Patent Application: US-84012501-A

Abstract:
long qt syndrome 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:
the present invention relates to alterations in the kvlqt1 , herg , scn5a , kcne1 and kcne2 genes and methods for detecting such alterations . the alterations in the kvlqt1 , herg , scn5a , kcne1 and kcne2 genes include mutations and polymorphisms . included among the mutations are frameshift , nonsense , splice , regulatory and missense mutations . any method which is capable of detecting the mutations and polymorphisms described herein can be used . such methods include , but are not limited to , dna sequencing , allele - specific probing , mismatch detection , single stranded conformation polymorphism detection and allele - specific pcr amplification . kvlqt1 , herg , scn5a , kcne1 and kcne2 mutations cause increased risk for lqts . many different mutations occur in kvlqt1 , herg , scn5a , kcne1 and kcne2 . in order to detect the presence of alterations in the kvlqt1 , herg , scn5a , kcne1 and kcne2 genes , a biological sample such as blood is prepared and analyzed for the presence or absence of a given alteration of kvlqt1 , herg , scn5a , kcne1 or kcne2 . in order to detect the increased risk for lqts or for the lack of such increased risk , a biological sample is prepared and analyzed for the presence or absence of a mutant allele of kvlqt1 , herg , scn5a , kcne1 or kcne2 . results of these tests and interpretive information are returned to the health care provider for communication to the tested individual . such diagnoses may be performed by diagnostic laboratories or , alternatively , diagnostic kits are manufactured and sold to health care providers or to private individuals for self - diagnosis . the presence of hereditary lqts may be ascertained by testing any tissue of a human for mutations of the kvlqt1 , herg , scn5a , kcne1 or kcne2 gene . for example , a person who has inherited a germline herg mutation would be prone to develop lqts . this can be determined by testing dna from any tissue of the person &# 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 , scnsa , kcnei or kcne2 allele , whether , for example , by point mutation or deletion , can be detected by any of the means discussed herein . there are several methods that can be used to detect dna sequence variation . direct dna sequencing , either manual sequencing or automated fluorescent sequencing can detect sequence variation . another approach is the single - stranded conformation polymorphism assay ( sscp ) ( orita et al ., 1989 ). this method does not detect all sequence changes , especially if the dna fragment size is greater than 200 bp , but can be optimized to detect most dna sequence variation . the reduced detection sensitivity is a disadvantage , but the increased throughput possible with sscp makes it an attractive , viable alternative to direct sequencing for mutation detection on a research basis . the fragments which have shifted mobility on sscp gels are then sequenced to determine the exact nature of the dna sequence variation . other approaches based on the detection of mismatches between the two complementary dna strands include clamped denaturing gel electrophoresis ( cdge ) ( sheffield et al ., 1991 ), heteroduplex analysis ( ha ) ( white et al ., 1992 ) and chemical mismatch cleavage ( cmc ) ( grompe et al ., 1989 ). none of the methods described above will detect large deletions , duplications or insertions , nor will they detect a regulatory mutation which affects transcription or translation of the protein . other methods which might detect these classes of mutations such as a protein truncation assay or the asymmetric assay , detect only specific types of mutations and would not detect missense mutations . a review of currently available methods of detecting dna sequence variation can be found in a recent review by grompe ( 1993 ). once a mutation is known , an allele specific detection approach such as allele specific oligonucleotide ( aso ) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation . such a technique can utilize probes which are labeled with gold nanoparticles to yield a visual color result ( elghanian et al ., 1997 ). a rapid preliminary analysis to detect polymorphisms in dna sequences can be performed by looking at a series of southern blots of dna cut with one or more restriction enzymes , preferably with a large number of restriction enzymes . each blot contains a series of normal individuals and a series of lqts cases . southern blots displaying hybridizing fragments ( differing in length from control dna when probed with sequences near or including the herg locus ) indicate a possible mutation . if restriction enzymes which produce very large restriction fragments