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WO2006019984A2 - Compositions, procedes et trousses pour le diagnostic et le traitement du syndrome de mort subite du nourrisson - Google Patents

Compositions, procedes et trousses pour le diagnostic et le traitement du syndrome de mort subite du nourrisson Download PDF

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Publication number
WO2006019984A2
WO2006019984A2 PCT/US2005/025099 US2005025099W WO2006019984A2 WO 2006019984 A2 WO2006019984 A2 WO 2006019984A2 US 2005025099 W US2005025099 W US 2005025099W WO 2006019984 A2 WO2006019984 A2 WO 2006019984A2
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sids
infant
scn5a
nucleic acid
seq
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WO2006019984A3 (fr
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Steve A. N. Goldstein
Peter N. Bowers
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Yale University
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Yale University
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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
    • C12Q1/6876—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • SIDS Sudden Infant Death Syndrome
  • SIDS is defined as sudden and unanticipated death in an infant under one year of age with no recognized lethal disorder after an investigation that includes an autopsy, examination of the death scene, and review of the clinical history (Willinger et al. , 1991 , Pediatr. Pathol. 11 : 677-684) . Only after all other alternatives have been exhausted is the diagnosis of SIDS made. Thus, SIDS is a diagnosis by exclusion. There is significant variation in the number of SIDS cases in different racial and ethnic groups; rates for African- Americans and American Indians are three-fold those for Caucasians and six times higher than for Hispanics and Asians (Mathews et al, 2003, Nat'l Vital Stat. Rep. 52: 1-28).
  • LQTS inherited long QT syndrome
  • the invention includes, for example, methods, compositions and kits useful for identifying, screening, diagnosing and treating individuals having, or at risk of having, SIDS. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figures IA- IB are a series of images depicting a polymorphism in SCN5A that encodes a variant cardiac sodium channel.
  • Figure IA is an image depicting the denaturing high-performance liquid chromatography (dHPLC) waveform and direct sequencing of wild type and an Sl 103 Y variant. The chromatogram displays the additional peak resulting from the heteroduplex assembly of S 1103 and Yl 103 amplicons.
  • dHPLC denaturing high-performance liquid chromatography
  • FIG. 1B is an image depicting a schematic diagram of the topology of the cardiac sodium channel encoded by SCN5A.
  • the cytoplasmic location of the Sl 103 Y missense change (solid circle), the four homologous membrane domains (DI-DIV), the pore- forming loops (P) and voltage sensing segments (+) are depicted.
  • Figures 2A-2I Figures 2 A through 21 are a series of images depicting that Yl 103 currents demonstrate a change in inactivation gating at low pH. Macroscopic currents were recorded in HEK-293 cells expressing SCN5A-S1103 (open square) or SCN5A-Y1103 (filled square) channel genes. Figures 2A through 2C depict representative current traces with Sl 103 channels. Arrows in protocols indicate points of measure.
  • Figures 2E and 2H are a series of images depicting a shift in voltage- dependent steady-state inactivation at low cytosolic pH (6.7) in Yl 103 channels whereas Sl 103 channels do not demonstrate the same shift.
  • Figure 3A depicts single S 1103 channels opening and inactivating rapidly with no re-opening in response to depolarization and that this behavior is unaltered when internal pH is lowered from 7.4 to 6.7.
  • Figure 3 A further depicts that, in contrast, Yl 103 channels behave like Sl 103 channels at pH 7.4 but demonstrate late re-openings at pH 6.7.
  • Figures 4A-4B Figures 4 A and 4B are a series of images depicting that abnormal channel re-openings were suppressed by mexiletine.
  • FIG. 4A depicts representative Sl 103 current traces evoked by a pulse in drug-free solution (control) and the first (pulse 1) to demonstrate tonic block and 50th (pulse 50) to demonstrate phasic block by 10 ⁇ M mexiletine. Scale bars represent 2 ms and 200 pA.
  • Figure 4B depicts single Yl 103 channels studied in inside-out off-cell patches demonstrating that late re-openings of variant channels are fully suppressed by 5 mM mexiletine. Scale bars represent 0.3 pA and 10 ms.
  • Figure 5 is an image depicting the topology of the SCN5A channel. Identified missense variants in African- American SIDS cases are indicated by closed circles, the transmembrane domains (DI-DIV), pore-forming domains (P) and voltage sensor domains (+) are depicted. Missense variants disclosed herein are S524Y, R688H, El 107K and Sl 103 Y. Missense variants disclosed previously are A997S and R1826H (Ackerman et al., 2001, JAMA 286: 2264-2269).
  • Figures 6A-6C are a series of images depicting an ancestry analysis of SIDS probands homozygous for SCN5A Sl 103 Y.
  • the triangle plot depicts the maximum likelihood estimate (center circle) and the 2 fold (inner ring), 5 fold (middle ring) and 10 fold (outer ring) likelihood confidence intervals, respectively.
  • SIDS Sudden Infant Death Syndrome
  • SCN5A a cardiac sodium channel that opens to initiate electrical activity in the ventricles and inactivates to end each heartbeat, can cause arrhythmia, chaotic rhythms and sudden death if the channel fails to inactivate.
  • the present data demonstrate that the phenotype of this polymorphism is a gain in function of a cardiac sodium ion channel at lower physiological pH, activity after channel depolarization, and in homozygous infants, a 24-fold increase in risk of SIDS. Therefore, the present invention includes methods for diagnosing the probability of SIDS in an infant and prophylactic methods of preventing SIDS in infants.
  • the term "amplification” refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction, and ligase chain reaction. Any method known in the art is intended to be used in the practice of the methods disclosed herein to copy the polynucleotide sequence into a larger number of polynucleotide molecules.
  • the term “antibody” refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) 2 , as well as single chain antibodies and humanized antibodies (Harlow et at, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et at, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et at, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et at, 1988, Science 242:423-426).
  • synthetic antibody refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • Any antibody of any form, including synthetic antibodies, binding specifically to the Sl 103 Y epitope or the Sl 103 epitope is intended to be used in the practice of the methods of the invention disclosed herein.
  • the term "antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand.
  • an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • assessing a risk refers to determining whether an individual is at a greater risk for a disease or condition than another individual.
  • biological sample refers to a sample obtained from an individual that can be used to assess the presence or absence of an SCN5A polymorphism in a nucleic acid or a polypeptide, the level of SCN5A polypeptide present, or both.
  • a sample includes, but is not limited to, a blood sample, a tissue sample, a bodily fluid sample and a cell.
  • both allelic copies refers to a copy of an allele from the mother and a copy of an allele from the father.
  • carrier refers to an individual that is at least heterozygous for a specified genetic trait.
  • the term "complementary to a portion or all of the nucleic acid encoding SCN5A” refers to a sequence of nucleic acid which does not encode an SCN5A protein. Rather, the sequence which is being expressed in the cells is identical to the non-coding strand of the nucleic acid encoding an SCN5A protein and thus, does not encode an SCN5 A protein.
  • Antisense refers particularly to the nucleic acid sequence of the non- coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand.
  • nucleic acids As used herein, the term "complementary" refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs). As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein.
  • antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • a "coding region" of a gene comprises the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
  • a "coding region" of an mRNA molecule also comprises the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon.
  • the coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g. amino acid residues in a protein export signal sequence).
  • the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent one another or if the two regions are separated by no more than about 1000 nucleotide residues, and preferably no more than about 100 nucleotide residues.
  • infant refers to a fer-s ⁇ n under the age of 18.
  • adult refers to a-per-son 18 years of age and older.
  • the term "fetus” as used herein refers to an infant in utero.
  • the term "individual” refers to any organism. While usually humans are the intended individuals, any other organism expressing SCN5A proteins, or having one or more copies of a nucleic acid encoding SCN5A proteins, can be screened, tested or diagnosed for expression of the SCN5A gene or gene product. Any organism can be screened, tested or diagnosed for expression for the mutant C3308A SNC5A gene or gene product using the methods or compositions disclosed herein. Any organism can be screened, tested or diagnosed for expression of the Sl 103 Y SCN5A protein using the methods or compositions disclosed herein. Any organism can be treated for any disease condition resulting from the expression of the C3308A SCN5A gene or Sl 103Y SCN5A protein.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the methods and compositions of the invention for their designated use.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the composition, and/or components thereof, to be shipped together with a container which contains the composition and/or components thereof.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the composition and/or components thereof be used cooperatively by the recipient.
  • an "isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • a marker refers to a specific nucleic acid or protein that can be identified in a mammal and used to indicate that an indivisual has a greater disposition towards a certain genetic trait.
  • a marker for SIDS is a polymorphism identified on a nucleic acid or the protein encoded by the nucleic acid that indicates a human, for example, has a greater disposition towards having SIDS when compared to a human without a marker for SIDS.
  • naturally-occurring refers to the fact that the object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man is naturally- occurring.
  • nucleic acid bases As used herein, the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenine, “C” refers to cytosine, “G” refers to guanine, “T” refers to thyine, and “U” refers to uracil. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • operably linked refers to a single-stranded or double- stranded nucleic acid moiety which comprises two polynucleotides arranged within the nucleic acid moiety such that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other.
  • a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.
  • polynucleotide refers to a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide may be either a single- stranded or a double-stranded nucleic acid.
  • nucleic acid typically refers to large polynucleotides.
