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  
       [0001]     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. 
     
    
       [[0002]]     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  
       [0003]     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.  
         [0004]     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.  
         [0005]     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).  
         [0006]     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.  
         [0007]     The present invention relates to alterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes and methods for detecting such alterations.  
         [0008]     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.  
         [0009]     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  
       [0010]     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  
       [0011]      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.  
         [0012]      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.  
         [0013]      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.  
         [0014]      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.  
         [0015]      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  
       [0016]     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.  
         [0017]     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.  
         [0018]     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&#39;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.  
         [0019]     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).  
         [0020]     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.  
         [0021]     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.  
         [0022]     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.  
         [0023]     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.  
         [0024]     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.  
         [0025]     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.  
         [0026]     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.  
         [0027]     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).  
         [0028]     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.  
         [0029]     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.  
         [0030]     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.  
         [0031]     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.  
         [0032]     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.  
         [0033]     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.  
         [0034]     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.  
         [0035]     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.  
         [0036]     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.  
         [0037]     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 10 3 -10 6  increase in sensitivity. For example, the preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes are well known.  
         [0038]     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.  
         [0039]     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.  
         [0040]     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.  
         [0041]     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.  
         [0042]     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.  
         [0043]     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).  
         [0044]     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.  
         [0045]     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.  
         [0046]     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.  
         [0047]     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.  
         [0048]     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.  
         [0049]     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.  
         [0050]     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.  
         [0051]     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.  
         [0052]     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.  
         [0053]     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).  
         [0054]     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%.  
         [0055]     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).  
         [0056]     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).  
         [0057]     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).  
         [0058]     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.  
         [0059]     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.  
         [0060]     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  
       [0061]     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  
       [0062]     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.  
         [0063]     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             
 
         [0064]     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)             
 
         [0065]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 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    
               
             
          
         
       
     
         [0066]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 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 I Na  were screened for all SCN5A exons. All individuals were screened for exons 23-28.    
               
             
          
         
       
     
         [0067]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 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    
               
             
          
         
       
     
         [0068]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0069]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 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    
               
             
          
         
       
     
         [0070]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0071]     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.  
       List of References  
       [0072]    
       