are used , then pulsed field gel electrophoresis ( pfge ) is employed . detection of point mutations may be accomplished by molecular cloning of the kvlqt1 , herg , scn5a , kcne1 or kcne2 alleles and sequencing the alleles using techniques well known in the art . also , the gene or portions of the gene may be amplified , e . g ., by pcr or other amplification technique , and the amplified gene or amplified portions of the gene may be sequenced . there are six well known methods for a more complete , yet still indirect , test for confirming the presence of a susceptibility allele : 1 ) single stranded conformation analysis ( sscp ) ( orita et al ., 1989 ); 2 ) denaturing gradient gel electrophoresis ( dgge ) ( wartell et al ., 1990 ; sheffield et al ., 1989 ); 3 ) rnase protection assays ( finkelstein et al ., 1990 ; kinszler et al ., 1991 ); 4 ) allele - specific oligonucleotides ( asos ) ( conner et al ., 1983 ); 5 ) the use of proteins which recognize nucleotide mismatches , such as the e . coli muts protein ( modrich , 1991 ); and 6 ) allele - specific pcr ( ruano and kidd , 1989 ). for allele - specific pcr , primers are used which hybridize at their 3 ′ ends to a particular kvlqt1 , herg , scn5a , kcne1 or kcne2 mutation . if the particular mutation is not present , an amplification product is not observed . amplification refractory mutation system ( arms ) can also be used , as disclosed in european patent application publication no . 0332435 and in newton et al ., 1989 . insertions and deletions of genes can also be detected by cloning , sequencing and amplification . in addition , restriction fragment length polymorphism ( rflp ) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment . such a method is particularly useful for screening relatives of an affected individual for the presence of the mutation found in that individual . other techniques for detecting insertions and deletions as known in the art can be used . in the first three methods ( sscp , dgge and rnase protection assay ), a new electrophoretic band appears . sscp detects a band which migrates differentially because the sequence change causes a difference in single - strand , intramolecular base pairing . rnase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments . dgge detects differences in migration rates of mutant sequences compared to wild - type sequences , using a denaturing gradient gel . in an allele - specific oligonucleotide assay , an oligonucleotide is designed which detects a specific sequence , and the assay is performed by detecting the presence or absence of a hybridization signal . in the muts assay , the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild - type sequences . mismatches , according to the present invention , are hybridized nucleic acid duplexes in which the two strands are not 100 % complementary . lack of total homology may be due to deletions , insertions , inversions or substitutions . mismatch detection can be used to detect point mutations in the gene or in its mrna product . while these techniques are less sensitive than sequencing , they are simpler to perform on a large number of samples . an example of a mismatch cleavage technique is the rnase protection method . in the practice of the present invention , the method involves the use of a labeled riboprobe which is complementary to the human wild - type kvlqt1 , herg , scn5a , kcnei or kcne2 gene coding sequence . the riboprobe and either mrna or dna isolated from the person are annealed ( hybridized ) together and subsequently digested with the enzyme rnase a which is able to detect some mismatches in a duplex rna structure . if a mismatch is detected by rnase a , it cleaves at the site of the mismatch . thus , when the annealed rna preparation is separated on an electrophoretic gel matrix , if a mismatch has been detected and cleaved by rnase a , an rna product will be seen which is smaller than the full length duplex rna for the riboprobe and the mrna or dna . the riboprobe need not be the full length of the mrna or gene but can be a segment of either . if the riboprobe comprises only a segment of the mrna or gene , it will be desirable to use a number of these probes to screen the whole mrna sequence for mismatches . in similar fashion , dna probes can be used to detect mismatches , through enzymatic or chemical cleavage . see , e . g ., cotton et al ., 1988 ; shenk et al ., 1975 ; novack et al ., 1986 . alternatively , mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes . see , e . g ., cariello , 1988 . with either riboprobes or dna probes , the cellular mrna or dna which might contain a mutation can be amplified using pcr ( see below ) before hybridization . changes in dna of the kvlqt1 , herg , scn5a , kcne1 or