US20050003445A1 - Alterations in the long QT syndrome genes KVLQT1 and SCN5A and methods for detecting same - Google Patents

Abstract

Long QT Syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on electrocardiogram and presence of syncope, seizures and sudden death. Five genes have been implicated in Romano-Ward syndrome, the autosomal dominant form of LQTS. These genes are KVLQT1, HERG, SCN5A, KCNE1 and KCNE2. Mutations in KVLQT1 and KCNE1 also cause the Jervell and Lange-Nielsen syndrome, a form of LQTS associated with deafness, a phenotypic abnormality inherited in an autosomal recessive fashion. Mutational analyses were used to screen 262 unrelated individuals with LQTS for mutations in the five defined genes. A total of 134 mutations were observed of which eighty were novel.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
• The present invention is a division of U.S. patent application Ser. No. 09/840,125 filed 24 Apr. 2001 which in turn is a division of U.S. patent application Ser. No. 09/634,920 filed 9 Aug. 2000. Ser. No. 09/634,920 is related and claims priority under 35 U.S.C. § 119(e) to provisional patent application Ser. No. 60/190,057 filed 17 Mar. 2000 and to provisional patent application Ser. No. 60/147,488 filed 9 Aug. 1999. Each application is incorporated herein by reference.
• This application was made with Government support from NHLBI under Grant Nos. RO1-HL46401, RO1-HL33843, RO1-HL51618, P50-HL52338 and MO1-RR000064. The federal government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
• Long QT Syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on electrocardiogram and presence of syncope, seizures and sudden death, usually in young, otherwise healthy individuals (Jervell and Lange-Nielsen, 1957; Romano et al., 1963; Ward, 1964). The clinical features of LQTS result from episodic ventricular tachyarrhythmias, such as torsade de pointes and ventricular fibrillation (Schwartz et al., 1975; Moss et al., 1991). Two inherited forms of LQTS exist. The more common form, Romano-Ward syndrome (RW), is not associated with other phenotypic abnormalities and is inherited as an autosomal dominant trait with variable penetrance (Roman et al., 1963; Ward, 1964). Jervell and Lange-Nielsen syndrome (JLN) is characterized by the presence of deafness, a phenotypic abnormality inherited as an autosomal recessive trait (Jervell and Lange-Nielsen, 1957). LQTS can also be acquired, usually as a result of pharmacologic therapy.
• In previous studies, we mapped LQTS loci to chromosomes 11p15.5 (LQT1) (Keating et al., 1991), 7 q35-36 (LQT2) (Jiang et al., 1994) and LQT3 to 3p21-24 (Jiang et al., 1994). A fourth locus (LQT4) was mapped to 4q25-27 (Schott et al., 1995). Five genes have been implicated in Romano-Ward syndrome, the autosomal dominant form of LQTS. These genes are KVLQT1 (LQT1) (Wang Q. et al., 1996a), HERG (LQT2) (Curran et al., 1995), SCN5A (LQT3) (Wang et al., 1995a), and two genes located at 21q22-KCNE1 (LQT5) (Splawski et al., 1997a) and KCNE2 (LQT6) (Abbott et al., 1999). Mutations in KVLQT1 and KCNE1 also cause the Jervell and Lange-Nielsen syndrome, a form of LQTS associated with deafness, a phenotypic abnormality inherited in an autosomal recessive fashion.
• KVLQT1, HERG, KCNE1 and KCNE2 encode potassium channel subunits. Four KVLQT1 α-subunits assemble with minK (β-subunits encoded by KCNE1, stoichiometry is unknown) to form I_K channels underlying the slowly activating delayed rectifier potassium current in the heart (Sanguinetti et al., 1996a; Barhanin et al., 1996). Four HERG α-subunits assemble with MiRP1 (encoded byKCNE2, stoichiometry unknown) to form I_Kr channels, which underlie the rapidly activating, delayed rectifier potassium current (Abbott et al., 1999). Mutant subunits lead to reduction of I_Ks or I_Kr by a loss-of-function mechanism, often with a dominant-negative effect (Chouabe et al., 1997; Shalaby et al., 1997; Wollnik et al., 1997; Sanguinetti et al., 1996b). SCN5A encodes the cardiac sodium channel that is responsible for I_Na, the sodium current in the heart (Gellens et al., 1992). LQTS-associated mutations in SCN5A cause a gain-of-function (Bennett et al., 1995; Dumaine et al., 1996). In the heart, reduced I_Ks or I_Kr or increased I_Na leads to prolongation of the cardiac action potential, lengthening of the QT interval and increased risk of arrhythmia. KVLQT1 and KCNE1 are also expressed in the inner ear (Neyroud et al., 1997; Vetter et al., 1996). Others and we demonstrated that complete loss of I_Ks causes the severe cardiac phenotype and deafness in JLN (Neyroud et al., 1997; Splawski et al., 1997b; Tyson et al., 1997; Schulze-Bahr et al., 1997).
• Presymptomatic diagnosis of LQTS is currently based on prolongation of the QT interval on electrocardiogram. Genetic studies, however, have shown that diagnosis based solely on electrocardiogram is neither sensitive nor specific (Vincent et al., 1992; Priori et al., 1999). Genetic screening using mutational analysis can improve presymptomatic diagnosis. However, a comprehensive study identifying and cataloging all LQTS-associated mutations in all five genes has not been achieved. To determine the relative frequency of mutations in each gene, facilitate presymptomatic diagnosis and enable genotype-phenotype studies, we screened a pool of 262 unrelated individuals with LQTS for mutations in the five defined genes. The results of these studies are presented in the Examples below.
• The present invention relates to alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes and methods for detecting such alterations.
• The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
• The present invention is directed to alterations in genes and gene products associated with long QT syndrome and to a process for the diagnosis and prevention of LQTS. LQTS is diagnosed in accordance with the present invention by analyzing the DNA sequence of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene of an individual to be tested and comparing the respective DNA sequence to the known DNA sequence of the normal gene. Alternatively, these genes of an individual to be tested can be screened for mutations which cause LQTS. Prediction of LQTS will enable practitioners to prevent this disorder using existing medical therapy.
SUMMARY OF THE INVENTION
• The present invention relates to alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes and methods for detecting such alterations. The alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes include mutations and polymorphisms. Included among the mutations are frameshift, nonsense, splice, regulatory and missense mutations. Any method which is capable of detecting the alterations described herein can be used. Such methods include, but are not limited to, DNA sequencing, allele-specific probing, mismatch detection, single stranded conformation polymorphism detection and allele-specific PCR amplification.
BRIEF DESCRIPTION OF THE FIGURES
• FIG. 1 is a schematic representation of the predicted topology of KVLQT1 and the locations of LQTS-associated mutations. KVLQT1 consists of six putative transmembrane segments (S1 to S6) and a pore (Pore) region. Each circle represents an amino acid. The approximate location of LQTS-associated mutations identified in our laboratory are shown with filled circles.
• FIG. 2 is a schematic representation of HERG mutations. HERG consists of six putative transmembrane segments (S1 to S6) and a pore (Pore) region. Location of LQTS-associated mutations are shown with filled circles.
• FIG. 3 is a schematic representation of SCN5A and locations of LQTS-associated mutations. SCN5A consists of four domain (DI to DIV), each of which has six putative transmembrane segments (S1 to S6) and a pore (Pore) region. Location of LQTS-associated mutations identified in our laboratory are shown with filled circles.
• FIG. 4 is a schematic representation of minK and locations of LQT-associated mutations. MinK consists of one putative transmembrane domain (S1). The approximate location of LQTS-associated mutations identified in our laboratory are shown with filled circles.
• FIG. 5 is a schematic representation of the predicted topology of MiRP1 and locations of arrhythmia-associated mutations. MiRP1 consists of one putative transmembrane domain (S1). The approximate location of arrhythmia-associated mutations identified in our laboratory are shown with filled circles.
DETAILED DESCRIPTION OF THE INVENTION
• The present invention relates to alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes and methods for detecting such alterations. The alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes include mutations and polymorphisms. Included among the mutations are frameshift, nonsense, splice, regulatory and missense mutations. Any method which is capable of detecting the mutations and polymorphisms described herein can be used. Such methods include, but are not limited to, DNA sequencing, allele-specific probing, mismatch detection, single stranded conformation polymorphism detection and allele-specific PCR amplification.
• KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 mutations cause increased risk for LQTS. Many different mutations occur in KVLQT1, HERG, SCN5A, KCNE1 and KCNE2. In order to detect the presence of alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes, a biological sample such as blood is prepared and analyzed for the presence or absence of a given alteration of KVLQT1, HERG, SCN5A, KCNE1 or KCNE2. In order to detect the increased risk for LQTS or for the lack of such increased risk, a biological sample is prepared and analyzed for the presence or absence of a mutant allele of KVLQT1, HERG, SCN5A, KCNE1 or KCNE2. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual. Such diagnoses may be performed by diagnostic laboratories or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis.
• The presence of hereditary LQTS may be ascertained by testing any tissue of a human for mutations of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene. For example, a person who has inherited a germline HERG mutation would be prone to develop LQTS. This can be determined by testing DNA from any tissue of the person's body. Most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene. Alteration of a wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein.
• There are several methods that can be used to detect DNA sequence variation. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation. Another approach is the single-stranded conformation polymorphism assay (SSCP) (Orita et al., 1989). This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCP makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis. The fragments which have shifted mobility on SSCP gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., 1991), heteroduplex analysis (HA) (White et al., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989). None of the methods described above will detect large deletions, duplications or insertions, nor will they detect a regulatory mutation which affects transcription or translation of the protein. Other methods which might detect these classes of mutations such as a protein truncation assay or the asymmetric assay, detect only specific types of mutations and would not detect missense mutations. A review of currently available methods of detecting DNA sequence variation can be found in a recent review by Grompe (1993). Once a mutation is known, an allele specific detection approach such as allele specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation. Such a technique can utilize probes which are labeled with gold nanoparticles to yield a visual color result (Elghanian et al., 1997).
