in the presence or in the absence of the alteration.
  • the primers will yield PCR products of a certain size in the presence of the alteration, and will yield no PCR products in the absence of the alteration; alternatively, the primers will yield PCR products of a certain size in the absence of the alteration, and will yield no PCR products in the presence of the alteration.
  • test sample is obtained as described above, and long range PCR is performed as described.
  • the PCR products, if any, are then assessed for the presence or absence of an alteration in the gene of interest, using the methods as described above.
  • the presence or absence of the PCR products, or the presence of PCR products of particular sizes, is indicative of the presence or absence of the alteration in the target gene of interest.
  • C4 protein allotypes were analyzed by high-voltage agarose electrophoresis on carboxypeptidase (Sigma Type I) and neuraminidase (Sigma Type VIII) treated serum samples followed by immunofixation with monoclonal antibodies (Incstar) (sim and Cross, Biochem. J. 239:163-161 (1986)).
  • C4A and C4B allotypes were determined by their positions on the gel in comparison to known standard samples.
  • C4A and C4B zygosity was determined by their relative band intensity.
  • HLA- A, B, and C Typing of MHC class allotypes was performed by serological methods using the lymphocytotoxicity test (Perdue et al, Tissue Antigens 9:259-266 (1977)).
  • Typing of MHC class II alleles was perfonned by PCR with sequence specific primers (PCR-SSP) (Dynal) (Olerup and Zetterquist, Tissue Antigens 30:225-235 (1992)).
  • PCR-SSP sequence specific primers
  • C4 protein allotyping and HLA typing was performed on a collection of DNA samples obtained in rheumatological disease studies performed at the Center for Rheumatology Research, Reykjavik, Iceland. This identified appropriate individuals for screening with a long PCR strategy. There were two individuals who were both C4A protein deficient and homozygous for B 8-C4AQ0-C4B 1 -DR3 , whilst 21 heterozygotes had protein deficiency of C4A. A total of 66 individuals did not have the B8-C4AQ0-C4B1-DR3 extended haplotype and were not deficient in C4A protein expression.
  • C4A Deletion Genotyping by Long PCR Two separate long PCR assays - one, C4A deletion-specific and the other, non-deletion-specific - were designed to determine the C4A genotype. Individuals having different genotypes (homozygous for C4AQ0 (the "deletion"); heterozygous for the deletion; homozygous for no deletion) were assessed to determine the presence or absence of C4AQ0 using long range PCR. Genotypes of the individuals had been previously determined using analysis of C4 protein and HLA typing as described above.
  • the amplification reaction mixture included the following components, for a total volume of 20 ⁇ l:
  • Template DNA (genomic) 1 ⁇ l (approximately 100 ng)
  • Reverse primer (5 ⁇ M) 2 ⁇ l (Final: 0.4 ⁇ M) rTth polymerase 0.4 ⁇ l
  • TCTAGCTTCAGTACTTCCAGCCTGT SEQ ID NO:l
  • primer 1 TCTAGCTTCAGTACTTCCAGCCTGT
  • reverse primer either GATGACACAAAATACCAGGATGTGA (SEQ ID NO:2) ("primer 2"), which corresponds to a DNA sequence in exon 10 of the C4A gene, or TGGTCCCCAACATGTCTGTGCATGCTG (SEQ ID NO:3) (“primer 3”), which corresponds to a DNA sequence in the junction between the retroviral sequence and intron 9 of the C4A gene, was used. Because rTth pol has exonuclease activity; PCR was started immediately after addition of rTth pol, which is added last to the mixture. The reaction mixture was subjected to the following cycling conditions: 1 cycle: 94°C for 30 sec
  • the PCR products were loaded with standard loading buffer (0.25%) bromophenol blue; 40% (w/v) sucrose in water) on a 0.8% ethidium bromide agarose gel (Sigma), run in Tris-borate-EDTA (lxTBE) at 100 V/hr, and visualized under ultraviolet light (Eagle Eye II, Stratagene).
  • Positive clones were sequenced approximately 500 bp from each end to confirm accurate amplification using Universal Ml 3 forward (-20) (16 mer) and reverse (17 mer) primers (Invitrogen) and the BigDye terminator cycle sequencing kit (Perkin Elmer). The samples were run on an ABI 377 and analyzed using sequence analysis softward 3.1 (Perkin Elmer).