          Abbott G W, et al. (1999).  Cell  97:175-187.  
          Ackerman M J, et al. (1998).  Pediatr. Res.  44:148-153.  
          Ackerman M J, et al. (1999a).  N. Engl. J. Med.  341:1121-1125.  
          Ackerman M J, et al. (1999b).  Mayo Clin. Proc.  74:1088-1094.  
          Akimoto K, et al. (1998).  Hum. Mutat.  1:S184-S186.  
          An R H, et al. (1998).  Circ. Res.  83:141-146.  
          Ausubel F M, et al. (1992).  Current Protocols in Molecular Biology , (John Wiley and Sons, New York, N.Y.).  
          Barhanin J, et al. (1996).  Nature  384:78-80.  
          Beaucage S L and Caruthers M H (1981).  Tetra. Letts.  22:1859-1862.  
          Bennett P B, et al. (1995).  Nature  376:683-685.  
          Benson D W, et al. (1996).  Circulation  93:1791-1795.  
          Berthet M, et al. (1999).  Circulation  99:1464-1470.  
          Bezzina C, et al. (1999).  Circ. Res.  85:1206-1213.  
          Bianchi L, et al. (1999).  Hum. Mol. Genet.  8:1499-1507.  
          Borman S (1996).  Chemical  &amp;  Engineering News , December 9 issue, pp. 42-43.  
          Cariello N F (1988).  Am. J. Human Genetics  42:726-734.  
          Chee M, et al. (1996).  Science  274:610-614.  
          Chen J, et al. (1999).  J. Biol. Chem.  274:10113-10118.  
          Chen Q, et al. (1999).  Circulation  99:1344-1347.  
          Chouabe C, et al. (1997).  EMBO J.  1:5472-5479.  
          Compton S J, et al. (1996).  Circulation  94:1018-1022.  
          Conner B J, et al. (1983).  Proc. Natl. Acad. Sci. USA  80:278-282.  
          Cotton R G, et al. (1988).  Proc. Natl. Acad. Sci. USA  85:4397-4401.  
          Curran M E, et al. (1995).  Cell  80:795-803.  
          Dausse E, et al. (1996).  J. Mol. Cell. Cardiol.  2:1609-1615.  
          DeRisi J, et al. (1996).  Nat. Genet.  14:457-460.  
          Donger C, et al. (1997).  Circulation  96:2778-2781.  
          Doyle D A, et al. (1998).  Science  280:69-77.  
          Duggal P, et al. (1998).  Circulation  97:142-146.  
          Dumaine R, et al. (1996).  Circ. Res.  78:914-924.  
          Editorial (1996).  Nature Genetics  14:367-370.  
          Elghanian R, et al. (1997).  Science  277:1078-1081.  
           Enhancers and Eukaryotic Gene Expression , Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1983).  
          Fiers W, et al. (1978).  Nature  273:113-120.  
          Finkelstein J, et al. (1990).  Genomics  7:167-172.  
          Fodor S P A (1997).  Science  277:393-395.  
          Franqueza L, et al. (1999).  J. Biol. Chem.  274:21063-21070.  
          Furutani M, et al. (1999).  Circulation  99:2290-2294.  
          Gellens M, et al. (1992).  Proc. Natl. Acad. Sci. USA  89:554-558.  
          Grompe M (1993).  Nature Genetics  5:111-117.  
          Grompe M, et al., (1989).  Proc. Natl. Acad. Sci. USA  86:5855-5892.  
          Hacia J G, et al. (1996).  Nature Genetics  14:441-447.  
          Hoorntje T, et al. (1999).  Circulation  100:1264-1267.  
          Itoh T, et al. (1998a).  Hum. Genet.  102:435-439.  
          Itoh T, et al. (1998b).  Hum. Genet.  103:290-294.  
          Jakoby W B and Pastan I H (eds.) (1979).  Cell Culture. Methods in Enzymology  volume 58 (Academic Press, Inc., Harcourt Brace Jovanovich (New York)).  
          Jervell A and Lange-Nielsen F (1957).  Am. Heart J.  54:59-68.  
          Jiang C, et al. (1994).  Nat. Genet.  8:141-147.  
          Jongbloed R J, et al. (1999).  Hum. Mutat.  13:301-310.  
          Kanters J (1998).  J. Cardiovasc. Electrophysiol.  9:620-624.  
          Keating M, et al. (1991).  Science  252:704-706.  
          Kinszler K W, et al. (1991).  Science  251:1366-1370.  
          Kubo T, et al. (1988).  FEBS Lett.  241:119.  
          Larsen L A, et al. (1999a).  Hum. Mutat.  13:318-327.  
          Larsen L A, et al. (1999b).  Eur. J. Hum. Genet.  7:724-728.  
          Li H, et al. (1998).  Circulation  97:1264-1269.  
          Lipshutz R J, et al. (1995).  Biotechniques  19:442-447.  
          Lockhart D J, et al. (1996).  Nature Biotechnology  19:1675-1680.  
          Ludwig J, et al. (1994).  EMBO J.  13:4451-4458.  
          MacKinnon R, et al. (1998).  Science  280:106-109.  
          Makita N, et al. (1998).  FEBS Lett.  423:5-9.  