• A rapid preliminary analysis to detect polymorphisms in DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably with a large number of restriction enzymes. Each blot contains a series of normal individuals and a series of LQTS cases. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or including the HERG locus) indicate a possible mutation. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis (PFGE) is employed.
• Detection of point mutations may be accomplished by molecular cloning of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 alleles and sequencing the alleles using techniques well known in the art. Also, the gene or portions of the gene may be amplified, e.g., by PCR or other amplification technique, and the amplified gene or amplified portions of the gene may be sequenced.
• There are six well known methods for a more complete, yet still indirect, test for confirming the presence of a susceptibility allele: 1) single stranded conformation analysis (SSCP) (Orita et al., 1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989); 3) RNase protection assays (Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specific oligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991); and 6) allele-specific PCR (Ruano and Kidd, 1989). For allele-specific PCR, primers are used which hybridize at their 3′ ends to a particular KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 mutation. If the particular mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Such a method is particularly useful for screening relatives of an affected individual for the presence of the mutation found in that individual. Other techniques for detecting insertions and deletions as known in the art can be used.
• In the first three methods (SSCP, DGGE and RNase protection assay), a new electrophoretic band appears. SSCP detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay, an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
• Mismatches, according to the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of samples. An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labeled riboprobe which is complementary to the human wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene coding sequence. The riboprobe and either mRNA or DNA isolated from the person are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
• In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
• DNA sequences of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene which have been amplified by use of PCR may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the gene sequence harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the gene sequence. By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the gene. Hybridization of allele-specific probes with amplified KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under high stringency hybridization conditions indicates the presence of the same mutation in the tissue as in the allele-specific probe.
• The newly developed technique of nucleic acid analysis via microchip technology is also applicable to the present invention. In this technique, literally thousands of distinct oligonucleotide probes are built up in an array on a silicon chip. Nucleic acid to be analyzed is fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique one can determine the presence of mutations or even sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest. The method is one of parallel processing of many, even thousands, of probes at once and can tremendously increase the rate of analysis. Several papers have been published which use this technique. Some of these are Hacia et al., 1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996; DeRisi et al., 1996; Lipshutz et al., 1995. This method has already been used to screen people for mutations in the breast cancer gene BRCA1 (Hacia et al., 1996). This new technology has been reviewed in a news article in Chemical and Engineering News (Borman, 1996) and been the subject of an editorial (Editorial, Nature Genetics, 1996). Also see Fodor (1997).
• The most definitive test for mutations in a candidate locus is to directly compare genomic KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 sequences from patients with those from a control population. Alternatively, one could sequence messenger RNA after amplification, e.g., by PCR, thereby eliminating the necessity of determining the exon structure of the candidate gene.
• Mutations from patients falling outside the coding region of KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the genes. An early indication that mutations in non-coding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in patients as compared to control individuals.
• Alteration of KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 mRNA expression can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type gene. Alteration of wild-type genes can also be detected by screening for alteration of wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 protein. For example, monoclonal antibodies immunoreactive with HERG can be used to screen a tissue. Lack of cognate antigen would indicate a mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant gene product. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 protein can be used to detect alteration of wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 genes. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 biochemical function. Finding a mutant KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene product indicates alteration of a wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene.
• Mutant KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 genes or gene products can also be detected in other human body samples, such as serum, stool, urine and sputum. The same techniques discussed above for detection of mutant genes or gene products in tissues can be applied to other body samples. By screening such body samples, a simple early diagnosis can be achieved for hereditary LQTS.
• Initially, the screening method involves amplification of the relevant KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 sequence. In another preferred embodiment of the invention, the screening method involves a non-PCR based strategy. Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity. Further details of these methods are briefly presented below and further descriptions can be found in PCT published application WO 96/05306, incorporated herein by reference.
• The most popular method used today is target amplification. Here, the target nucleic acid sequence is amplified with polymerases. One particularly preferred method using polymerase-driven amplification is the polymerase chain reaction (PCR). The polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million-fold increase in copy number through the use of polymerase-driven amplification cycles. Once amplified, the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.
• When the probes are used to detect the presence of the target sequences, the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic acids. The sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting. The targeted region of the analyte nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.
• Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte. The region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of the genes. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide. Under certain circumstances, the formation of higher order hybrids, such as triplexes, quadraplexes, etc., may be desired to provide the means of detecting target sequences.
• Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes. Alternatively, the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly. Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies and the like. Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety. A number of these variations are well known.
• As noted above, non-PCR based screening assays are also contemplated in this invention. This procedure hybridizes a nucleic acid probe (or an analog such as a methyl phosphonate backbone replacing the normal phosphodiester), to the low level DNA target. This probe may have an enzyme covalently linked to the probe, such that the covalent linkage does not interfere with the specificity of the hybridization. This enzyme-probe-conjugate-target nucleic acid complex can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection. Enzymatic activity is observed as a change in color development or luminescent output resulting in a 10³-10⁶ increase in sensitivity. For example, the preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes are well known.
• Two-step label amplification methodologies are known in the art. These assays work on the principle that a small ligand (such as digoxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specifically binding the target gene. Allele specific probes are also contemplated within the scope of this example.
• In one example, the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate. In one embodiment of this example, digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate. In a second example, the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand. A well known embodiment of this example is the biotin-avidin type of interactions. Methods for labeling nucleic acid probes and their use in biotin-avidin based assays are well known.
• It is also contemplated within the scope of this invention that the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting the gene or genes. Thus, in one example to detect the presence of KVLQT1 in a cell sample, more than one probe complementary to KVLQT1 is employed and in particular the number of different probes is alternatively 2, 3, or 5 different nucleic acid probe sequences. In another example, to detect the presence of mutations in the KVLQT1 gene sequence in a patient, more than one probe complementary to KVLQT1 is employed where the cocktail includes probes capable of binding to the allele-specific mutations identified in populations of patients with alterations in KVLQT1. In this embodiment, any number of probes can be used.
• Large amounts of the polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the polynucleotide constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with and without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention are described, e.g., in Sambrook et al., 1989 or Ausubel et al., 1992.
• The polynucleotides of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers (1981) or the triester method according to Matteucci and Caruthers (1981) and may be performed on commercial, automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
• Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al. (1989) or Ausubel et al. (1992).
• An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may include, when appropriate, those naturally associated with the KVLQT1 or other gene. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989) or Ausubel et al. (1992); see also, e.g., Metzger et al. (1988). Many useful vectors are known in the art and may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A. Appropriate non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al., 1978) or promoters derived from murine Molony leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. Insect promoters may be derived from baculovirus. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene maybe made. For appropriate enhancer and other expression control sequences, see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1983). See also, e.g., U.S. Pat. Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.