kcne2 gene can also be detected using southern hybridization , especially if the changes are gross rearrangements , such as deletions and insertions . dna sequences of the kvlqt1 , herg , scn5a , kcne1 or kcne2 gene which have been amplified by use of pcr may also be screened using allele - specific probes . these probes are nucleic acid oligomers , each of which contains a region of the gene sequence harboring a known mutation . for example , one oligomer may be about 30 nucleotides in length , corresponding to a portion of the gene sequence . by use of a battery of such allele - specific probes , pcr amplification products can be screened to identify the presence of a previously identified mutation in the gene . hybridization of allele - specific probes with amplified kvlqt1 , herg , scn5a , kcne1 or kcne2 sequences can be performed , for example , on a nylon filter . hybridization to a particular probe under high stringency hybridization conditions indicates the presence of the same mutation in the tissue as in the allele - specific probe . the newly developed technique of nucleic acid analysis via microchip technology is also applicable to the present invention . in this technique , literally thousands of distinct oligonucleotide probes are built up in an array on a silicon chip . nucleic acid to be analyzed is fluorescently labeled and hybridized to the probes on the chip . it is also possible to study nucleic acid - protein interactions using these nucleic acid microchips . using this technique one can determine the presence of mutations or even sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest . the method is one of parallel processing of many , even thousands , of probes at once and can tremendously increase the rate of analysis . several papers have been published which use this technique . some of these are hacia et al ., 1996 ; shoemaker et al ., 1996 ; chee et al ., 1996 ; lockhart et al ., 1996 ; derisi et al ., 1996 ; lipshutz et al ., 1995 . this method has already been used to screen people for mutations in the breast cancer gene brca1 ( hacia et al ., 1996 ). this new technology has been reviewed in a news article in chemical and engineering news ( borman , 1996 ) and been the subject of an editorial ( editorial , nature genetics , 1996 ). also see fodor ( 1997 ). the most definitive test for mutations in a candidate locus is to directly compare genomic kvlqt1 , herg , scn5a , kcne1 or kcne2 sequences from patients with those from a control population . alternatively , one could sequence messenger rna after amplification , e . g ., by pcr , thereby eliminating the necessity of determining the exon structure of the candidate gene . mutations from patients falling outside the coding region of kvlqt1 , herg , scn5a , kcne1 or kcne2 can be detected by examining the non - coding regions , such as introns and regulatory sequences near or within the genes an early indication that mutations in noncoding regions are important may come from northern blot experiments that reveal messenger rna molecules of abnormal size or abundance in patients as compared to control individuals . alteration of kvlqt1 , herg , scn5a , kcne1 or kcne2 mrna expression can be detected by any techniques known in the art . these include northern blot analysis , pcr amplification and rnase protection . diminished mrna expression indicates an alteration of the wild - type gene . alteration of wild - type genes can also be detected by screening for alteration of wild - type kvlqt1 , herg , scn5a , kcne1 or kcne2 protein . for example , monoclonal antibodies immunoreactive with herg can be used to screen a tissue . lack of cognate antigen would indicate a mutation . antibodies specific for products of mutant alleles could also be used to detect mutant gene product . such immunological assays can be done in any convenient formats known in the art . these include western blots , immunohistochemical assays and elisa assays . any means for detecting an altered kvlqt1 , herg , scn5a , kcne1 or kcne2 protein can be used to detect alteration of wild - type kvlqt1 , herg , scn5a , kcne1 or kcne2 genes . functional assays , such as protein binding determinations , can be used . in addition , assays can be used which detect kvlqt1 , herg , scn5a , kcne1 or kcne2 biochemical function . finding a mutant kvlqt1 , herg , scn5a , kcne1 or kcne2 gene product indicates alteration of a wild - type kvlqt1 , herg , scn5a , kcne1 or kcne2 gene . mutant kvlqt1 , herg , scn5a , kcne1 or kcne2 genes or gene products can also be detected in other human body samples , such as serum , stool , urine and sputum . the same techniques discussed above for detection of mutant genes or gene products in tissues can be applied to other body samples . by