  • oligonucleotide are used interchangeably here. Where is a difference is intended, such will be explicitly stated.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences;” sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • a "portion" of a polynucleotide means at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.
  • the term "primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide.
  • Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase.
  • a primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications.
  • a primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions.
  • Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • probe refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide.
  • a probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions.
  • Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • One of skill in the art can determine the conditions which comprise "stringent hybridization" conditions.
  • the term "recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together.
  • An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.
  • a recombinant polynucleotide may serve a non- coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • a host cell that comprises a recombinant polynucleotide is referred to as a
  • recombinant host cell A gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide, produces a "recombinant polypeptide.”
  • a “recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.
  • polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides and the term “peptide” typically refers to short polypeptides. However, the terms are used interchangeably herein unless explicitly indicated otherwise.
  • polypeptide sequences the left- hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be involved in the expression of the gene product in a tissue specific manner.
  • SNP single nucleotide polymorphism refers to nucleotide sequence variations that occur when a single nucleotide (A, T, C or G) in the genome sequence is altered.
  • SNPs can occur in both coding (gene) and noncoding regions of the genome. Many SNPs have no effect on cell function while other SNPs predispose people to a disease or influence their response to a drug or are linked to a locus that predisposes a person to a disease or influences their response to a drug. Such linkages can be determined by any commonly available means such as those procedures that produce linkage disequilibrium maps for a given SNP or a group of SNPs.
  • telomere binding an antibody which recognizes and binds an epitope of an SCN5 A polypeptide or a nucleic acid which recognizes and specifically binds to (hybridizes with) a nucleic acid that encodes an SCN5A polypeptide, but does not substantially recognize or bind other molecules in a sample.
  • testing a human individual refers to testing a human fetus, infant, child or adult.
  • human includes any one or more of a human fetus, a human infant, a human child and a human adult.
  • a “therapeutic” treatment is a treatment administered to an individual who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
  • a “therapeutically effective amount" of a compound refers to that amount of compound which is sufficient to provide a beneficial effect to the individual to which the compound is administered.
  • transgene refers means an exogenous nucleic acid sequence comprising a nucleic acid which encodes a promoter/regulatory sequence operably linked to nucleic acid which encodes an amino acid sequence, which exogenous nucleic acid is encoded by an animal or cell.
  • a "vector” refers to a composition comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • an "expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
  • variant 1 has Accession No. NM_198056 and variant 2 has Accession No. NM_000335.
  • SEQ ID NO: 67 is the nucleotide sequence of the human SCN5A (GenBank ACCESSION No. NM_198056) used herein.
  • SEQ ID NO: 68 is the amino acid sequence of the human SCN5A (GenBank ACCESSION No. NM_198056) used herein.
  • the protein, splice variant 1, has 2016 amino acids.
  • Splice variant 2 has 2015 amino acids. Amino acid sequence alignment of variants 1 and 2 reveals a glutamine residue (Q) at position 1078 in splice variant 1 but a deletion in the sequence of splice variant 2 at position number 1078.
  • Variant proteins are described by the symbol XnY in which n is the position of an amino acid within the reference sequence, X is the amino acid occupying that position in the reference sequence and Y is the amino acid occupying that position in a variant protein.
  • n is the position of an amino acid within the reference sequence
  • X is the amino acid occupying that position in the reference sequence
  • Y is the amino acid occupying that position in a variant protein.
  • the human SCN5A variant protein having a mutation at amino acid position 1103 where the naturally occurring serine residue S is substituted with a tyrosine residue Y would be designated "Sl 103 Y.”
  • the codons in the variant protein are assigned the same numbers as corresponding codons in the reference protein when the variant and reference protein are maximally aligned.
  • an amino acid position of the variant protein is said to "correspond" to an amino acid position of the reference protein.
  • splice variant 1 has a serine residue at position no. 1103 of SEQ ID NO: 68.
  • Splice variant 2 having a deletion at amino acid position 1078 therefore has the corresponding serine residue occurring at position 1102.
  • the serine in position 1102 of splice variant 2 corresponds to the serine in position 1103 of splice variant 1, the reference protein.
  • variant nucleotides within the gene are described by the symbol WnZ in which n is the position of a nucleotide within the reference sequence, W is the nucleotide occupying that position in the reference sequence and Z is the nucleotide occupying that position in a variant gene.
  • the numbering used in this nomenclature within the present specification refers to the position of the nucleotide within the coding sequence with the adenosine nucleotide of the start ATG codon assigned nucleotide number one.
  • the variant SCN5A gene has the nucleotide sequence comprising the change at C3308A, indicating the cytosine nucleotide at position 3308 was substituted by an adenine.
  • nucleic acid sequences encoding SCN5A proteins can maximally align nucleic acid sequences encoding SCN5A proteins from samples to the nucleic acid sequence of SEQ ID NO: 67 (nucleic acid sequence of splice variant 1) and determine whether a nucleotide in a position of interest in the sample sequence corresponds to the cytosine in nucleic acid position 3308 of the nucleic acid sequence encoding the SCN5A protein. See, for example, published US patent Appl. No. 2003/0162192 Al, incorporated herein by reference in its entirety.
  • the invention includes a method of testing a human individual for a marker for
  • SIDS Sudden Infant Death Syndrome
  • the testing is post-mortem.
  • the invention also includes a method of testing a human individual for a marker for Sudden Infant Death Syndrome (SIDS) comprising identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO:68 in a sample from the individual comprising one or both allelic copies of a protein encoded by the individual's SCN5A nucleic acid; wherein the individual is an infant or a fetus, wherein if the amino acid in the individual's SCN5A protein is tyrosine at a position corresponding to position 1103 of SEQ ID NO:68, then the individual has a marker for SIDS.
  • the testing is post-mortem.
  • the invention includes a method of assessing a risk for Sudden Infant Death Syndrome (SIDS) in a human individual comprising identifying a nucleotide at a position corresponding to position 3308 in SEQ ID NO:67 in a nucleic acid sample from the individual, wherein the sample comprises both allelic copies of a nucleic acid encoding a SCN5A protein, wherein the individual is an infant or a fetus, wherein if the nucleotide in both copies of the nucleic acid is adenine at a position corresponding to position 3308 of SEQ ID NO:67, then the individual is assessed to be at an increased risk for SIDS relative to an individual in which a nucleic acid encoding the SCN5A protein comprises a nucleotide other than adenine at a position corresponding to position 3308 in SEQ ID NO.67.
  • the testing is post-mortem.
  • Another method included in the invention is a method of assessing a risk for Sudden Infant Death Syndrome (SIDS) in a human individual comprising identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO:68 in a sample from the individual comprising one or both allelic copies of a protein encoded by the individual's SCN5A nucleic acid; wherein the individual is an infant or a fetus, wherein if the amino acid in the protein is tyrosine at a position corresponding to position 1103 of SEQ ID NO:68, then the individual is assessed to be at an increased risk for SIDS relative to an individual in which a protein encoded by the SCN5 A nucleic acid comprises an amino acid other than tyrosine at a position corresponding to position 1103 in SEQ ID NO:68.
  • the testing is post-mortem.
  • Yet another method included in the invention is a method of identifying a human carrier of a marker for Sudden Infant Death Syndrome (SIDS) comprising identifying a nucleotide at a position corresponding to position 3308 in SEQ ID NO:67 in a nucleic acid sample from the carrier, wherein the sample comprises one or both allelic copies of a nucleic acid encoding an SCN5A protein; wherein if the nucleotide in one or both copies of the nucleic acid is adenine at a position corresponding to position 3308 of SEQ ID NO:67, then the human is a carrier of a marker for SIDS.
  • SIDS Sudden Infant Death Syndrome
  • a method of identifying a human carrier of a marker for Sudden Infant Death Syndrome comprising identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO:68 in a sample from the carrier comprising one or both allelic copies of a protein encoded by the individual's SCN5A nucleic acid; wherein if the amino acid in one or both copies of the protein has a tyrosine at a position corresponding to position 1103 of SEQ ID NO:68, then the human is a carrier of a marker for SIDS.
  • SIDS Sudden Infant Death Syndrome
  • kits for testing a human infant or fetus for a marker for Sudden Infant Death Syndrome comprising a) at least one reagent for identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO:68; and b) instructional material for the use thereof, wherein if the amino acid at a position corresponding to position 1103 in SEQ ID NO:2 is Y/Y, then the infant or fetus has a marker for SIDS.
  • the invention includes a kit for assessing a risk for Sudden Infant Death Syndrome (SIDS) in a human infant or fetus comprising a) at least one reagent for identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO:68; and b) instructional material for the use thereof, wherein if the amino acid at a position corresponding to position 1103 in SEQ ID NO:68 is Y/Y, then the infant or fetus is assessed to be at increased risk for SIDS relative to an infant or fetus in which the amino acid at a position corresponding to position 1103 in SEQ ID NO:68 is not Y/Y.
  • SIDS Sudden Infant Death Syndrome
  • kits for identifying a human carrier of a marker for Sudden Infant Death Syndrome comprising a) at least one reagent for identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO:68; and b) instructional material for the use thereof, wherein if the amino acid at a position corresponding to position 1103 in SEQ ID NO:68 is Yl-, then the human is identified as a carrier of a marker for SIDS.