• While such expression vectors may replicate autonomously, they may also replicate by being inserted into the genome of the host cell, by methods well known in the art.
• Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of only those host cells which express the inserts. Typical selection genes encode proteins that a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc., b) complement auxotrophic deficiencies, or c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.
• The vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection (see, Kubo et al. (1988)), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al. (1989) and Ausubel et al. (1992). The introduction of the polynucleotides into the host cell by any method known in the art, including, inter alia, those described above, will be referred to herein as “transformation.” The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
• Large quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the KVLQT1 nucleic acid or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
• Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, or amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Jakoby and Pastan (eds.) (1979). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and W138, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features. An example of a commonly used insect cell line is SF9.
• Clones are selected by using markers depending on the mode of the vector construction. The marker maybe on the same or a different DNA molecule, preferably the same DNA molecule. In prokaryotic hosts, the transformant may be selected, e.g., by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
• Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of KVLQT1 or other polypeptides.
• The probes and primers based on the KVLQT1 or other gene sequences disclosed herein are used to identify homologous KVLQT1 or other gene sequences and proteins in other species. These gene sequences and proteins are used in the diagnostic/prognostic, therapeutic and drug screening methods described herein for the species from which they have been isolated.
• The studies described in the Examples below resulted in the determination of many novel mutations. Previous studies had defined 126 distinct disease causing mutations in the LQTS genes KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 (Wang Q. et al., 1996a; Curran et al., 1995; Wang et al., 1995a; Splawski et al., 1997a; Abbott et al., 1999; Chouabe et al., 1997; Wollnik et al., 1997; Neyroud et al., 1997; Splawski et al., 1997b; Tyson et al., 1997; Schulze-Bahr et al., 1997; Priori et al., 1999; Splawski et al., 1998; Wang et al., 1995b; Russell et al., 1996; Neyroud et al., 1998; Neyroud et al., 1999; Donger et al., 1997; Tanaka et al., 1997; Jongbloed et al., 1999; Priori et al., 1998; Itoh et al., 1998a; Itoh et al., 1998b; Mohammad-Panah et al., 1999; Saarinen et al., 1998; Ackerman et al., 1998; Berthet et al., 1999; Kanters, 1998; van den Berg et al., 1997; Dausse et al., 1996; Benson et al., 1996; Akimoto et al., 1998; Satler et al., 1996; Satler et al., 1998; Makita et al., 1998, An et al., 1998; Schulze-Bahr et al., 1995; Duggal et al., 1998; Chen Q. et al., 1999; Li et al., 1998; Wei et al., 1999; Larsen et al., 1999a; Bianchi et al., 1999; Ackerman et al., 1999a; Ackerman et al., 1999b; Murray et al., 1999; Larsen et al., 1999b; Yoshida et al., 1999; Wattanasirichaigoon et al., 1999; Bezzina et al., 1999; Hoorntje et al., 1999). The sequence of each wild-type gene has been published. The KVLQT1 can be found in Splawski et al. (1998) and the coding region of the cDNA is shown herein as SEQ ID NO:1 and the encoded KVLQT1 is shown as SEQ ID NO:2. SCN5A was reported by Gellens et al. (1992) and its sequence is provided by GenBank Accession No. NM_—000335. The coding sequence of SCN5A is shown herein as SEQ ID NO:3 and the encoded SCN5A is shown as SEQ ID NO:4. Most of the mutations were found in KVLQT1 (Yoshida et al., 1999) and HERG (Itoh et al., 1998b), and fewer in SCN5A (Wang Q. et al., 1996a), KCNE1 (Jiang et al., 1994) and KCNE2 (Ward, 1964). These mutations were identified in regions with known intron/exon structure, primarily the transmembrane and pore domains. In this study, we screened 262 individuals with LQTS for mutations in all known arrhythmia genes. We identified 134 mutations, 80 of which were novel. Together with 43 mutations reported in our previous studies, we have now identified 177 mutations in these 262 LQTS individuals (68%). The failure to identify mutations in 32% of the individuals may result from phenotypic errors, incomplete sensitivity of SSCP or presence of mutations in regulatory sequences. However, it is also clear that additional LQTS genes await discovery (Jiang et al., 1994; Schott et al., 1995).
• Missense mutations were most common (72%), followed by frameshift mutations (10%), in-frame deletions, nonsense and splice site mutations (5-7% each). Most mutations resided in intracellular (52%) and transmembrane (30%) domains; 12% were found in pore and 6% in extracellular segments. One hundred one of the 129 distinct LQTS mutations (78%) were identified in single families or individuals. Most of the 177 mutations were found in KVLQT1 (75 or 42%) and HERG (80 or 45%). These two genes accounted for 87% of the identified mutations, while mutations in SCN5A (14 or 8%), KCNE1 (5 or 3%) and KCNE2 (3 or 2%) accounted for the other 13%.
• Multiple mutations were found in regions encoding S5, S5/P, P and S6 of KVLQT1 and HERG. The P region of potassium channels forms the outer pore and contains the selectivity filter (Doyle et al., 1998). Transmembrane segment 6, corresponding to the inner helix of KcsA, forms the inner ⅔ of the pore. This structure is supported by the S5 transmembrane segment, corresponding to the outer helix of KcsA, and is conserved from prokaryotes to eukaryotes ((MacKinnon et al., 1998). Mutations in these regions will likely disrupt potassium transport. Many mutations were identified in the C-termini of KVLQT1 and HERG. Changes in the C-terminus of HERG could lead to anomalies in tetramerization as it has been proposed that the C-terminus of eag, which is related to HERG, is involved in this process (Ludwig et al, 1994).
• Multiple mutations were also identified in regions that were different for KVLQT1 and HERG. In KVLQT1, multiple mutations were found in the sequences coding for the S2/S3 and S4/S5 linkers. Coexpression of S2/S3 mutants with wild-type KVLQT1 in Xenopus oocytes led to simple loss of function or dominant-negative effect without significantly changing the biophysical properties of I_Ks channels (Chouabe et al., 1997; Shalaby et al., 1997; Wang et al., 1999). On the other hand, S4/S5 mutations altered the gating properties of the channels and modified KVLQT1 interactions with minK subunits (Wang et al., 1999; Franqueza et al., 1999). In HERG, more than 20 mutations were identified in the N-terminus. HERG channels lacking this region deactivate faster and mutations in the region had a similar effect (Chen J. et al., 1999).
• Mutations in KCNE1 and KCNE2, encoding minK and MiRP 1, the respective I_Ks and I_Kr β-subunits, altered the biophysical properties of the channels (Splawski et al., 1997a; Abbott et al., 1999; Sesti and Goldstein, 1998). A MiRPI mutant, involved in clarithromyocin-induced arrhythmia, increased channel blockade by the antibiotic (Abbott et al., 1999). Mutations in SCN5A, the sodium channel α-subunit responsible for cardiac I_Na, destabilized the inactivation gate causing delayed channel inactivation and dispersed reopenings (Bennett et al., 1995; Dumaine et al., 1996; Wei et al., 1999; Wang D W et al., 1996). One SCN5A mutant affected the interactions with the sodium channel β-subunit (An et al., 1998).
• It is interesting to note that probands with KCNE1 and KCNE2 mutations were older and had shorter QTc than probands with the other genotypes. The significance of these differences is unknown, however, as the number of probands with KCNE1 and KCNE2 genotypes was small.
• This catalogue of mutations will facilitate genotype-phenotype analyses. It also has clinical implications for presymptomatic diagnosis and, in some cases, for therapy. Patients with mutations in KVLQT1, HERG, KCNE1 and KCNE2, for example, may benefit from potassium therapy (Compton et al., 1996). Sodium channel blockers, on the other hand, might be helpful in patients with SCN5A mutations (Schwartz et al. (1995). The identification of mutations is of importance for ion channel studies as well. The expression of mutant channels in heterologous systems can reveal how structural changes influence the behavior of the channel or how mutations affect processing (Zhou et al., 1998; Furutani et al., 1999). These studies improve our understanding of channel function and provide insights into mechanisms of disease. Finally, mutation identification will contribute to the development of genetic screening for arrhythmia susceptibility.
• The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described in the Examples were utilized.
EXAMPLE 1 Ascertainment and Phenotyping
• Individuals were ascertained in clinics from North America and Europe. Individuals were evaluated for LQTS based on QTc (the QT interval corrected for heart rate) and for the presence of symptoms. In this study, we focused on the probands. Individuals show prolongation of the QT interval (QTc≧460 ms) and/or documented torsade de pointes, ventricular fibrillation, cardiac arrest or aborted sudden death. Informed consent was obtained in accordance with local institutional review board guidelines. Phenotypic data were interpreted without knowledge of genotype. Sequence changes altering coding regions or predicted to affect splicing that were not detected in at least 400 control chromosomes were defined as mutations. No changes except known polymorphisms were detected ina ny of the genes in the control population. This does not exclude the possibility that some mutations are rare variants not associated with disease.
EXAMPLE 2 Mutational Analyses
• To determine the spectrum of LQTS mutations, we used SSCP (Single Stand Conformation Polymorphism) and DNA sequence analyses to screen 262 unrelated individuals with LQTS. Seventeen primer pairs were used to screen KVLQT1 (Splawski et al., 1998), twenty-one primer pairs were used for HERG (Splawski et al., 1998) and three primer pairs were used for KCNE1 (Splawski et al., 1997a) and KCNE2 (Abbott et al., 1999). Thirty-three primer pairs (Wang Q. et al., 1996b) were used in SSCP analysis to screen all SCN5A exons in 50 individuals with suspected abnormalities in I_Na. Exons 23-28, in which mutations were previously identified, were screened in all 262 individuals.
• Gender, age, QTc and presence of symptoms are summarized in Table 1. The average age at ascertainment was 29 with a corrected QT interval of 492 ms. Seventy-five percent had a history of symptoms and females predominated with an˜2:1 ratio. Although the numbers were small, corrected QT intervals for individuals harboring KCNE1 and KCNE2 mutations were shorter at 457 ms.