screening such body samples , a simple early diagnosis can be achieved for hereditary lqts . initially , the screening method involves amplification of the relevant kvlqt1 , herg , scn5a , kcne1 or kcne2 sequence . in another preferred embodiment of the invention , the screening method involves a non - pcr based strategy . such screening methods include two - step label amplification methodologies that are well known in the art . both pcr and non - pcr based screening strategies can detect target sequences with a high level of sensitivity . further details of these methods are briefly presented below and further descriptions can be found in pct published application wo 96 / 05306 , incorporated herein by reference . the most popular method used today is target amplification . here , the target nucleic acid sequence is amplified with polymerases . one particularly preferred method using polymerase - driven amplification is the polymerase chain reaction ( pcr ). the polymerase chain reaction and other polymerase - driven amplification assays can achieve over a million - fold increase in copy number through the use of polymerase - driven amplification cycles . once amplified , the resulting nucleic acid can be sequenced or used as a substrate for dna probes . when the probes are used to detect the presence of the target sequences , the biological sample to be analyzed , such as blood or serum , may be treated , if desired , to extract the nucleic acids . the sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence ; e . g . denaturation , restriction digestion , electrophoresis or dot blotting . the targeted region of the analyte nucleic acid usually must be at least partially single - stranded to form hybrids with the targeting sequence of the probe . if the sequence is naturally single - stranded , denaturation will not be required . however , if the sequence is double - stranded , the sequence will probably need to be denatured . denaturation can be carried out by various techniques known in the art . analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte . the region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of the genes . therefore , high stringency conditions are desirable in order to prevent false positives . however , conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome . the stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure , including temperature , ionic strength , base composition , probe length , and concentration of formamide . under certain circumstances , the formation of higher order hybrids , such as triplexes , quadraplexes , etc ., may be desired to provide the means of detecting target sequences . detection , if any , of the resulting hybrid is usually accomplished by the use of labeled probes . alternatively , the probe may be unlabeled , but may be detectable by specific binding with a ligand which is labeled , either directly or indirectly . suitable labels , and methods for labeling probes and ligands are known in the art , and include , for example , radioactive labels which may be incorporated by known methods ( e . g ., nick translation , random priming or kinasing ), biotin , fluorescent groups , chemiluminescent groups ( e . g ., dioxetanes , particularly triggered dioxetanes ), enzymes , antibodies nd the like . variations of this basic scheme known in the art , and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and / or that amplify the signal from the labeled moiety . a number of these variations are well known . as noted above , non - pcr based screening assays are also contemplated in this invention . this procedure hybridizes a nucleic acid probe ( or an analog such as a methyl phosphonate backbone replacing the normal phosphodiester ), to the low level dna target . this probe may have an enzyme covalently linked to the probe , such that the covalent linkage does not interfere with the specificity of the hybridization . this enzyme - probe - conjugate - target nucleic acid complex can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection . enzymatic activity is observed as a change in color development or luminescent output resulting in a 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 . two - step label amplification methodologies are known in the art . these assays work on the principle that a small ligand ( such as digoxigenin , biotin , or the like ) is attached to a nucleic acid probe capable of specifically binding the target gene . allele specific probes are also contemplated within the scope of this example . in one example , the small ligand attached to the nucleic acid probe is specifically recognized by an antibody - enzyme conjugate . in one embodiment of this example , digoxigenin is attached to the nucleic acid