  • SIDS Sudden Infant Death Syndrome
  • kits for testing a human infant or fetus for a marker for Sudden Infant Death Syndrome comprising a) a reagent for identifying an amino acid at a position corresponding to position 1103 in SEQ ID NO: 68; and b) an instructional material for the use thereof, wherein if the amino acid at a position corresponding to position 1103 in SEQ ID NO:68 is Y/Y, then the infant or fetus has a marker for SIDS.
  • SIDS Sudden Infant Death Syndrome
  • the invention further includes a method of screening for Sudden Infant Death Syndrome (SIDS) in a human infant or fetus comprising contacting a sample from the infant or fetus with a reagent that detects the presence or absence of a C3308A mutation in a nucleic acid encoding SCN5A; assessing the presence or absence of the mutation in the sample, wherein the presence of the mutation is an indication that the human infant is a candidate for SIDS, thereby screening SIDS in a human infant.
  • the reagent is a nucleic acid.
  • the reagent is detectably labeled.
  • the reagent is detectably labeled with a label selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.
  • the reagent is a polymerase chain reaction (PCR) primer comprising at least one nucleic acid sequence set forth in SEQ ID NOs: 1-66.
  • the primer nucleic acid sequence is SEQ ID NO. 37 or 38.
  • a method for diagnosing SIDS in an infant or fetus comprising contacting a sample from the infant or fetus with a reagent that detects the presence or absence of a mutant SCN5A protein, wherein the mutant protein comprises a Sl 103 Y mutation; and, assessing the presence or absence of the mutation in the sample; wherein the presence of the mutation is an indication that the infant or fetus has SIDS, thereby diagnosing SIDS in infant or fetus.
  • the reagent is an antibody or antibody fragment binding to the Sl 103 epitope.
  • a method for preventing SIDS in a human infant or fetus comprising contacting a sample from the infant or fetus with a reagent that detects the presence or absence of a mutation in a nucleic acid encoding SCN5A, wherein the mutation encodes an Sl 103 Y mutant SCN5A protein; and, assessing the presence or absence of the mutation in the sample, wherein the presence of the mutation is an indication that the human infant or fetus is a candidate for SIDS, the method further comprising administering to the human infant an effective amount of an anti-arrhythmic, thereby preventing SIDS in a human infant.
  • the anti-arrhythmic is selected from the group consisting of mexiletine, propranolol or amiodarone.
  • the reagent is a nucleic acid.
  • the reagent is detectably labeled.
  • the reagent is detectably labeled with a label selected from the group consisting of a radioisotope, a bioluminescent compound, a chemilurm ' nescent compound, a fluorescent compound, a metal chelate, and an enzyme.
  • the reagent is a polymerase chain reaction (PCR) primer comprising one or more of the nucleic acid sequences set forth in SEQ ID NO: 1-66.
  • the primer nucleic acid sequence is SEQ ID NO. 37 or 38.
  • the present invention encompasses methods for diagnosing SIDS in a human individual, testing a human individual for SIDS, assessing the risk of SIDS in a human individual, determining if a human individual is a carrier of a marker for SIDS, and screening a human individual for a SIDS marker.
  • the present invention provides a method of testing a human individual for a marker for SIDS. This is because, as demonstrated by the data disclosed herein, a Sl 103 Y mutation in SCN5A leads to, inter alia, a gain in function mutation in the SCN5 A cardiac sodium channel that causes abnormal openings of the sodium channel, leading to arrhythmia and other cardiac anomalies, hi one aspect of the method, a sample is obtained from a human infant or fetus wherein the biological sample comprises both allelic copies of a nucleic acid encoding an SCN5A polypeptide (SEQ ED NO: 68; Accession No. NM_198056).
  • the method further comprises determining in the biological sample the identity of the nucleotide at, or in a position corresponding to, position 3308 in a nucleic acid encoding an SCN5A polypeptide (SEQ ID NO: 67; Accession No. NM_198056).
  • SCN5A polypeptide SEQ ID NO: 67; Accession No. NM_198056
  • the invention further comprises a method of assessing an individual for the risk of SIDS. Ln one aspect of the method, the individual is a human fetus or infant.
  • the method comprises collecting or obtaining from the individual a biological sample comprising both allelic copies of a nucleic acid encoding an SCN5A polypeptide in the individual and determining in each of the nucleic acids encoding an SCN5A polypeptide the identity of the nucleotide at a position corresponding to position 3308 in SEQ ED NO: 67.
  • the individual preferably a human infant, is at an increased risk of SEDS when compared to an individual having a nucleic acid encoding an SCN5A polypeptide wherein the nucleotide at position 3308 is not an adenine, and is, for example, a cytosine, a guanine or a thymine.
  • the present invention also encompasses a method of determining the presence of a marker for SIDS in a human individual, and thus determining if the human individual is a carrier of a marker for SIDS.
  • the method comprises collecting a biological sample from a human wherein the biological sample comprises one or both allelic copies of a nucleic acid encoding an SCN5A polypeptide and determining the identity of the nucleic acid at position 3308 in a nucleic acid encoding an SCN5A polypeptide.
  • a nucleic acid encoding an SCN5A polypeptide is represented by the nucleic acid sequence set forth in SEQ ID NO: 67.
  • the human is a carrier of a marker for SIDS and thus, the presence of a marker for SIDS is determined.
  • a human is a carrier for a marker for SIDS, i.e.
  • the human is heterozygous or homozygous for a missense mutation in a nucleic acid encoding an SCN5A polypeptide
  • the human is at an 8-fold increased risk for arrhythmia in middle age
  • children of a human having a missense mutation in a nucleic acid encoding an SCN5A polypeptide can possibly have a 24-fold increase in the risk for SIDS.
  • the methods of the present invention comprise a method for identifying the possibility of arrhythmia in adults and possibly SIDS in the children of such adults.
  • the present invention further comprises a method for testing a human fetus for a marker for SIDS.
  • the method comprises collecting or otherwise obtaining a biological sample from a fetus wherein the biological sample comprises both allelic copies of a nucleic acid encoding an SCN5 A polypeptide.
  • nucleic acid so collected in then analyzed using methods well known in the art and disclosed elsewhere herein to determine the identity of the nucleotide at position 3308, or at a position corresponding to position 3308, of a nucleic acid encoding an SCN5A polypeptide.
  • the fetus is determined to have a marker for SIDS.
  • the present invention also includes a method for assessing whether a fetus is at risk for SIDS.
  • the method disclosed herein comprises obtaining a biological sample from the fetus wherein the biological sample comprises both allelic copies of a nucleic acid encoding an SCN5A polypeptide and assaying the nucleic acid encoding an SCN5A polypeptide to determine the identity of the nucleotide at position 3308, or at a position corresponding to position 3308, in the nucleic acid encoding an SCN5A polypeptide.
  • the nucleotide at position 3308, or at a position corresponding to position 3308 is an adenine in both copies of a nucleic acid encoding an SCN5A polypeptide, i.e., the fetus is homozygous for the presence of an adenine at position 3308 (or at a position corresponding to position 3308), then the fetus is assessed to be at risk for SIDS in relation to a fetus in which the nucleotide at position 3308, or at a position corresponding to position 3308, in a nucleic acid encoding an SCN5A polypeptide is not adenine.
  • the present invention includes a method for assessing the risk of SIDS in a fetus.
  • the skilled artisan when equipped with the present disclosure and the methods detailed herein, can readily determine that a fetus is at risk for SIDS once born, and therefore, the skilled artisan can take preventive steps and administer preventive therapies as disclosed elsewhere herein.
  • the methods of the present invention can be used to test a human infant or fetus for a marker for SIDS in a live infant. That is, the present method can be used to test an infant for a SIDS marker where the infant is healthy but susceptible to SIDS or the infant is ill with symptoms resembling pre-SIDS, such as cyanosis, apnea and diminished or no detectable pulse.
  • the present invention can further be used to assess the risk of SIDS in a fetus or in a human infant.
  • the present method can also be used in the post-mortem diagnosis of SIDS, either as a replacement or as a supplement to current methods for diagnosing SIDS in a post-mortem infant as disclosed elsewhere herein.
  • the methods of the present invention can be applied to both living and non-living infants.
  • the method disclosed herein are germane to the present invention with the addition of collecting or obtaining a biological sample from a fetus.
  • the skilled artisan is aware of various methods for obtaining a biological sample from a fetus and further, one of skill in the art can perform various genetic tests on such a biological sample to determine the presence of various markers associated with genetic diseases and other conditions.
  • the skilled artisan in order to test a human fetus for a marker for SDDS or to assess the risk of SIDS in a human fetus, the skilled artisan must obtain a biological sample from a fetus, preferably through the use of amniocentesis.
  • Amniocentesis is a diagnostic procedure performed by inserting a hollow needle through the abdominal wall into the uterus and withdrawing a small amount of fluid from the sac surrounding the fetus. Typically, the exact location of the fetus is determined, usually by ultrasound. The skin of the abdomen is disinfected and a topical anesthetic may be applied, or a local anesthetic may be injected into the skin.
  • a long, thin needle is inserted through the abdomen and into the uterus.
  • a small amount of fluid is taken from the fluid-filled sac surrounding the fetus (usually about 1 cubic centimeter per week of gestation).
  • the amniotic fluid is then assayed as described herein.
  • the biological sample obtained from the human infant or fetus may be any fluid or tissue in which SCN5A nucleic acid or protein can be detected.
  • SCN5A can be detected in body fluids such as, for example, blood, and in solid tissue samples as well.
  • the sample comprises blood obtained from a human infant or amniotic fluid obtained from the sac surrounding a human fetus.