  TABLE 1
  Age, QTc, Gender and Presence of Symptoms

  	Age*, y		QTc, ms
  Genotype	(mean ± SD)	Gender (F/M)	(mean ± SD)	Symptoms†
  KVLQT1	32 ± 19	52/23	493 ± 45	78%
  HERG	31 ± 19	51/29	498 ± 48	71%
  SCN5A	32 ± 24	8/6	511 ± 42	55%
  KCNE1	43 ± 16	3/2	457 ± 25	40%
  KCNE2	54 ± 20	3/0	457 ± 05	67%
  unknown	25 ± 16	56/29	484 ± 46	81%
  all	29 ± 19	173/89	492 ± 47	75%
  *-age at ascertainment
  †-symptoms include syncope, cardiac arrest or sudden death

• The SSCP analyses revealed many mutations. KVLQT1 mutations associated with LQTS were identified in 52 individuals (FIG. 1 and Table 2). Twenty of the mutations were novel. HERG mutations were identified in 68 LQTS individuals (FIG. 2 and Table 3). Fifty-two of these mutations were novel. SCN5A mutations were identified in eight cases (FIG. 3 and Table 4). Five of the mutations were novel. Three novel KCNE1 mutations were identified (FIG. 4 and Table 5) and three mutations were identified in KCNE2 (FIG. 5 and Table 6) (Abbott et al., 1999). None of the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 mutations was observed in 400 control chromosomes.