probe . hybridization is detected by an antibody - alkaline phosphatase conjugate which turns over a chemiluminescent substrate . in a second example , the small ligand is recognized by a second ligand - enzyme conjugate that is capable of specifically complexing to the first ligand . a well known embodiment of this example is the biotin - avidin type of interactions . methods for labeling nucleic acid probes and their use in biotin - avidin based assays are well known . it is also contemplated within the scope of this invention that the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting the gene or genes . thus , in one example to detect the presence of kvlqt1 in a cell sample , more than one probe complementary to kvlqt1 is employed and in particular the number of different probes is alternatively 2 , 3 , or 5 different nucleic acid probe sequences . in another example , to detect the presence of mutations in the kvlqt1 gene sequence in a patient , more than one probe complementary to kvlqt1 is employed where the cocktail includes probes capable of binding to the allele - specific mutations identified in populations of patients with alterations in kvlqt1 . in this embodiment , any number of probes can be used . large amounts of the polynucleotides of the present invention may be produced by replication in a suitable host cell . natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs , usually dna constructs , capable of introduction into and replication in a prokaryotic or eukaryotic cell . usually the polynucleotide constructs will be suitable for replication in a unicellular host , such as yeast or bacteria , but may also be intended for introduction to ( with and without integration within the genome ) cultured mammalian or plant or other eukaryotic cell lines . the purification of nucleic acids produced by the methods of the present invention are described , e . g ., in sambrook et al ., 1989 or ausubel et al ., 1992 . the polynucleotides of the present invention may also be produced by chemical synthesis , e . g ., by the phosphoramidite method described by beaucage and caruthers ( 1981 ) or the triester method according to matteucci and caruthers ( 1981 ) and may be performed on commercial , automated oligonucleotide synthesizers . a double - stranded fragment may be obtained from the single - stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using dna polymerase with an appropriate primer sequence . polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host , including the intended polynucleotide fragment encoding the desired polypeptide , and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment . expression vectors may include , for example , an origin of replication or autonomously replicating sequence ( ars ) and expression control sequences , a promoter , an enhancer and necessary processing information sites , such as ribosome - binding sites , rna splice sites , polyadenylation sites , transcriptional terminator sequences , and mrna stabilizing sequences . such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed , for example , in sambrook et al . ( 1989 ) or ausubel et al . ( 1992 ). an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host , and may include , when appropriate , those naturally associated with the kvlqt1 or other gene . examples of workable combinations of cell lines and expression vectors are described in sambrook et al . ( 1989 ) or ausubel et al . ( 1992 ); see also , e . g ., metzger et al . ( 1988 ). many useful vectors are known in the art and may be obtained from such vendors as stratagene , new england biolabs , promega biotech , and others . promoters such as the trp , lac and phage promoters , trna promoters and glycolytic enzyme promoters may be used in prokaryotic hosts . useful yeast promoters include promoter regions for metallothionein , 3 - phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde - 3 - phosphate dehydrogenase , enzymes responsible for maltose and galactose utilization , and others . vectors and promoters suitable for use in yeast expression are further described in hitzeman et al ., ep 73 , 675a . appropriate non - native mammalian promoters might include the early and late promoters from sv40 ( fiers et al ., 1978 ) or promoters derived from murine molony leukemia virus , mouse tumor virus , avian sarcoma viruses , adenovirus ii , bovine papilloma virus or polyoma . insect promoters may be derived from baculovirus . in addition , the construct may be joined to an amplifiable gene ( e . g ., dhfr ) so that multiple copies of the gene may be made . for appropriate enhancer and other expression control sequences , see also enhancers and eukaryotic gene expression , cold spring harbor press , cold spring harbor , new york ( 1983 ). see also , e . g ., u . s . pat . nos . 