          Matteucci M D and Caruthers M H (1981).  J. Am. Chem. Soc.  103:3185.  
          Metzger D, et al. (1988).  Nature  334:31-36.  
          Modrich P (1991).  Ann. Rev. Genet.  25:229-253.  
          Mohammad-Panah R, et al. (1999).  Am. J. Hum. Genet.  64:1015-1023.  
          Moss A, et al. (1991).  Circulation  84:1136-1144.  
          Murray A, et al. (1999).  Circulation  100: 1077-1084.  
          Newton C R, et al. (1989).  Nucl. Acids Res.  17:2503-2516.  
          Neyroud N, et al. (1997).  Nat. Genet.  15:186-189.  
          Neyroud N, et al. (1998).  Eur. J. Hum. Genet.  6:129-133.  
          Neyroud N, et al. (1999).  Circ. Res.  84:290-297.  
          Novack D F, et al. (1986).  Proc. Natl. Acad. Sci. USA  83:586-590.  
          Orita M, et al. (1989).  Proc. Natl. Acad. Sci. USA  86:2766-2770.  
          Priori S G, et al. (1998).  Circulation  97:2420-2425.  
          Priori S G, et al. (1999).  Circulation  99:529-533.  
          Romano C, et al. (1963).  Clin. Pediatr.  45:656-683.  
          Ruano G and Kidd K K (1989).  Nucl. Acids Res.  12:8392.  
          Russell M W, et al. (1996).  Hum. Mol. Genet.  5:1319-1324.  
          Saarinen K, et al. (1998).  Hum. Mutat.  11:158-165.  
          Sambrook J, et al. (1989).  Molecular Cloning: A Laboratory Manual,  2nd Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).  
          Sanguinetti M C, et al. (1996a).  Nature  384:80-83.  
          Sanguinetti M C, et al. (1996b).  Proc. Natl. Acad. Sci. USA  93:2208-2212.  
          Satler C A, et al. (1996).  Am. J. Med. Genet.  65:27-35.  
          Satler C A, et al. (1998).  Hum. Genet.  102:265-272.  
          Schott J, et al. (1995).  Am. J. Hum. Genet.  57:1114-1122.  
          Schulze-Bahr E, et al. (1995).  N. Engl. J. Med.  333:1783-1784.  
          Schulze-Bahr E, et al. (1997).  Nat. Genet.  17:267-268.  
          Schwartz P J, et al. (1975).  Am. Heart J.  89:378-390.  
          Schwartz P J, et al. (1995).  Circulation  92:3381-3386.  
          Sesti F and Goldstein S A (1998).  J. Gen. Physiol.  112:651-663.  
          Shalaby F Y, et al. (1997).  Circulation  96:1733-1736.  
          Sheffield V C, et al. (1989).  Proc. Natl. Acad. Sci. USA  86:232-236.  
          Sheffield V C, et al. (1991).  Am. J. Hum. Genet.  49:699-706.  
          Shenk T E, et al. (1975).  Proc. Natl. Acad. Sci. USA  72:989-993.  
          Shoemaker D D, et al. (1996).  Nature Genetics  14:450-456.  
          Splawski I, et al. (1997a).  Nat. Genet.  17:338-340.  
          Splawski I, et al. (1997b).  N. Engl. J. Med.  336:1562-1567.  
          Splawski I, et al. (1998).  Genomics  51:86-97.  
          Tanaka T, et al. (1997).  Circulation  95:565-567.  
          Tyson J, et al. (1997).  Hum. Mol. Genet.  6:2179-2185.  
          van den Berg M H, et al. (1997).  Hum. Genet.  100:356-361.  
          Vetter D E, et al. (1996).  Neuron  17:1251-1264.  
          Vincent G M, et al. (1992).  N. Engl. J. Med.  327:846-852.  
          Wang D W (1996).  Proc. Natl. Acad. Sci. USA  93:13200-13205.  
          Wang Q, et al. (1995a).  Cell  80:805-811.  
          Wang Q, et al. (1995b).  Hum. Mol. Genet.  4:1603-1607.  
          Wang Q, et al. (1996a).  Nat. Genet.  12:17-23.  
          Wang Q, et al. (1996b).  Genomics  34:9-16.  
          Wang Z, et al. (1999).  J. Cardiovasc. Electrophysiol.  10:817-826.  
          Ward O C (1964).  J. Ir. Med. Assoc.  54:103-106.  
          Wartell R M, et al. (1990).  Nucl. Acids Res.  18:2699-2705.  
          Wattanasirichaigoon D, et al. (1999).  Am. J. Med. Genet.  86:470-476.  
          Wei J, et al. (1999).  Circulation  99:3165-3171.  
          White M B, et al. (1992).  Genomics  12:301-306.  
          Wollnik B, et al. (1997).  Hum. Mol. Genet.  6:1943-1949.  
          Yoshida H, et al. (1999).  J. Cardiovasc. Electrophysiol.  10:1262-1270.  
          Zhou Z, et al. (1998).  J. Biol. Chem.  21:21061-21066.  
          Hitzeman et al., EP 73,675A.  
          European Patent Application Publication No. 0332435.  
          U.S. Pat. No. 5,436,146  
          U.S. Pat. No. 5,691,198  
          U.S. Pat. No. 5,735,500  
          U.S. Pat. No. 5,747,469