  TABLE 2
  Summary of All KVLQT1 Mutations*

  				Number
  Nucleotide	Coding			of
  Change†	Effect	Position	Exon	familles‡	Study

  del211-219	del71-73	N-terminus	1	1	Ackerman et al., 1999a
  A332G†	Y111C	N-terminus	1	1	This
  de1451-452	A150fs/132	S2	2	1 JLN	Chen Q. et al., 1999
  T470G	F157C	S2	1	1	Larsen et al., 1999a
  G477 + 1A	M159sp	S2	2	1 JLN,	This; Donger et al., 1997
  				1 UK
  G477 + 5A	M159sp	S2	1	1	Ackerman et al., 1999b
  G478A†	E160K	S2	3	1	This
  del500-502	F167W/del	S2	3	1	Wang Q. et al., 1996a
  	G168
  G502A	G168R	S2	3	7	This; Splawski et al.,
  					1998; Donger et al., 1997
  C520T	R174C	S2/S3	3	1	Donger et al., 1997
  G521A†	R174H	S2/S3	3	1	This
  G532A	A178T	S2/S3	3	1	Tanaka et al., 1997
  G532C	A178P	S2/S3	3	1	Wang Q. et al., 1996a
  G535A†	G179S	S2/S3	3	1	This
  A551C	Y184S	S2/S3	3	2	This; Jongbloed et al.,
  					1999
  G565A	G189R	S2/S3	3	3	Wang Q. et al., 1996a;
  					Jongbloed et al., 1999
  insG567-568	G189fs/94	S2/S3	3	1 (RW + JLN)	Splawski et al., 1997b
  G569A	R190Q	S2/S3	3	2	Splawski et al., 1998;
  					Donger et al., 1997
  del572-576	L191fs/90	S2/S3	3	1 JLN,	Tyson et al., 1997;
  				1 RW	Ackerman et al., 1999b
  				2 (JLN + RW)
  G580C†	A194P	S2/S3	3	1	This
  C674T	S225L	S4	4	2	This; Priori et al., 1999
  G724A	D242N	S4/S5	5	1	Itoh et al., 1998b
  C727T†	R243C	S4/S5	5	2	This
  G728A	R243H	S4/S5	5	1 JLN	Saarinen et al., 1998
  T742C†	W248R	S4/S5	5	1	This
  T749A	L250H	S4/S5	5	1	Itoh et al., 1998a
  G760A	V254M	S4/S5	5	4	This; Wang Q. et al.,
  					1996a; Donger et al.,
  					1997
  G781A	E261K	S4/S5	6	1	Donger et al., 1997
  T797C†	L266P	S5	6	1	This
  G805A	G269S	S5	6	1	Ackerman et al., 1999b
  G806A	G269D	S5	6	3	This; Donger et al., 1997
  C817T	L273F	S5	6	2	This; Wang Q. et al.,
  					1996a
  A842G	Y281C	S5	6	1	Priori et al., 1999
  G898A	A300T	S5/Pore	6	1	Priori et al., 1998
  G914C	W305S	Pore	6	1 JLN	Chouabe et al., 1997
  G916A	G306R	Pore	6	1	Wang Q. et al, 1996a
  de1921-	V307sp	Pore	6	1	Li et al., 1998
  (921 + 2)
  G921 + 1T†	V307sp	Pore	6	1	This
  A922-2C†	V307sp	Pore	7	1	This
  G922-1C	V307sp	Pore	7	1	Murray et al., 1999
  C926G	T309R	Pore	7	1	Donger et al., 1997
  G928A†	V310I	Pore	7	1	This
  C932T	T311I	Pore	7	1	Saarinen et al., 1998
  C935T	T312I	Pore	7	2	This; Wang Q. et al.,
  					1996a
  C939G	I313M	Pore	7	1	Tanaka et al., 1997
  G940A	G314S	Pore	7	7	Splawski et al., 1998;
  					Russell et al., 1996;
  					Donger et al., 1997;
  					Jongbloed et al., 1999;
  					Itoh et al., 1998b
  A944C	Y315S	Pore	7	3	Donger et al., 1997;
  					Jongbloed et al., 1999
  A944G	Y315C	Pore	7	2	Priori et al., 1999;
  					Splawski et al., 1998
  G949A	D317N	Pore	7	2	Wollnik et al., 1997;
  					Saarinen et al., 1998
  G954C	K318N	Pore	7	1	Splawski et al., 1998
  C958G	P320A	Pore	7	1	Donger et al., 1997
  G973A	G325R	S6	7	4	This; Donger et al., 1997;
  					Tanaka et al., 1997
  del1017-1019	delF340	S6	7	2	This; Ackerman et al.,
  					1998
  C1022A	A341E	S6	7	5	This; Wang Q. et al.,
  					1996a; Berthet et al., 1999
  C1022T	A341V	S6	7	7	This; Wang Q. et al.,
  					1996a; Russell et al.,
  					1996; Donger et al., 1997;
  					Li et al., 1998
  C1024T	L342F	S6	7	1	Donger et al., 1997
  C1031T	A344V	S6	7	1	Donger et al., 1997
  G1032A	A344sp	S6	7	9	This; Kanters, 1998; Li et
  					al., 1998; Ackerman et al.,
  					1999b; Murray et al.,
  					1999
  G1032C	A344sp	S6	7	1	Murray et al, 1999
  G1033C	G345R	S6	8	1	van den Berg et al., 1997
  G1034A	G345E	S6	8	1	Wang Q. et al., 1996a
  C1046G†	S349W	S6	8	1	This
  T1058C	L353P	S6	8	1	Splawski et al., 1998
  C1066T†	Q356X	C-terminus	8	1	This
  C1096T	R366W	C-terminus	8	1	Splawski et al., 1998
  G1097A†	R366Q	C-terminus	8	1	This
  G1097C	R366P	C-terminus	8	1	Tanaka et al., 1997
  G1111A	A371T	C-terminus	8	1	Donger et al., 1997
  T1117C	S373P	C-terminus	8	1	Jongbloed et al., 1999
  C1172T†	T391I	C-terminus	9	1	This
  T1174C	W392R	C-terminus	9	1	Jongbloed et al., 1999
  C1343G†	P448R	C-terminus	10	2	This
  C1522T	R518X	C-terminus	12	1 JLN,	This; Larsen et al., 1999
  				3 RW
  G1573A	A525T	C-terminus	12	1	Larsen et al., 1999b
  C1588T†	Q530X	C-terminus	12	1 JLN,	This
  				1 RW
  C1615T	R539W	C-terminus	13	1	Chouabe et al., 1997
  de16/ins7	E543fs/107	C-terminus	13	1 JLN	Neyroud et al., 1997
  C1663T	R555C	C-terminus	13	3	Donger et al., 1997
  C1697T†	S566F	C-terminus	14	3	This
  C1747T†	R583C	C-terminus	15	1	This
  C1760T	T587M	C-terminus	15	1 JLN,	Donger et al., 1997;
  				1 RW	Itoh et al., 1998b
  G1772A	R591H	C-terminus	15	1	Donger et al., 1997
  G1781A†	R594Q	C-terminus	15	3	This
  del1892-1911	P630fs/13	C-terminus	16	1 JLN	Donger et al., 1997
  insC1893-1894	P631fs/19	C-terminus	16	1	Donger et al., 1997
  *—ins denotes insertion; del denotes deletion; sp denotes the last unaffected amino acid before the predicted splice mutation; fs denotes the last amino acid unaffected by a frameshift, following fs is the number of amino acids before termination; X denotes a stop codon occurred.
  †—denotes novel mutation
  ‡—Number of Romano-Ward families unless otherwise indicated (UK —unknown)