5 , 691 , 198 ; 5 , 735 , 500 ; 5 , 747 , 469 and 5 , 436 , 146 . while such expression vectors may replicate autonomously , they may also replicate by being inserted into the genome of the host cell , by methods well known in the art . expression and cloning vectors will likely contain a selectable marker , a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector . the presence of this gene ensures growth of only those host cells which express the inserts . typical selection genes encode proteins that a ) confer resistance to antibiotics or other toxic substances , e . g . ampicillin , neomycin , methotrexate , etc ., b ) complement auxotrophic deficiencies , or c ) supply critical nutrients not available from complex media , e . g ., the gene encoding d - alanine racemase for bacilli . the choice of the proper selectable marker will depend on the host cell , and appropriate markers for different hosts are well known in the art . the vectors containing the nucleic acids of interest can be transcribed in vitro , and the resulting rna introduced into the host cell by well - known methods , e . g ., by injection ( see , kubo et al . ( 1988 )), or the vectors car be introduce 1 directly into host cells by methods well known in the art , which vary depending on the type of cellular host , including electroporation ; transfection employing calcium chloride , rubidium chloride calcium phosphate , deae - dextran , or other substances ; microprojectile bombardment ; lipofection ; infection ( where the vector is an infectious agent , such as a retroviral genome ); and other methods . see generally , sambrook et al . ( 1989 ) and ausubel et al . ( 1992 ). the introduction of the polynucleotides into the host cell by any method known in the art , including , inter alia , those described above , will be referred to herein as “ transformation .” the cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells . large quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the kvlqt1 nucleic acid or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells . the most commonly used prokaryotic hosts are strains of escherichia coli , although other prokaryotes , such as bacillus subtilis or pseudomonas may also be used . mammalian or other eukaryotic host cells , such as those of yeast , filamentous fungi , plant , insect , or amphibian or avian species , may also be useful for production of the proteins of the present invention . propagation of mammalian cells in culture is per se well known . see , jakoby and pastan ( eds .) ( 1979 ). examples of commonly used mammalian host cell lines are vero and hela cells , chinese hamster ovary ( cho ) cells , and wi38 , bhk , and cos cell lines , although it will be appreciated by the skilled practitioner that other cell lines may be appropriate , e . g ., to provide higher expression , desirable glycosylation patterns , or other features . an example of a commonly used insect cell line is sf9 . clones are selected by using markers depending on the mode of the vector construction . the marker may be on the same or a different dna molecule , preferably the same dna molecule . in prokaryotic hosts , the transformant may be selected , e . g ., by resistance to ampicillin , tetracycline or other antibiotics . production of a particular product based on temperature sensitivity may also serve as an appropriate marker . prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention , but also , for example , in studying the characteristics of kvlqt1 or other polypeptides . the probes and primers based on the kvlqt1 or other gene sequences disclosed herein are used to identify homologous kvlqt1 or other gene sequences and proteins in other species . these gene sequences and proteins are used in the diagnostic / prognostic , therapeutic and drug screening methods described herein for the species from which they have been isolated . the studies described in the examples below resulted in the determination of many novel mutations . previous studies had defined 126 distinct disease causing mutations in the lqts genes kvlqt1 , herg , scn5a , kcne1 and kcne2 ( wang q . et al ., 1996a ; curran et al ., 1995 ; wang et al ., 1995a ; splawski et al ., 1997a ; abbott et al ., 1999 ; chouabe et al ., 1997 ; wollnik et al ., 1997 ; neyroud et al ., 1997 ; splawski et al ., 1997b ; tyson et al ., 1997 ; schulze - bahr et al ., 1997 ; priori et al ., 1999 ; splawski et al ., 1998 ; wang et al ., 1995b ; russell et al ., 1996 ; neyroud et al ., 1998 ; neyroud et al ., 1999 ; donger et al ., 1997 ; tanaka et al ., 1997 ; jongbloed et al ., 1999 ; priori et al ., 1998 ; itoh et al ., 1998a ; itoh et al ., 1998b ; mohammad - panah et al ., 1999 ; saarinen