  • Nucleic acid is isolated from biological sample using techniques well known in the art and disclosed elsewhere herein.
  • DNA- based assays such as those described herein can be conducted on any subject sample from which DNA can be obtained.
  • any biological sample is suitable for an assay of genomic DNA.
  • tissue samples examples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • Nucleic acid molecules can be obtained according to procedures well-known in the art.
  • biological samples include, but are not limited to cardiac tissue, brain tissue, kidney tissue, and the like. Such solid tissue samples can be processed according to DNA analysis techniques well known in the art and described elsewhere herein. However, the invention should not be construed to be limited to any particular biological sample obtained from the subject.
  • Preferred reagents for detection of mutant SCN5A nucleic acid include, but are not limited to, a nucleic acid complementary to a nucleic acid encoding a mutant SCN5A.
  • analysis of SCN5A mutations can be conducted using polymerase chain reaction (PCR) amplification of genomic DNA to generate templates of human infant DNA for direct sequencing of a nucleic acid encoding an SCN5A polypeptide.
  • PCR polymerase chain reaction
  • PCR Protocols A Guide to Methods and Applications (eds. Innis et at, Academic Press, San Diego, Calif. (1990); Mattila et al. Nucleic Acids Res. 19:4967 (1991); Eckert & Kunkel PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson et al, IRL Press, Oxford), each of which is incorporated by this reference in its entirety.
  • the nucleic acid sequence must be accessible to the various enzymes, nucleotides and other components of the PCR amplification system.
  • the nucleic acid must often be isolated from the remaining tissues and cells in a biological sample using techniques well known in the art and described herein.
  • the sample can be disrupted with some ease, the nucleic acid need not be purified prior to amplification by PCR, i. e. , if the sample comprises cells, particularly peripheral blood lymphocytes or monocytes, lysis and dispersion of the intracellular components may be accomplished merely by suspending the cells in hypotonic buffer.
  • a first step is the synthesis of a DNA copy (cDNA) of the region to be amplified by reverse transcription.
  • Reverse transcription is the polymerization of deoxynucleoside triphosphates to form primer extension products that are complementary to a ribonucleic acid template. This is effected by reverse transcriptase, which initiates synthesis at the 3 '-end of the primer and proceeds toward the 5'-end of the template until synthesis terminates.
  • suitable polymerizing agents that convert the RNA nucleic acid molecule into a complementary, copy-DNA (cDNA) sequence are avian myeloblastosis virus reverse transcriptase and Thermus thermophilous DNA polymerase.
  • Reverse transcription can be carried out as a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR).
  • Polymerizing agents suitable for synthesizing a cDNA sequence from the RNA template are reverse transcriptase (RT), such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, Thermus thennophilous DNA polymerase, and other such reverse transcriptases known in the art.
  • Primers for PCR amplification are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer.
  • the primers are selected to be substantially complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. However, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer with the remainder of the primer sequence being complementary to the strand. Alternatively, complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer. Paired primers for amplification of a given segment of DNA are designated forward and reverse primers.
  • the forward primer hybridizes to a double-stranded DNA molecule at a position 5', or upstream, from the reverse primer.
  • the forward primer hybridizes to the complement of the coding strand of the double stranded sequence, Le., the antisense strand, and the reverse primer hybridizes to the coding strand.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from about 10 to about 100, preferably about 15 to about 50, more preferably about 15 to about 35, or more preferably about 20 to about 30 nucleotides in length.
  • the spacing of primers determines the length of segment to be amplified. The spacing is not usually critical and amplified segments can range in size from about 25 bases to at least about 35 kilobases in length. Segments from about 25 to about 2000, preferably about 50 to about 1000, more preferably about 100 to about 500 nucleotides in length are typical.
  • a primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in an ELISA), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • a label can also be used to "capture" the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
  • An allele-specific primer can be used in a PCR amplification.
  • the allele-specific primer hybridizes to a site on a nucleic acid molecule that overlaps with a polymorphism and extension will only occur if an allelic form complementary to the primer is present.
  • This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present. Thus, the presence or absence of an amplification product is detected using standard methods. Controls can be used that test the efficacy of the amplification reaction itself or that allow the experimental results to be compared with known wildtype or polymorphic SCN5A nucleic acid molecule samples.
  • a number of PCR primers can be used and can be designed to amplify the coding region of SCN5A, splice junctions and flanking regions.
  • Such primers can include, for example, SEQ ID NOS: 1-66, as disclosed herein.
  • the primers used for PCR amplification of a nucleic acid encoding an SCN5A polypeptide are SEQ ID NOS:37 and 38, as disclosed elsewhere herein.
  • the present invention is not limited to the primers disclosed herein, and can encompass any primer for the amplification of a nucleic acid encoding an SCN5A polypeptide.
  • Such primers can be designed and generated using techniques well known in the art for the skilled artisan equipped with the present disclosure and the methods and nucleic acid sequences disclosed herein.
  • LCR ligase chain reaction
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the amplicon is sequenced to determine the identity of the nucleotide at position 3308, or corresponds to the position 3308.
  • Such sequencing can be accomplished using various sequencing techniques, such as the Sanger method (Sanger and Coulson, 1975, J. MoI. Biol. 94: 441- 448), dye-termination sequencing, and other methods of sequencing well known in the art.
  • sequence of the mutation in the nucleic acid that encodes SCN5A can also be determined using, for example, denaturing high-performance liquid chromatography heteroduplex analysis, as described elsewhere herein.
  • Hybridization using allele-specific probes can be used to determine the presence or absence of a polymorphism by, for example Southern blot, dot blots, and the like.
  • An allele-specific probe can be designed that hybridizes to a segment of a nucleic acid molecule from one individual but does not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the two individuals.
  • Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles.
  • the hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on a solid support can also be used as a means of detecting the presence of variant sequences.
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis (DGGE). Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7.
  • DGGE denaturing gradient gel electrophoresis
  • Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis (SSCP), which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., 1989, Proc. Nat. Acad. Sci. 86: 2766-2770.
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of target sequences.
  • the identity of a polymorphism disclosed in the present invention can also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al, 1985, Proc. Natl. Acad. Sci. USA 82: 7575; Meyers et al, 1985, Science 230: 1242) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991, Ann. Rev. Genet. 25: 229- 253). Kits for RNase protection assays are commercially available and are performed essentially according to the manufacturer's directions (BD RiboQuantTM, BD Biosciences, San Jose, CA).
  • the present invention further encompasses the use of polymerase-mediated primer extension to identify a polymorphism in SCN5 A.
  • polymerase-mediated primer extension to identify a polymorphism in SCN5 A.
  • Such methods include, for example, "Genetic Bit Analysis” method (WO 92/15712) and ligase/polymerase mediated genetic bit analysis (U.S. Pat. No. 5,679,524, WO 91/02087, WO 90/09455, WO 95/17676 and 5,945,283).
  • extended primers comprising a polymorphism can be detected by mass spectrometry as described in U.S. Pat. No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Turki et al, 1995, J. Clin. Invest. 95: 1635-1641; Ruao etal, 1989, Nucl. Acids Res. 17: 8392; WO 93/22456).
  • Other methods of detection encompassed by the present invention include, but are not limited to, mismatch cleavage detection and heteroduplex analysis in gel matrices. These methods are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility.
  • a polymorphism creates or destroys a recognition site for a restriction endonuclease, referred to as restriction fragment length polymorphism, or RFLP
  • the sample is digested with that endonuclease and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.
  • the present invention further encompasses the use of an array of oligonucleotides, where discrete positions on the array are complementary to one or more of the provided polymorphic sequences, e.g. oligonucleotides of at least 12 nucleotides, preferably about 20 nucleotides, even more preferably about 25 nucleotides or larger and including the sequence flanking the SCN5A polymorphic position.
  • an array can comprise a series of oligonucleotides, each of which can specifically hybridize to a different polymorphism. For examples of arrays, see Hacia et al, 1996, Nat. Genet., 14: 441-447 and DeRisi et al, 1996, Nat. Genet, 14: 457-460.
  • Arrays of interest may further comprise sequences, including polymorphisms, of other genetic sequences, particularly other sequences of interest for pharmacogenetic screening.
  • Such arrays can include nucleic acid sequences disclosed elsewhere herein, including other polymorphisms identified in SCN5A, such as, for example, S524Y, R668H, Sl 103 Y and E1107K.
  • the present invention further includes a method of testing a human infant for a marker of SIDS.
  • the method comprises obtaining a biological sample from an infant wherein the biological sample comprises both allelic copies of the polypeptide encoded by the SCN5A locus.
  • the method further comprises identifying the amino acid at position 1103 as set forth in SEQ ID NO:68, the polypeptide sequence of SCN5A. If the amino acid at a position corresponding to position 1103 in SEQ ID NO:68 is homozygous for tyrosine, then, as demonstrated by the data disclosed herein, the infant has a marker for SIDS.
  • the invention further encompasses a method of assessing the risk for SIDS in a human infant.
  • the method comprises obtaining a biological sample comprising both allelic copies of the polypeptide encoded by the SCN5A locus in the infant and determining the identity of the amino acid at a position at, or corresponding to, position 1103 in the SCN5A polypeptide, SEQ ID NO:68.
  • the infant Upon identification of the amino acid at, or corresponding to, position 1103 of the SCN5A polypeptide, if both copies of the amino acid are a tyrosine, the infant, as demonstrated by the data disclosed herein, has an increased risk of SIDS.