• TABLE 3
  Summary of All HERG Mutations*

  				Number
  Nucleotide	Coding			of RW
  Change	Effect	Position	Exon	Families	Study

  C87A†	F29L	N-terminus	2	1	This
  A98C†	N33T	N-terminus	2	2	This
  C132A†	C44X	N-terminus	2	1	This
  G140T†	G47V	N-terminus	2	1	This
  G157C†	G53R	N-terminus	2	1	This
  G167A†	R56Q	N-terminus	2	1	This
  T196G†	C66G	N-terminus	2	1	This
  A209G†	H70R	N-terminus	2	2	This
  C215A†	P72Q	N-terminus	2	2	This
  del221-251†	R73fs/31	N-terminus	2	1	This
  G232C†	A78P	N-terminus	2	1	This
  dupl234-250†	A83fs/37	N-terminus	2	1	This
  C241T†	Q81X	N-terminus	2	1	This
  T257G†	L86R	N-terminus	2	1	This
  insC422-423†	P141fs/2	N-terminus	3	1	This
  insC453-454†	P151fs/	N-terminus	3	1	This
  	179
  dupl558-600	L200fs/	N-terminus	4	1	Hoorntje et al., 1999
  	144
  insC724-725†	P241fs/89	N-terminus	4	1	This
  del885†	V295fs/63	N-terminus	4	1	This
  C934T†	R312C	N-terminus	5	1	This
  C1039T†	P347S	N-terminus	5	1	This
  G1128A†	Q376sp	N-terminus	5	1	This
  A1129-2G†	Q376sp	N-terminus	6	1	This
  del1261	Y420fs/12	S1	6	1	Curran et al., 1995
  C1283A	S428X	S1/S2	6	1	Priori et al., 1999
  C1307T	T436M	S1/S2	6	1	Priori et al., 1999
  A1408G	N470D	S2	6	1	Curran et al., 1995
  C1421T	T474I	S2/S3	6	1	Tanaka et al., 1997
  C1479G	Y493X	S2/S3	6	1	Itoh et al., 1998a
  del1498-1524	del500-508	S3	6	1	Curran et al., 1995
  G1592A†	R531Q	S4	7	1	This
  C1600T	R534C	S4	7	1	Itoh et al., 1998a
  T1655C†	L552S	S5	7	1	This
  delT1671	T556fs/7	S5	7	1	Schulze-Bahr et al., 1995
  G1672C	A558P	S5	7	1	Jongbloed et al., 1999
  G1681A	A561T	S5	7	4	This; Dausse et al., 1996
  C1682T	A561V	S5	7	4	This; Curran et al., 1995;
  					Priori et al., 1999
  G1714C	G572R	S5/Pore	7	1	Larsen et al., 1999a
  G1714T	G572C	S5/Pore	7	1	Splawski et al., 1998
  C1744T	R582C	S5/Pore	7	1	Jongbloed et al., 1999
  G1750A†	G584S	S5/Pore	7	1	This
  G1755T†	W585C	S5/Pore	7	1	This
  A1762G	N588D	S5/Pore	7	1	Splawski et al., 1998
  T1778C†	I593T	S5/Pore	7	1	This
  T1778G	I593R	S5/Pore	7	1	Benson et al., 1996
  G1801A	G601S	S5/Pore	7	1	Akimoto et al., 1998
  G1810A	G604S	S5/Pore	7	2	This; Jongbloed et al.,
  					1999
  G1825A†	D609N	S5/Pore	7	1	This
  T1831C	Y611H	S5/Pore	7	1	Tanaka et al., 1997
  T1833 (A or	Y611X	S5/Pore	7	1	Schulze-Bahr et al., 1995
  G)
  G1834T	V612L	Pore	7	1	Satler et al., 1998
  C1838T	T613M	Pore	7	4	This; Jongbloed et aL,
  					1999
  C1841T	A614V	Pore	7	6	Priori et al., 1999;
  					Splawski et al., 1998;
  					Tanaka et al., 1997,
  					Satler et al., 1998
  C1843G†	L615V	Pore	7	1	This
  G1876A†	G626S	Pore	7	1	This
  C1881G†	F627L	Pore	7	1	This
  G1882A	G628S	Pore	7	2	This; Curran et al., 1995
  A1885G	N629D	Pore	7	1	Satler et al., 1998
  A1886G	N629S	Pore	7	1	Satler et al., 1998
  C1887A	N629K	Pore	7	1	Yoshida et al., 1999
  G1888C	V630L	Pore	7	1	Tanaka et al., 1997
  T1889C	V630A	Pore	7	1	Splawski et al., 1998
  C1894T†	P632S	Pore	7	1	This
  A1898G	N633S	Pore	7	1	Satler et al., 1998
  A1912G†	K638E	S6	7	1	This
  del1913-1915†	delK638	S6	7	1	This
  C1920A	F640L	S6	7	1	Jongbloed et al., 1999
  A1933T†	M645L	S6	7	1	This
  del1951-1952	L650fs/2	S6	8	1	Itoh et al., 1998a
  G2044T†	E682X	S6/cNBD	8	1	This
  C2173T	Q725X	S6/cNBD	9	1	Itoh et al., 1998a
  insT2218-2219†	H739fs/63	S6/cNBD	9	1	This
  C2254T†	R752W	S6/cNBD	9	1	This
  dupl2356-2386	V796fs/22	cNBD	9	1	Itoh et al., 1998a
  del2395†	I798fs/10	cNBD	9	1	This
  G2398 + 1C	L799sp	cNBD	9	2	This; Curran et al., 1995
  T2414C†	F805S	cNBD	10	1	This
  T2414G†	F805C	cNBD	10	1	This
  C2453T	S818L	cNBD	10	1	Berthet et al., 1999
  G2464A	V822M	cNBD	10	2	Berthet et al., 1999;
  					Satler et al., 1996
  C2467T†	R823W	cNBD	10	2	This
  A2582T†	N861I	C-terminus	10	1	This
  G2592 + 1A	D864sp	C-terminus	10	2	This; Berthet et al., 1999
  del2660†	K886fs/85	C-terminus	11	1	This
  C2750T†	P917L	C-terminus	12	1	This
  del2762†	R920fs/51	C-terminus	12	1	This
  C2764T†	R922W	C-terminus	12	1	This
  insG2775-2776†	G925fs/13	C-terminus	12	1	This
  del2906†	P968fs/4	C-terminus	12	1	This
  del2959-2960†	P986fs/	C-terminus	12	1	This
  	130
  C3040T†	R1014X	C-terminus	13	2	This
  de13094†	G1031fs/	C-terminus	13	1	This
  	24
  insG3107-3108	G1036fs/	C-terminus	13	1	Berthet et al., 1999
  	82
  insC3303-3304†	P1101fs	C-terminus	14	1	This
  *—all characters same as in Table 2