et al ., 1998 ; ackerman et al ., 1998 ; berthet et al ., 1999 ; kanters , 1998 ; van den berg et al ., 1997 ; dausse et al ., 1996 ; benson et al ., 1996 ; akimoto et al ., 1998 ; satler et al ., 1996 ; satler et al ., 1998 ; makita et al ., 1998 , an et al ., 1998 ; schulze - bahr et al ., 1995 ; duggal et al ., 1998 ; chen q . et al ., 1999 ; li et al ., 1998 ; wei et al ., 1999 ; larsen et al ., 1999a ; bianchi et al ., 1999 ; ackerman et al ., 1999a ; ackerman et al ., 1999b ; murray et al ., 1999 ; larsen et al ., 1999b ; yoshida et al ., 1999 ; wattanasirichaigoon et al ., 1999 ; bezzina et al ., 1999 ; hoorntje et al ., 1999 ). the sequence of each wild - type gene has been published . the kvlqt1 can be found in splawski et al . ( 1998 ) and the coding region of the cdna is shown herein as seq id no : 1 and the encoded kvlqt1 is shown as seq id no : 2 . scn5a was reported by gellens et al . ( 1992 ) and its sequence is provided by genbank accession no . nm — 000335 . the coding sequence of scn5a is shown herein as seq id no : 3 and the encoded scn5a is shown as seq id no : 4 . most of the mutations were found in kvlqt1 ( yoshida et al ., 1999 ) and herg ( itoh et al ., 1998b ), and fewer in scnsa ( wang q . et al ., 1996a ), kcnei ( jiang et al ., 1994 ) and kcne2 ( ward , 1964 ). these mutations were identified in regions with known intron / exon structure , primarily the transmembrane and pore domains . in this study , we screened 262 individuals with lqts for mutations in all known arrhythmia genes . we identified 134 mutations , 80 of which were novel . together with 43 mutations reported in our previous studies , we have now identified 177 mutations in these 262 lqts individuals ( 68 %). the failure to identify mutations in 32 % of the individuals may result from phenotypic errors , incomplete sensitivity of sscp or presence of mutations in regulatory sequences . however , it is also clear that additional lqts genes await discovery ( jiang et al ., 1994 ; schott et al ., 1995 ). missense mutations were most common ( 72 %), followed by frameshift mutations ( 10 %), in - frame deletions , nonsense and splice site mutations ( 5 - 7 % each ). most mutations resided in intracellular ( 52 %) and transmembrane ( 30 %) domains ; 12 % were found in pore and 6 % in extracellular segments . one hundred one of the 129 distinct lqts mutations ( 78 %) were identified in single families or individuals . most of the 177 mutations were found in kvlqti ( 75 or 42 %) and herg ( 80 or 45 %). these two genes accounted for 87 % of the identified mutations , while mutations in scn5a ( 14 or 8 %), kcne1 ( 5 or 3 %) and kcne2 ( 3 or 2 %) accounted for the other 13 %. multiple mutations were found in regions encoding s5 , s5 / p , p and s6 of kvlqt1 and herg . the p region of potassium channels forms the outer pore and contains the selectivity filter ( doyle et al ., 1998 ). transmembrane segment 6 , corresponding to the inner helix of kcsa , forms the inner 2 / 3 of the pore . this structure is supported by the s5 transmembrane segment , corresponding to the outer helix of kcsa , and is conserved from prokaryotes to eukaryotes (( mackinnon et al ., 1998 ). mutations in these regions will likely disrupt potassium transport . many mutations were identified in the c - termini of kvlqt1 and herg . changes in the c - terminus of herg could lead to anomalies in tetramerization as it has been proposed that the c - terminus of eag , which is related to herg , is involved in this process ( ludwig et al , 1994 ). multiple mutations were also identified in regions that were different for kvlqt1 and herg . in kvlqt1 , multiple mutations were found in the sequences coding for the s2 / s3 and s4 / s5 linkers . coexpression of s2 / s3 mutants with wild - type kvlqt1 in xenopus oocytes led to simple loss of function or dominant - negative effect without significantly changing the biophysical properties of i ks channels ( chouabe et al ., 1997 ; shalaby et al ., 1997 ; wang et al ., 1999 ). on the other hand , s4 / s5 mutations altered the gating properties of the channels and modified kvlqt1 interactions with mink subunits ( wang et al ., 1999 ; franqueza et al ., 1999 ). in herg , more than 20 mutations were identified in the n - terminus . herg channels lacking this region deactivate faster and mutations in the region had a similar effect ( chen j . et al ., 1999 ). mutations in kcne1 and kcne2 , encoding mink and mirp1 , 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 mir p1 mutant , involved in clarithromyocin - induced arrhythmia , increased channel blockade by the antibiotic ( abbott et al ., 1999 ). mutations in scn5a , the sodium channel α - subunit responsible for cardiac i na , destabilized the inactivation gate