  • the invention disclosed herein also encompasses a method for testing a human fetus for a marker of SIDS.
  • the method comprises collecting a biological sample from a fetus wherein the biological comprises both allelic copies of the polypeptide encoded by the SCN5A locus and determining the identity of the amino acid at, or at a position that corresponds to, position 1103 in SEQ ID NO:68. If the polypeptide encoded by both allelic copies of the SCN5A locus comprises a tyrosine at, or that corresponds to, position 1103 of SEQ ID NO:68 in both copies, the human fetus has a marker for SIDS.
  • the present invention further includes a method of assessing the risk for SIDS in a human fetus.
  • the method comprises obtaining a biological sample from the fetus wherein the biological sample comprises both allelic copies of the polypeptide encoded by the SCN5A locus in the chromosome of the human fetus.
  • the method further comprises identifying the amino acid at, or at a position corresponding to, position 1103 in SEQ ID NO:68.
  • the amino acid at, or at a position corresponding to, position 1103 in SEQ ID NO:68 in both copies of the polypeptide encoded by the SCN5A locus is a tyrosine
  • the fetus is assessed as having a greater risk of SIDS in comparison to a fetus in which the polypeptide encoded by the SCN5A locus comprises an amino acid other than tyrosine at a position, or at a position corresponding to, position 1103 in SEQ ID NO:68.
  • a biological sample for used in the methods of the present invention can be obtained according to the methods disclosed elsewhere herein. Such methods include, but are not limited to, obtaining tissue samples, drawing blood via venipuncture and obtaining fetal cells by amniocentesis. Any methods known in the art for obtaining other types of biological samples from the human individual are intended for use in the methods of the invention disclosed herein.
  • Preferred reagents for detection of the identity of an amino acid at, or corresponding to, position 1103 in the polypeptide encoded by the SCN5A locus include, but are not limited to, an antibody. It is further preferred that these reagents be labeled to facilitate detection of mutant SCN5A protein.
  • reagents for detection of mutant SCN5A can be labeled using a variety of suitable labels including a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme. Any method known in the art for detecting a single amino acid substitution is intended to be included in the practice of the methods of the invention disclosed herein.
  • an antibody that specifically binds a mutant SCN5A polypeptide, or binds a wild-type SCN5A, preferably at, or at a position that corresponds to, position 1103, is useful in determining if a polymorphism has changed the identity of the amino acid at or about position 1103 of the SCN5A polypeptide.
  • an antibody that binds an SCN5A polypeptide with a tyrosine at position 1103 (or at a position corresponding to position 1103) and does not bind an SCN5A polypeptide with a serine or other amino acid at position 1103 can be used to determine if an infant or fetus is at risk for SIDS or has a marker for SIDS.
  • the presence of a tyrosine at position 1103, or at a position corresponding to position 1103, of both allelic copies of an SCN5A polypeptide results in, among other things, a 24-fold increased risk for SIDS.
  • polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom using standard antibody production methods such as those described in, for example, Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY).
  • Such techniques include immunizing an animal with a chimeric protein comprising a portion of another protein such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and/or a moiety such that the SCN5 A polypeptide is rendered immunogenic (e.g., SCN5A conjugated with keyhole limpet hemocyanin,
  • the chimeric proteins are produced by cloning the appropriate nucleic acids encoding SCN5A (e.g., SEQ ID NO: 67 into a plasmid vector suitable for this purpose, such as but not limited to, pMAL-2 or pCMX.
  • SCN5A e.g., SEQ ID NO: 67
  • a plasmid vector suitable for this purpose such as but not limited to, pMAL-2 or pCMX.
  • the invention should not be construed as being limited solely to polyclonal antibodies that bind SCN5A or mutant SCN5A (SCNA5 protein having the Sl 103Y codon). Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to SCN5A, specifically the region at or about position 1103.
  • the present invention should be construed to encompass antibodies that, among other things, bind to SCN5A and are able to bind SCN5A present on Western blots, in immunohistochemical staining of tissues thereby localizing SCNSA in the tissues, and in immunofluorescence microscopy of a cell transiently or stably transfected with a nucleic acid encoding at least a portion of SCN5 A.
  • the antibody can specifically bind with any portion of the protein and the full-length protein can be used to generate antibodies specific therefor.
  • the present invention is not limited to using the full-length protein as an immunogen.
  • the present invention includes using an immunogenic portion of the protein to produce an antibody that specifically binds with SCN5A. That is, the invention includes immunizing an animal using an immunogenic portion, or antigenic determinant, of the SCN5A protein, for example, the epitope comprising position 1103, or a position corresponding to position 1103.
  • the antibodies can be produced by immunizing an animal such as, but not limited to, a rabbit or a mouse, with an SCN5A protein, or a portion thereof, or by immunizing an animal using a protein comprising at least a portion of SCN5A, or a fusion protein including a tag polypeptide portion comprising, for example, a maltose binding protein tag polypeptide portion, covalently linked with a portion comprising the appropriate SCN5A amino acid residues.
  • an animal such as, but not limited to, a rabbit or a mouse
  • an SCN5A protein or a portion thereof
  • an isolated SCN5 A polypeptide can be used to generate antibodies to epitopes comprising a mutant SCN5A, i.e. an SCN5A with a tyrosine at position 1103, or wild-type SCN5A, i.e. an SCN5A with a serine at position 1103.
  • a mutant SCN5A i.e. an SCN5A with a tyrosine at position 1103, or wild-type SCN5A, i.e. an SCN5A with a serine at position 1103.
  • the antibodies can be used to localize the relevant protein in a cell and to study the role(s) of the antigen recognized thereby in cell processes. Moreover, the antibodies can be used to detect and or measure the amount and/or the identity of a protein present in a biological sample using well-known methods such as, but not limited to, Western blotting and enzyme-linked immunosorbent assay (ELISA). Moreover, the antibodies can be used to immunoprecipitate and/or immuno-affinity purify their cognate antigen using methods well-known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • the invention encompasses polyclonal, monoclonal, synthetic antibodies, and the like.
  • the crucial feature of the antibody of the invention is that the antibody bind specifically with SCN5A, more specifically position 1103 in the SCN5A polypeptide. That is, the antibody of the invention recognizes SCN5A, or a fragment thereof (e.g., an immunogenic portion or antigenic determinant thereof), on Western blots, in immunostaining of cells, and immunoprecipitates SCN5A using standard methods well- known in the art.
  • the antibody of the present invention can thus be used to identify the polypeptide encoded by the SCN5A locus.
  • an antibody that specifically binds a mutant SCN5A polypeptide having a tyrosine at position 1103 or at a position corresponding to position 1103, will indicate, in the methods of the present invention, that the SCN5A polypeptide is associated with an increased risk for SIDS.
  • an antibody that specifically binds a wild-type SCN5A polypeptide, but does not specifically bind a mutant SCN5A polypeptide can be used to determine that the SCN5A polypeptide is not associated with an increased risk of SIDS.
  • the present invention further comprises other types of antibodies, as that term is defined herein, such as monoclonal antibodies.
  • Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72:109-115). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
  • Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12:125-168), and the references cited therein. etaletaletal
  • the antibody of the invention may be "humanized” using the technology described in, for example, Wright (1992, Critical Rev. Immunol. 12:125-168), and in the references cited therein, and in Gu (1997, Thrombosis and Hematocyst 77:755-759).
  • the present invention also includes the use of humanized antibodies specifically reactive with epitopes of SCN5A. Such antibodies are capable of specifically binding SCN5A, or a fragment thereof.
  • the humanized antibodies of the invention have a human framework and have one or more complementarity determining regions (CDRs) from an antibody, typically, but not limited to a mouse antibody, specifically reactive with SCN5A, or a fragment thereof.
  • CDRs complementarity determining regions
  • the antibody used in the invention when the antibody used in the invention is humanized, the antibody may be generated as described in Queen, (U.S. Patent No. 6,180,370), Wright et ah, (1992, Critical Rev. Immunol. 12:125-168) and in the references cited therein, or in Gu et ah (1997, Thrombosis and Hematocyst 77(4):755-759).
  • the method disclosed in Queen et ah is directed in part toward designing humanized immunoglobulins that are produced by expressing recombinant DNA segments encoding the heavy and light chain complementarity determining regions (CDRs) from a donor immunoglobulin capable of binding to a desired antigen, such as SCN5A or mutant SCN5A, attached to DNA segments encoding acceptor human framework regions.
  • CDRs complementarity determining regions
  • the invention in the Queen patent has applicability toward the design of substantially any humanized immunoglobulin. Queen explains that the DNA segments will typically include an expression control DNA sequence operably linked to the humanized immunoglobulin coding sequences, including naturally-associated or heterologous promoter regions.
  • the expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells or the expression control sequences can be prokaryotic promoter systems in vectors capable of transforming or transfecting prokaryotic host cells.
  • Human constant region (CDR) DNA sequences from a variety of human cells can be isolated in accordance with well known procedures.
  • the human constant region DNA sequences are isolated from immortalized B-cells as described in WO 87/02671, which is herein incorporated by reference.
  • CDRs useful in producing the antibodies of the present invention may be similarly derived from DNA encoding monoclonal antibodies capable of binding to SCN5A.
  • Such humanized antibodies may be generated using well known methods in any convenient mammalian source capable of producing antibodies, including, but not limited to, mice, rats, rabbits, or other vertebrates.