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Abstract

Cette invention se rapporte à des procédés servant à détecter une altération dans un gène d'intérêt, par exemple une délétion dans le gène C4A (notamment pour détecter C4AQ0), en effectuant une amplification du type réaction en chaîne de polymérase (PCR) longue portée sur un échantillon test comprenant un ADN génomique. Ces procédés amplifient un ADN cible comprenant la totalité ou une partie d'un élément mobile humain (par exemple un insert rétro-viral dans l'intron 9 du gène C4A) en utilisant des amorces conçues pour que les produits PCR soient formés uniquement si l'échantillon test comprend un ADN génomique comportant l'altération dans le gène d'intérêt; dans une variante, ces procédés amplifient l'ADN cible en utilisant des amorces conçues pour que les produits PCR soient formés uniquement si l'échantillon test comprend un ADN génomique qui ne comporte pas l'altération dans le gène d'intérêt. Dans une variante, les amorces sont conçues pour que les produits PCR aient des tailles différentes détectables, selon que l'échantillon test comprend ou ne comprend pas un ADN génomique comportant l'altération dans le gène d'intérêt. Ces procédés peuvent servir à identifier si un individu présente des risques de développer une maladie ou un état associé à l'altération dans le gène d'intérêt, dès lors que la présence de cette altération est à mettre en corrélation avec le risque de développer ladite maladie.
PCT/IB2000/001283 1999-09-07 2000-09-06 Detection des alterations dans un gene par amplification pcr longue portee a l'aide d'elements mobiles humains Ceased WO2001018245A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
MXPA02002507A MXPA02002507A (es) 1999-09-07 2000-09-06 Deteccion de alteraciones en un gen por pcr de largo alcance mediante el uso de elementos moviles humanos.
BR0014017-1A BR0014017A (pt) 1999-09-07 2000-09-06 Método para detectar alterações em um gene através de pcr de ampla abrangência usando elementos humanos móveis
JP2001521779A JP2003508082A (ja) 1999-09-07 2000-09-06 ヒト可動性因子を使用する長領域ｐｃｒによる遺伝子における変更の検出
CA002383857A CA2383857A1 (fr) 1999-09-07 2000-09-06 Detection des alterations dans un gene par amplification pcr longue portee a l'aide d'elements mobiles humains
NZ517641A NZ517641A (en) 1999-09-07 2000-09-06 Detection of alterations in a gene by long range PCR using human mobile elements
EP00954856A EP1214449A2 (fr) 1999-09-07 2000-09-06 Detection des alterations dans un gene par amplification pcr longue portee a l'aide d'elements mobiles humains
AU67207/00A AU6720700A (en) 1999-09-07 2000-09-06 Detection of alterations in a gene by long range pcr using human mobile elements

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US39124499A 1999-09-07 1999-09-07
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US47159899A 1999-12-23 1999-12-23
US09/470,673 1999-12-23
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US09/470,673 US6225093B1 (en) 1999-09-07 1999-12-23 Detection of C4A deletion by long range PCR

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Cited By (3)

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WO2002062197A3 (fr) * 2000-12-19 2002-10-31 Hospital For Special Surgery Marqueurs de predisposition pour des maladies et cibles pour therapie
EP1475445A1 (fr) * 2003-05-07 2004-11-10 Tosoh Corporation Méthode de détection de micro-métastase
EP2397561A1 (fr) 2010-06-18 2011-12-21 Progenika Biopharma, S.A. Sondes et procédés pour déterminer la présence ou l'absence de segments génétiques

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ES2833299T3 (es) 2014-02-04 2021-06-14 Jumpcode Genomics Inc Fraccionamiento del genoma
EP3943615A1 (fr) * 2014-02-27 2022-01-26 Jumpcode Genomics, Inc. Procédés pour l'analyse d'éléments mobiles somatiques, et leurs utilisations
US10968536B2 (en) 2015-02-25 2021-04-06 Jumpcode Genomics, Inc. Methods and compositions for sequencing
US11339427B2 (en) 2016-02-12 2022-05-24 Jumpcode Genomics, Inc. Method for target specific RNA transcription of DNA sequences
CN113820499B (zh) * 2021-10-26 2024-06-25 深圳临研医学有限公司 用于诊断系统性红斑狼疮的蛋白标志物

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US5096557A (en) * 1990-07-11 1992-03-17 Genetype A.G. Internal standard for electrophoretic separations

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002062197A3 (fr) * 2000-12-19 2002-10-31 Hospital For Special Surgery Marqueurs de predisposition pour des maladies et cibles pour therapie
EP1475445A1 (fr) * 2003-05-07 2004-11-10 Tosoh Corporation Méthode de détection de micro-métastase
CN100335654C (zh) * 2003-05-07 2007-09-05 东曹株式会社 检测微转移的方法
US7393641B2 (en) 2003-05-07 2008-07-01 Tosoh Corporation Method of detecting micrometastasis
EP2397561A1 (fr) 2010-06-18 2011-12-21 Progenika Biopharma, S.A. Sondes et procédés pour déterminer la présence ou l'absence de segments génétiques

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