• TABLE 4
  Summary of All SCN5A Mutations

  				Number
  Nucleotide	Coding			of RW
  Change	Effect	Position	Exon	Families	Study
  G3340A†	D1114N	DII/DIII	18	1	This
  C3911T	T1304M	DIII/S4	22	1	Wattanasirichaigoon et al.,
  					1999
  A3974G	N1325S	DIII/S4/S5	23	1	Wang et al., 1995b
  C4501G†	L1501V	DIII/DIV	26	1	This
  del4511-4519	del1505-1507	DIII/DIV	26	4	Wang et al., 1995a; Wang et
  					al., 1995b
  del4850-4852†	delF1617	DIV/S3/S4	28	1	This
  G4868A	R1623Q	DIV/S4	28	2	This; Makita et al., 1998
  G4868T†	R1623L	DIV/S4	28	1	This
  G4931A	R1644H	DIV/S4	28	2	This; Wang et al., 1995b
  C4934T	T1645M	DIV/S4	28	1	Wattanasirichaigoon et al.,
  					1999
  G5350A†	E1784K	C-terminus	28	2	This; Wei et al., 1999
  G5360A†	S1787N	C-terminus	28	1	This
  A5369G	D1790G	C-terminus	28	1	An et al., 1998
  insTGA	insD1795-1796	C-terminus	28	1	Bezzina et al., 1999
  5385-5386
  *—all characters same as in Table 2. Fifty individuals with suspected abnormalities in INa were screened for all SCN5A exons. All individuals were screened for exons 23-28.

• TABLE 5
  Summary of All KCNE1 Mutations*

  Nucleotide	Coding			Number of
  Change	Effect	Position	Exon	Families	Study
  C20T	T7I	N-terminus	3	1 JLN	Schulze-
  					Bahr et al.,
  					1997
  G95A†	R32H	N-terminus	3	1	This
  G139T	V47F	S1	3	1 JLN	Bianchi et
  					al., 1999
  TG151-152AT	L51H	S1	3	1 JLN	Bianchi et
  					al., 1999
  A172C/TG	TL58-	S1	3	1 JLN	Tyson et al.,
  176-177CT	59PP				1997
  C221T	S74L	C-terminus	3	1	Splawski et
  					al., 1997a
  G226A	D76N	C-terminus	3	1 JLN,	Splawski et
  				1 RW,	al., 1997a;
  				1 (JLN +	Tyson et al.,
  				RW)	1997;
  					Duggal et
  					al., 1998
  T259C	W87R	C-terminus	3	1	Bianchi et
  					al., 1999
  C292T†	R98W	C-terminus	3	1	This
  C379A†	P127T	C-terminus	3	1	This
  *—all characters same as in Table 2

• TABLE 6
  Summary of All KCNE2 Mutations

  Nucle-				Number
  otide	Coding			of
  Change	Effect	Position	Exon	Families	Study
  C25G	Q9E	N-terminus	1	1	Abbott et al., 1999
  T161T	M54T	S1	1	1	Abbott et al., 1999
  T170C	I57T	S1	1	1	Abbott et al., 1999

• TABLE 7
  Mutations by Type

  Type	KVLQT1	HERG	SCN5A	KCNE1	KCNE2	Total

  Missense	59	52	9	5	3	128
  Nonsense	6	5	0	0	0	11
  AA deletion*	2	2	5	0	0	9
  Frameshift	1	16	0	0	0	17
  Splice	7	5	0	0	0	12
  Total	75	80	14	5	3	177
  *—AA denotes amino acid

• TABLE 8
  Mutations by Position

  Gene
  Protein	KVLQT1	HERG	SCN5A	KCNE1	KCNE2
  Position	KVLQT1	HERG	SCN5A	minK	MiRP1	Total

  Extracellular	0	7	1	1	1	10
  Trans-	33	13	5	0	2	53
  membrane
  Pore	9	12	0	N/A	N/A	21
  Intracellular	33	48	8	4	0	93
  Total	75	80	14	5	3	177

• While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.
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Claims (16)

1. An isolated DNA comprising a sequence of SEQ ID NO:1 as altered by one or more mutations selected from the group consisting of C1172T, C1343G, C 1588T, C1697T, C1747T and G1781A.

2. An isolated nucleic acid probe which hybridizes to the isolated DNA of claim 1 under conditions at which it will not hybridize to wild-type KVLQT1 DNA.

3. A method for detecting a mutation in KVLQT1 said mutation selected from the group consisting of C1172T, C1343G, C1588T, C1697T, C1747T and G1781A which comprises analyzing a sequence of said KVLQT1 DNA or RNA from a human sample or analyzing the sequence of cDNA made from mRNA from said sample for said mutation.