  • Suitable cells for constant region and framework DNA sequences and host cells in which the antibodies are expressed and secreted can be obtained from a number of sources, for example, American Type Culture Collection, Manassas, VA.
  • a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
  • the procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al.
  • Bacteriophage which encode the desired antibody may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed.
  • the bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell.
  • Bacteriophage which do not express the antibody will not bind to the cell.
  • panning techniques are well known in the art and are described for example, in Wright et al. (992, Critical Rev. Immunol. 12:125-168).
  • a cDNA library is generated from mRNA obtained from a population of antibody-producing cells.
  • the mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same.
  • Amplified cDNA is cloned into Ml 3 expression vectors creating a library of phage which express human Fab fragments on their surface.
  • Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin.
  • this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.
  • Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHl) of the heavy chain.
  • Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment.
  • An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein.
  • Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al. (1991, J. MoI. Biol. 222:581-597). Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.
  • the invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1:837- 839; de Kruif et al. 1995, J. MoI. Biol. 248:97-105).
  • the antibodies of the present invention can be used in various assays to determine the identity of an amino acid at position 1103, or at a position corresponding to position 1103, in the polypeptide encoded by the SCN5A locus. More specifically, an antibody that specifically binds with either mutant SCN5A or wild-type SCN5A described herein is used in an immunoassay to detect this presence of SCN5A 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 mutant SCN5A or a wild-type SCN5A and the antibody is detected.
  • suitable immunoassays include a radioimmunoassay, a Western blot assay, an immunofluorescent assay, an enzyme linked immunoassay (ELISA), a chemiluminescent assay, an immunohistocheniical assay, an immunocytochemical assay, and the like (see, e.g., Principles and Practice of Inmunoassay, 1991, Eds. Christopher P. Price and David J. Neoman, Stockton Press, New York, N. Y.; Current Protocols in Immunology, 2001, Eds.
  • Proteins maybe isolated from test specimens and biological samples by conventional methods, as known in the art and described elsewhere herein.
  • a mutant SCN5A assay is a competitive assay designed to measure a specific peptide corresponding to a portion of mutant SCN5A as well as mutant SCN5A.
  • the assay is based upon the competition of labeled 125 I-mutant SCN5A peptide and unlabeled peptide (either standard or an unknown quantity of bodily fluid containing mutant SCN5A) binding to the limited quantity of antibodies specific for the mutant SCN5A peptide in each reaction mixture. As the quantity of standard or unknown in the reaction increases, the amount of I25 I-mutant SCN5A peptide able to bind to the peptide in decreased.
  • a standard curve is constructed from which the concentration of mutant SCN5A in the biological sample can be determined.
  • the competition assay described above is designed to quantitate the level of mutant SCN5A present in a patient's biological sample.
  • the presence of mutant SCN5A polypeptide in a given patient's sample to that of a patient or a standard reagent known to not have a tyrosine at position 1103, or at a position corresponding to position 1103, the presence of mutant SCN5A can be used as an indication that a given patient is at risk for SIDS or has a SIDS marker.
  • This assay can be useful to many fetuses and infants for the detection of the risk of SIDS or the presence of a SIDS marker.
  • a polypeptide encoded by the SCN5A locus can be isolated from a biological sample using methods described elsewhere herein and sequenced in order to identify the amino acid at position 1103. That is, protein sequencing can be used to determine if an infant or a fetus is at risk for SIDS or has a marker for SIDS by identifying the amino acid at position 1103 in the polypeptide encoded by SCN5A.
  • a polymorphism leading to the substitution of a tyrosine for a serine at position 1103 in SCN5A results in, among other things, a 24-fold increase in risk for SIDS and late re- openings in the SCN5A sodium channel, which is known to be associated with arrhythmia in LQTS patients.
  • Edman degradation In this method, one amino terminal amino acid residue at a time is removed from a polypeptide to be analyzed. That amino acid is normally identified by reverse phase high performance liquid chromatography (HPLC), but mass spectrometric procedures have been described for this purpose (Alexander et ah, 1990, Int. J. Mass Spectrom. Ion Processes 98: 107-134). The Edman degradation cycle is repeated for each successive terminal amino acid residue until the complete polypeptide has been degraded.
  • HPLC reverse phase high performance liquid chromatography
  • the Edman degradation process can be accomplished using automated processors available commercially (Applied Biosystems, Foster City, CA) or through protein sequencing services.
  • sequencing proteins by collision-induced dissociation mass spectrometry (MS) methods can also be used in the methods of the present invention and often requires less protein than Edman techniques (Shevchenko et al, 1996, Proc. Natl. Acad. Sci. (USA), 93: 14440-14445; WiIm, et al, 1996, Nature, 379: 466-469; and Bieman, 1990, Methods in Enzymology, 193: 455-479).
  • MS sequencing is accomplished either by using higher voltages in the ionization zone of the MS to randomly fragment a single peptide isolated from a protein digest, or more typically by tandem MS using collision-induced dissociation in the ion trap.
  • Several techniques can be used to select the peptide fragment used for MS/MS sequencing, including accumulation of the parent peptide fragment ion in the quadrapole MS unit (Bieman, 1990, Methods in Enzymology, 193: 455-479), capillary electrophoretic separation coupled to ES-TOF MS detection (Smith, et al, in: CRC Handbook of Capillary Electrophoresis: A Practical Approach, Chp.
  • mass spectrophotometer identification of proteins can be accomplished after their separation from a protein mixture by mass profile fingerprinting (MS fingerprinting; James, et al, 1993, Biochem. Biophys. Res. Commun., 195:58-64) and sequencing of one or more peptide domains by MS/MS (MS/MS sequencing; WiIm et al, 1996, Nature, 379: 466-469; Chart et al, 1993, Science 262: 89-92).
  • MS fingerprinting is achieved by accurately measuring the masses of several peptides generated by a proteolytic digest of the intact protein and searching a database for a known protein with that peptide mass fingerprint.
  • MS/MS sequencing involves actual determination of one or more PSTs of the protein by generation of sequence-specific fragmentation ions in the quadrapole of an MS/MS instrument.
  • the present invention comprises methods for determining the identity of the amino acid at position 1103 (or at a position corresponding to position 1103) in a polypeptide encoded by the SCN5A locus in order to assess the risk or test for a marker of SIDS in a human infant or fetus.
  • Methods of Prophylactic Treatment demonstrate that a polymorphism in SCN5A, specifically a C3308A missense mutation resulting in an Sl 103 Y mutation in the SCN5A polypeptide results in, among other things, a 24-fold increase in risk for SEDS in homozygous infants, late re-openings in the SCN5A sodium channel and an abnormal gain in function at lowered physiological pH.
  • the present invention includes a method of testing for a marker or assessing the risk of SIDS in a human infant and, if the marker is present, administering a pharmaceutical agent in order to prevent SEDS.
  • mutant SCN5A channels were inhibited similarly at normal physiological pH (7.4) using propranolol and amiodarone and mutant SCN5A channels were even more sensitive to blockage when mexiletine was administered.
  • pH normal physiological pH
  • mexiletene resulted in almost complete suppression of late channel re-openings, which are associated with, among other things, arrhythmia and SIDS.
  • the methods of the present invention encompasses testing a human individual, for example, an infant or fetus, for a marker for SIDS as disclosed elsewhere herein.
  • the methods of the present invention further comprise assessing the risk of SIDS in a human individual, for example, an infant or fetus.
  • the risk of SIDS or a marker for SIDS can be identified by determining the identity of the nucleotide at position 1103, or at a position corresponding to position 3308, in the nucleic acid encoding the SIDS polypeptide.
  • the present invention includes assessing the risk of SIDS or a testing a human infant, fetus or other individuals for a marker for SIDS by determining the identity of an amino acid at position 1103 (or at a position corresponding to position 1103) of the polypeptide encoded by the SCN5A locus.
  • the individual is administered a pharmaceutical composition comprising mexiletine, propranolol and/or amiodarone in a pharmaceutically-acceptable carrier.
  • mexiletine suppresses the pathological late re-openings of mutant SCN5A channels which have been associated with, among other things, arrhythmia and sudden death.
  • propranolol and amiodarone inhibits both wild-type and mutant SCN5A channels at normal physiological pH.
  • pharmaceutically-acceptable carrier means a chemical composition with which a pharmaceutical composition may be combined and which, following the combination, can be used to administer the appropriate pharmaceutical composition to the human individual.
  • the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.
  • the invention envisions administration of a dose which results in a concentration of the compound of the present invention between 1 ⁇ M and 10 ⁇ M in a human individual.
  • mexiletene otherwise known as MexitilTM
  • MexitilTM is initiated with a dose of 200 mg every eight hours when rapid control of arrhythmia is not essential.
  • a minimum of two to three days between dose adjustments is recommended.
  • the dose may be adjusted in 50 or 100 mg increments up or down. However, dosing of mexiletene should be adjusted according to individualized tolerance and response. In infants, the recommended dose is 10 mg per kilogram of body weight.
  • propranolol is usually initiated with a dosage of 80 mg propranolol once daily.
  • the dosage may be increased to 120 mg once daily or higher.
  • the usual maintenance dosage is 120 to 160 mg once daily, hi infants, the suggested dose of propranolol is 4 mg per kilogram of body weight.
  • Amiodarone (CordaroneTM) is administered at a loading dose of 800 to 1 ,600 mg for 1 to 3 weeks with dose of 600 to 800 mg after one month and maintenance dose of 400 mg. However, the dose should be monitored based on patient tolerance and response and improvement or decline in arrhythmia.
  • compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations. They can be administered intrathecally, intraventricularly, intraparenchymally, via direct injection, or via bioengineered polymers.
  • such pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration.
  • Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer an appropriate hypericin derivative according to the methods of the invention.
  • the invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of the diseases disclosed herein as an active ingredient.
  • a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • the active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the term "pharmaceutically acceptable carrier” means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
  • physiologically acceptable ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
  • compositions of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi- dose unit.
  • Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intraventricular, intraparenchymal, or another route of administration.
  • Other formulations include in the invention include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • Additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • a formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient.
  • Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
  • an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • a tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent.
  • Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • compositions used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents.
  • Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate.
  • Known surface active agents include, but are not limited to, sodium lauryl sulfate.
  • Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.
  • Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid.
  • binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.
  • Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
  • Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.
  • Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
  • Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • Liquid solutions of the active ingredient in aqueous or oily solvents maybe prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
  • Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20° C) and which is liquid at the rectal temperature of the subject (i.e. about 37° C in a healthy human).
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides.
  • Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject.
  • Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration.
  • a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or gel or cream or a solution for vaginal irrigation.
  • Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject.
  • Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Topically- administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65° F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
  • compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution, suspension, or slow-release polymer.
  • Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
  • formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Another formulation is the activate ingredient incorporated in a slow-release polymer.
  • Such polymers are well known in the pharmaceutical arts, and are detailed in, for example, U.S. Patent Nos. (4,728,512; 4,728,513; 5,084,287; 5,285,186).
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations, when dispersed preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier.
  • Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein.
  • Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
  • a pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for direct CNS administration.
  • Such formulations may, for example, be in the form of liquid administered by an Ommaya reservoir, by intrathecal or intraventricular administration, by direct intraparenchymal injection, by slow-release polymers, or other such methods well known in the pharmaceutical and neurological fields.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.
  • dosages of the compound of the invention which may be administered to a human individual range in amount from 1 ⁇ g to about 100 g per kilogram of body weight.
  • the precise dose administered will vary depending upon any number of factors, including but not limited to, the disease state being treated, the age of the individual and the route of administration.
  • the dose of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the individual. More preferably, the dosage will vary from about 2 mg to about 1 g per kilogram of body weight of the individual.
  • the compound may be administered as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan.
  • kits of the present invention encompasses various kits for accomplishing the methods of the present invention.
  • the kits of the present invention comprise nucleic acids, antibodies and other reagents for testing a human infant or fetus has a marker for SIDS.
  • the kits of the present invention further comprise nucleic acid, antibodies and other reagents for assessing whether a human infant or fetus is at risk for SIDS.
  • the kits of the present invention can comprise, among other things, a nucleic acid, such as a primer for PCR amplification, a polypeptide, such as an antibody, and other reagents described elsewhere, as well as various other implements for collecting tissue samples, blood, and the like.
  • the kits of the present invention further comprise an instructional material which describes use of the kit to perform the methods of the invention.
  • model kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the present invention.
  • kits for testing a human individual such as for example, an infant, for a marker for SIDS
  • the kit comprises a reagent for determining whether the infant is homozygous for tyrosine (Y/Y) at position 1103, or at a position corresponding to position 1103 in SEQ ID NO: 68.
  • the kit further comprises an instructional material for the use of the kit. According to the methods described in the accompanying instructional material, if the infant is homozygous for tyrosine at position 1103, or at a position corresponding to position 1103, of the SCN5A polypeptide, the infant has a marker for SIDS.
  • the kit described herein can be used to test a living human infant for a marker for SIDS.
  • the kit of the present invention can further be used in the post-mortem examination of an infant to determine if the infant died of SIDS.
  • the invention disclosed herein further comprises a kit for assessing the risk of SIDS in a human infant.
  • the kit comprises a reagent, such as a primer or a probe, to determine if the infant is homozygous for tyrosine (Y/Y) at position 1103, or at a position corresponding to position 1103 in the SCN5A polypeptide (SEQ ID NO: 68).
  • the kit further comprises an instructional material for the use of the kit. The instructional material simply embodies the methods of the present invention.
  • the infant Upon use of the kit described herein, if the infant is Y/Y for an amino acid at position 1103 or at a position that corresponds to position 1103 in the SCN5A polypeptide, the infant is assessed to be at an increased risk for SIDS when compared to an infant that is not Y/Y at position 1103 on the SCN5 A polypeptide or at a position corresponding to position 1103 on the SCN5A polypeptide.
  • the present invention further comprises a kit for determining whether a human, for example a parent of a child or those considering having children, is a carrier of a marker for SEDS.
  • the kit comprises a reagent for the detection of whether the human is heterozygous for tyrosine (Y/-) with respect to an amino acid at position 1103, or at a position corresponding to position 1103, in SEQ ID NO: 68, the SCN5A polypeptide.
  • the kit further comprises an instructional material for the use of the kit. The instructional material simply embodies the methods disclosed elsewhere herein for determining if a human is a carrier of a marker for SIDS.
  • a human is heterozygous for tyrosine (Y/-) at, or at a position corresponding to, position 1103 in an SCN5A polypeptide, the human is carrier for a marker of SIDS.
  • a kit included in the present invention is a kit for testing a human fetus or an infant for a marker for SIDS.
  • the kit comprises at least one reagent, such as a PCR primer, a probe, an antibody, or other reagent disclosed herein, that permits the determination of the identity of the amino acid at position 1103, or at a position corresponding to position 1103, of the SCN5A polypeptide.
  • the kit further comprises an instructional material for the use of the kit. The instructional material provides that if the fetus is homozygous for tyrosine at position 1103 or at a position on the SCN5A polypeptide corresponding to position 1103 on SEQ ID NO.68, the fetus has a marker for SIDS.
  • mutant channels are stable at baseline, the stress of acidosis provokes late re-openings that predispose to lethal arrhythmia, a gain-in-function not seen with wild type channels. Abnormal channel activity is fully suppressed in vitro by low doses of a drug now prescribed to infants for arrhythmia.
  • the results of the present invention indicate that genetic counseling and testing, with trials of prophylactic drug therapy for identified homozygous infants can prevent SEDS in the susceptible infant population.
  • Postmortem solid organ tissue was collected from 224 unrelated cases of autopsy- confirmed SIDS from 1985 to 1992 by the NIH-sponsored Brain and Tissue Banks for Developmental Disorders unit at the University of Maryland (Baltimore, MD). Medical information was limited to sex, ethnicity, age at death, time of autopsy, and for most cases a brief clinical history. Both parental DNA and family history were unavailable. Investigators were blinded to the identities of the affected individuals and their families. Adult African- Americans were used as controls without specifically questioning for a family history of sudden cardiac death or SIDS and include 100 previously described samples from Coriell (Camden, NJ) (Fraser and Froggatt, 1996, Lancet 2: 56).
  • Genomic DNA was extracted from frozen heart, brain or kidney ( ⁇ 1 cm 3 ). Solid organ tissue was macerated on ice, resuspended (3 mL of 75mM NaCl and 24mM EDTA), subjected to alkaline lysis (300 mL of 5% SDS) and proteinase K digestion (100 ⁇ L of 10 mg/mL) at 37° C overnight. Genomic DNA was isolated by equal volume Tris saturated phenol-chloroform extraction, two chloroform-isoamyl alcohol (49:1) washes, precipitated with 1.5 mL of 7.5M sodium acetate and two volumes of 100% ethanol.
  • Thermocycler conditions were an initial 5 minute denaturation at 94°C, followed by 40 cycles of: 30 seconds denaturation at 96°C, 30 seconds annealing at either 55 0 C, 58.7°C, 65 0 C, 60 seconds extension at 72°C and a single 10 minute terminal extension at 72 0 C.
  • PCR was completed with 50 ng DNA in a PCR volume of 30-50 ⁇ L.
  • the Yl 103 change was introduced to SCN5A-pcDNAl plasmid using the Pfu quick change kit (Stratagene, La Jolla, CA). Wild type or mutant constructs were transiently co-transfected with GFP-tagged SCNlB (the ⁇ -subunit of cardiac SCN5A channel) in HEK-293 cells using LipofectamineTM (Invitrogen, Carlsbad, CA). Twenty- four to 60 hours after transfection, patch-clamp whole-cell recording was performed using an Axon 200-B amplifier (Axon Instruments, Claremont, CA) at filter and sampling frequencies of 5 kHz and 20 kHz, respectively. Voltage errors were minimized using 80% series resistance compensation.
  • Internal (pipette) solution contained: 60 mM CsCl, 80 mM CsF, 10 mM EGTA, 1 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Na 2 ATP, 10 mM HEPES (pH as indicated with CsOH).
  • the bath solution contained: 130 mM NaCl, 5 mM CsCl, 2 mM CaCl 2 , 1.2 mM MgCl 2 , 10 mM HEPES, 5 mM glucose (pH 7.4 with NaOH), as described in (Fraser and Froggatt, 1966, Lancet 2: 56).
  • G-V conductance-voltage
  • EK sodium reversal potential
  • Mexiletine, propranolol and amiodarone were obtained from Sigma Chemical Company (St. Louis, MO). Stock solutions were made by dissolving the compounds in bath solution (mexiletine) or dimethyl sulfoxide (propranolol and amiodarone).