4. The method of claim 3 wherein said mutation is detected by a method selected from the group consisting of:

a) hybridizing a probe specific for one of said mutations to RNA isolated from said human sample and detecting the presence of a hybridization product, wherein the presence of said product indicates the presence of said mutation in the sample;

b) hybridizing a probe specific for one of said mutations to cDNA made from RNA isolated from said sample and detecting the presence of a hybridization product, wherein the presence of said product indicates the presence of said mutation in the sample;

c) hybridizing a probe specific for one of said mutations to genomic DNA isolated from said sample and detecting the presence of a hybridization product, wherein the presence of said product indicates the presence of said mutation in the sample;

d) amplifying all or part of said KVLQT1 DNA in said sample using a set of primers to produce amplified nucleic acids and sequencing the amplified nucleic acids;

e) amplifying part of said KVLQT1 DNA in said sample using a primer specific for one of said mutations and detecting the presence of an amplified product, wherein the presence of said product indicates the presence of said mutation in the sample;

f) molecularly cloning all or part of said KVLQT1 DNA in said sample to produce a cloned nucleic acid and sequencing the cloned nucleic acid;

g) amplifying said KVLQT1 DNA to produce amplified nucleic acids, hybridizing the amplified nucleic acids to a DNA probe specific for one of said mutations and detecting the presence of a hybridization product, wherein the presence of said product indicates the presence of said mutation;

h) forming single-stranded DNA from a KVLQT1 DNA fragment of said gene from said human sample and single-stranded DNA from a corresponding fragment of a wild-type gene, electrophoresing said single-stranded DNAs on a non-denaturing polyacrylamide gel and comparing the mobility of said single-stranded DNAs on said gel to determine if said single-stranded DNA from said sample is shifted relative to wild-type and sequencing said single-stranded DNA having a shift in mobility;

i) forming a heteroduplex consisting of a first strand of nucleic acid selected from the group consisting of a genomic DNA fragment isolated from said sample, an RNA fragment isolated from said sample and a cDNA fragment made from mRNA from said sample and a second strand of a nucleic acid consisting of a corresponding human wild-type gene fragment, analyzing for the presence of a mismatch in said heteroduplex, and sequencing said first strand of nucleic acid having a mismatch;

j) forming single-stranded DNA from said KVLQT1 DNA of said human sample and from a corresponding fragment of an allele specific for one of said mutations, electrophoresing said single-stranded DNAs on a non-denaturing polyacrylamide gel and comparing the mobility of said single-stranded DNAs on said gel to determine if said single-stranded DNA from said sample is shifted relative to said allele, wherein no shift in electrophoretic mobility of the single-stranded DNA relative to the allele indicates the presence of said mutation in said sample; and

k) forming a heteroduplex consisting of a first strand of nucleic acid selected from the group consisting of a genomic DNA fragment of said KVLQT1 DNA isolated from said sample, an RNA fragment isolated from said sample and a cDNA fragment made from mRNA from said sample and a second strand of a nucleic acid consisting of a corresponding gene allele fragment specific for one of said mutations and analyzing for the presence of a mismatch in said heteroduplex, wherein no mismatch indicates the presence of said mutation.

5. A method according to claim 4 wherein hybridization is performed in situ.

6. A method of assessing a risk in a human subject for long QT syndrome which comprises screening said subject for a mutation in KVLQT1 selected from T3911 , P448R, Q530X, S566F, R583C and R594Q by comparing the sequence of said KVLQT1 or its expression products isolated from a tissue sample of said subject with a wild-type sequence of said KVLQT1 or its expression products, wherein a mutation selected from T3911, P448R, Q530X, S566F, R583C and R594Q in the sequence of the subject indicates a risk for long QT syndrome.

7. The method of claim 6 wherein said expression product is selected from mRNA of KVLQT1 DNA or a polypeptide encoded by said gene.

8. The method of claim 6 wherein one or more of the following procedures is carried out:

(a) observing shifts in electrophoretic mobility of single-stranded DNA from said sample on non-denaturing polyacrylamide gels;

b) hybridizing a probe to genomic DNA isolated from said sample under conditions suitable for hybridization of said probe to said gene;

(c) determining hybridization of an allele-specific probe to genomic DNA from said sample;

(d) amplifying all or part of said KVLQT1 DNA from said sample to produce an amplified sequence and sequencing the amplified sequence;

(e) determining by nucleic acid amplification the presence of a specific mutant allele in said sample;

(f) molecularly cloning all or part of said KVLQT1 DNA from said sample to produce a cloned sequence and sequencing the cloned sequence;

(g) determining whether there is a mismatch between molecules (1) said KVLQT1 DNA or mRNA isolated from said sample, and (2) a nucleic acid probe complementary to the human wild-type gene DNA, when molecules (1) and (2) are hybridized to each other to form a duplex;

(h) amplification of said KVLQT1 DNA sequence in said sample and hybridization of the amplified sequence to nucleic acid probes which comprise wild-type gene sequences;

(i) amplification of said KVLQT1 DNA sequence in said tissue and hybridization of the amplified sequence to nucleic acid probes which comprise mutant gene sequence;

(j) screening for a deletion mutation;

(k) screening for a point mutation;

(l) screening for an insertion mutation;

(m) determining in situ hybridization of said KVLQT1 DNA in said sample with one or more nucleic acid probes which comprise said KVLQT1 DNA sequence or a mutant sequence of said KVLQT1 DNA;

(n) immunoblotting;

(o) immunocytochemistry;

(p) assaying for binding interactions between said protein isolated from said tissue and a binding partner capable of specifically binding the polypeptide expression product of a mutant allele and/or a binding partner for the polypeptide and assaying for the inhibition of biochemical activity of said binding partner.

9. A method for diagnosing a mutation which causes long QT syndrome comprising hybridizing a probe which hybridizes to isolated DNA comprising a sequence of SEQ ID NO:1 as altered by one or more mutations selected from the group consisting of C1172T, C1343G, C1588T, C1697T, C1747T and G1781A and not to wild-type KVLQT1 DNA, to a patient's sample of DNA or RNA, the presence of a hybridization signal being indicative of long QT syndrome.

10. A method according to claim 9 wherein the patient's DNA or RNA has been amplified and said amplified DNA or RNA is hybridized with said probe.

11. A method according to claim 9 wherein said hybridization is performed in situ.

12. A method according to claim 9 wherein said assay is performed using nucleic acid microchip technology.

13. A method for diagnosing a mutation which causes long QT syndrome comprising amplifying a region of gene or RNA for KVLQT1 and sequencing the amplified gene or RNA wherein long QT syndrome is indicated by any one or more mutations selected from the group consisting of C1172T, C1343G, C1588T, C1697T, C1747T and G1781A.

14. A method for diagnosing a mutation which causes long QT syndrome comprising identifying a mismatch between a patient's DNA or RNA and a wild-type DNA or RNA probe wherein said probe hybridizes to a region of DNA or RNA wherein said region comprises a mutation of SEQ ID NO:1 selected from the group consisting of C1172T, C1343G, C1588T, C1697T, C1747T and G1781A.

15. The method of claim 14 wherein the mismatch is identified by an RNase assay.

16. An isolated DNA encoding a KVLQT1 polypeptide of SEQ ID NO:2 having a mutation selected from the group consisting of T3911, P448R, Q530X, S566F, R583C and R594Q.

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