  • the use- dependent block of sodium currents was assessed using a train of 10 millisecond depolarization pulses from -100 mV to -30 mV applied at 2.5 Hz as described above. Only cells demonstrating stable current magnitude for three minutes before drug application were studied. Inhibition of the first pulse after drug exposure (tonic block) or inhibition at equilibrium during a repetitive pulse protocol (phasic block; here simulating a heart rate of 150 beats per minute) were used to study channel function. Half- maximum values were assessed by fitting the percentage block to the Hill equation with a coefficient of 1 (as this was close to the determined value in each case). Single channels were recorded from inside-out membrane patches excised from Sigma Chemical Company (St. Louis, MO). Stock solutions were made by
  • HEK293 cells Currents were stimulated every 2.5 seconds by a 50 millisecond depolarizing pulse to -30 mV from a holding potential of -120 mV. Data were recorded with an Axopatch 200B amplifier and pCLAMP software (Axon Instruments, Claremont, CA) at filter and sampling frequencies of 5 kHz and 50 kHz, respectively. Borosilicate pipettes (Clark, Kent U.K.) were coated with Sylgard (Dow Corning, Midland MI) and filled with bath solution as described above. Cells were perfused with the pipette solution described above. For each cell null sweeps, with no channel activity, were identified offline, averaged and subtracted away from data sweeps before analysis. For display purposes data were re-filtered offline using a 2kHz Bessel filter (pCLAMP, Axon Instruments). AU experiments were performed at between 20-22 0 C.
  • the probability term is the probability, given SIDS, that the infant was exposed to the YY genotype.
  • RR relative risk
  • SCN5A the cardiac sodium channel that opens to initiate electrical activity in the ventricles (the chambers that pump blood to the systemic circulation) and inactivates to allow each beat to end, were examined.
  • Rare SCN5A variants cause arrhythmia if the channels fail to inactivate fully and continue to operate late in the cardiac cycle when normal channels are silent; such late activity is a substrate for chaotic rhythms and sudden death (Spooner et al , 2001 , Circulation 103 : 2447-2452; Gutstein et al, 2003, Am. J. Physiol. Heart Circ. Physiol., 285: H1091-1096; Kleber and Rudy, 2004, Physiol. Rev., 84: 431-488).
  • sporadic mutations in SCN5A were also observed in other African- American SIDS cases.
  • SCN5 A-Yl 103 channels operate normally at baseline but show late-re-openings when challenged by lowered intracellular pH, an effect not seen with wild type channels. Abnormal openings are suppressed by low levels of the medication mexiletine that have little effect on normal first openings of either SCN5A- Sl 103 or Yl 103 channels.
  • the 224 autopsy confirmed cases of SIDS in the present case-control study were retrospectively ascertained to reflect the expected distribution of SIDS cases in the United States based on sex and ethnicity, with higher number of African- Americans (133) than Caucasians (86) and males (106) than females (79).
  • the control population for the African- American cases was 1,056 African- American adults with no known health problems.
  • DNA was extracted from postmortem solid tissue or control blood samples, subjected to denaturing high-performance liquid chromatography (dHPLC) for spectroscopic heteroduplex analysis in the presence and absence of control DNA, and the identified variants were sequenced on both strands to identify nucleotide changes (Figure IA).
  • the SCN5A gene encodes a 2016 residue ⁇ -subunit with four homologous domains (Figure IB) that fold in pseudo-symmetric fashion to form a single, central ion permeation pathway lined by four pore (P) loops.
  • Position 1103 is in the intracellular linker between DII and Dili of the sodium channel.
  • the pore-forming ⁇ -subunit assembles with an accessory (or ⁇ ) subunit encoded by SCNlB (Xiao at al., 2000, Am. J. Physiol. Heart Circ. Physiol. 279: H35-46).
  • wild type Sl 103 or Yl 103 channel ⁇ -subunits were expressed with the ⁇ -subunit in mammalian tissue culture cells (HEK-293). Channels were first studied using whole-cell patch-clamp mode so currents passed by many channels were assessed simultaneously.
  • Single wild type cardiac sodium channels are closed at rest, open briefly with membrane depolarization and then rapidly inactivate, staying closed despite maintained depolarization; (the channels stay inactivated until the membrane repolarizes when they return to the closed state in preparation for the next heart beat).
  • a shift in the voltage- dependence of inactivation with low pH indicates that single Yl 103 channels will be active after depolarization at times when wild type channels remain inactivated.
  • single Sl 103 channels studied in inside-out excised patch mode open just once on depolarization and then remain inactive at both pH 7.4 and pH 6.7 ( Figure 3A); conversely, Yl 103 channels operate normally atpH 7.4 but show abnormal re-openings during depolarization at pH 6.7.
  • Mexiletine Suppresses Pathological Re-Openings
  • the SCN5A-Y1103 allele is associated with arrhythmia and sudden death by three observations.
  • the 11.4% of adult African- Americans with a single copy of the Yl 103 allele show an 8-fold increase in risk for arrhythmia as they reach middle age (Splawski et al, 2002, Science 297: 1333-1336).
  • a Caucasian kindred with heterozygous SCN5A-S1103/Y1103 has been reported with syncope, ventricular tachycardia and fibrillation, QT prolongation and sudden death (Chen et al, 2002, J. Med. Genet. 39: 913-915).
  • African-Americans infants homozygous for Yl 103 show a 24-fold increased risk for SIDS .
  • the data disclosed herein indicate that the allele is a substrate for arrhythmia in the setting of a secondary challenge that is well-tolerated by individuals with wild type channels, a mechanism that mediates some cases of drug-induced arrhythmia where variant ion channel activity does not come to light until revealed by a secondary stress (Abbott et al, 1999, Cell 97: 175-187; Sesti et al, 2000, Proc. Nat'l. Acad. Sci. USA 97: 10613-10618).
  • the present disclosure demonstrates that in Yl 103 sodium channels, acidosis can produce a gain in function yielding late re-openings that are associated with dangerous arrhythmia in patients with inherited LQTS .
  • many of the known risk factors for SIDS are stressors that can lead to prolonged apnea and respiratory acidosis - a common event in normal infants that is exacerbated by diminished respiratory drive during sleep, respiratory infection, multiple siblings (increasing infectious exposures), premature birth, and second-hand cigarette smoke.
  • cardiac sodium channel is formed with a single pore-forming ⁇ -subunit
  • individual cells in patients with one Yl 103 allele are expected to have both wild type Sl 103 and altered channels.
  • Gain in function can allow the single altered gene to place these individuals at risk for arrhythmia as adults whereas two alleles confers a risk for SIDS, perhaps via a lethal arrhythmia induced more easily in the homozygous state.
  • SIDS may be diagnosed and prevented by screening individuals, especially those in populations with a higher incidence of the altered 1103 genotype, in at least three situations: infants who have acute life-threatening events (ALTEs), siblings of SIDS victims, and couples who experience infertility or fetal demise (Miller et al, 2004, Circulation 109: 3029-3024). Given a potential absolute risk of ⁇ 5%, prospective screening of a large cohort of susceptible infants may prevent SIDS, as demonstrated by the data disclosed herein. Should such a screening process identify YY infants, prophylactic mexiletine therapy would be indicated. Finally, it is important to support public health efforts to decrease known extrinsic risk factors, such as prone sleeping position. The evidence presented herein demonstrates that SIDS results, like many common disorders, from a genetic predisposition that yields poor tolerance of common challenges to physiological homeostasis.

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Abstract

La présente invention a trait à des procédés, des compositions et des trousses permettant l'identification de la présence d'un polymorphisme dans la protéine SCN5A du canal sodique cardiaque, en vue de l'identification de sujets à risque du syndrome de mort subite du nourrisson et l'identification de sujets portant le polymorphisme dans la protéine SCN5A de le canal sodique cardiaque.
PCT/US2005/025099 2004-07-15 2005-07-15 Compositions, procedes et trousses pour le diagnostic et le traitement du syndrome de mort subite du nourrisson Ceased WO2006019984A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063226A3 (fr) * 2007-11-12 2010-01-07 Karolinska Institutet Innovations Ab Procédés se rapportant à des troubles respiratoires
WO2015067857A1 (fr) 2013-11-08 2015-05-14 Modern Diagnostics Oy Procédé pour le criblage de facteurs de risques génétiques constituant une prédisposition ou une cause de décès chez le foetus ou le nourrisson
US12117453B2 (en) 2018-12-07 2024-10-15 Washington University Predicting patient response to sodium channel blockers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN. S. ET AL.: 'SNP S 1103Y In The Cardiac Sodium Channel Gene SCN5A Is Associated With Cardiac Arrhythmias And Sudden Death In A White Family.' JOURNAL OF MEDICAL GENETICS. vol. 39, 2002, pages 913 - 915 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063226A3 (fr) * 2007-11-12 2010-01-07 Karolinska Institutet Innovations Ab Procédés se rapportant à des troubles respiratoires
CN102036713A (zh) * 2007-11-12 2011-04-27 圣莎拉医学股份公司 涉及呼吸障碍的方法
WO2015067857A1 (fr) 2013-11-08 2015-05-14 Modern Diagnostics Oy Procédé pour le criblage de facteurs de risques génétiques constituant une prédisposition ou une cause de décès chez le foetus ou le nourrisson
US12117453B2 (en) 2018-12-07 2024-10-15 Washington University Predicting patient response to sodium channel blockers

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