causing delayed channel inactivation and dispersed reopenings ( bennett et al ., 1995 ; dumaine et al ., 1996 ; wei et al ., 1999 ; wang d w et al ., 1996 ). one scn5a mutant affected the interactions with the sodium channel β - subunit ( an et al ., 1998 ). it is interesting to note that probands with kcne1 and kcne2 mutations were older and had shorter qtc than probands with the other genotypes . the significance of these differences is unknown , however , as the number of probands with kcne1 and kcne2 genotypes was small . this catalogue of mutations will facilitate genotype - phenotype analyses . it also has clinical implications for presymptomatic diagnosis and , in some cases , for therapy . patients with mutations in kvlqt1 , herg , kcne1 and kcne2 , for example , may benefit from potassium therapy ( compton et al ., 1996 ). sodium channel blockers , on the other hand , might be helpful in patients with scn5a mutations ( schwartz et al . ( 1995 ). the identification of mutations is of importance for ion channel studies as well . the expression of mutant channels in heterologous systems can reveal how structural changes influence the behavior of the channel or how mutations affect processing ( zhou et al ., 1998 ; furutani et al ., 1999 ). these studies improve our understanding of channel function and provide insights into mechanisms of disease . finally , mutation identification will contribute to the development of genetic screening for arrhythmia susceptibility . the present invention is described by reference to the following examples , which are offered by way of illustration and are not intended to limit the invention in any manner . standard techniques well known in the art or the techniques specifically described in the examples were utilized . 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 depointes , 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 . to determine the spectrum of lqts mutations , we used sscp ( single stand conformation polymorphism ) and dna sequence analyses to screen 262 unrelated individuals with lqts . seventeen primer pairs were used to screen kvlqt1 ( splawski et al ., 1998 ), twenty - one primer pairs were used for herg ( splawski et al ., 1998 ) and three primer pairs were used for kcne1 ( splawski et al ., 1997a ) and kcne2 ( abbott et al ., 1999 ). thirty - three primer pairs ( wang q . et al ., 1996b ) were used in sscp analysis to screen all scn5a exons in 50 individuals with suspected abnormalities in i na . exons 23 - 28 , in which mutations were previously identified , were screened in all 262 individuals . gender , age , qtc and presence of symptoms are summarized in table 1 . the average age at ascertainment was 29 with a corrected qt interval of 492 ms . seventy - five percent had a history of symptoms and females predominated with an ˜ 2 : 1 ratio . although the numbers were small , corrected qt intervals for individuals harboring kcne1 and kcne2 mutations were shorter at 457 ms . the sscp analyses revealed many mutations . kvlqt1 mutations associated with lqts were identified in 52 individuals ( fig1 and table 2 ). twenty of the mutations were novel . herg mutations were identified in 68 lqts individuals ( fig2 and table 3 ). fifty - two of these mutations were novel . scn5a mutations were identified in eight cases ( fig3 and table 4 ). five of the mutations were novel . three novel kcne1 mutations were identified ( fig4 and table 5 ) and three mutations were identified in kcne2 fig5 and table 6 ) ( abbott et al ., 1999 ). none of the kvlqt1 , herg , scn5a , kcne1 and kcne2 mutations was observed in 400 control chromosomes . [ 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 † r73sf / 31 n - terminus 2 1 this g232c † a78p n - terminus 2 1 this dupl234 - a83fs / 37 n - terminus 2 1 this 250 † c241t † q81x n - terminus 2 1 this t257g † l86r n - terminus 2 1 this insc422 - p141sf / 2 n - terminus 3 1 this 423 † insc453 - p151fs / n - terminus 3 1 this 454 † 179 dupl558 - 600 l200sf / n - terminus 4 1 hoorntje 144 et al ., 1999 insc724 - p241fs / 89 n - terminus 4 1 this 725 † 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 - del500 - s3 6 1 curran et al ., 1524 508 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 - g ) bahr et al ., 1995 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 - delk638 s6 7 1 this 1915 † c1920a f640l s6 7 1 jongbloed et al ., 1999 a1933t † m645l s6 7 1 this del1951 - l650fs / 2 s6 8 1 itoh et al ., 1952 1998a g2044t † e682x s6 / cnbd 8 1 this c2173t q725x s6 / cnbd 9 1 itoh et al ., 1998a inst2218 - h739fs / 63 s6 / cnbd 9 1 this 2219 † c2254t † r752w s6 / cnbd 9 1 this dupl2356 - v796fs / 22 cnbd 9 1 itoh et al ., 2386 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 - g925fs / 13 c - terminus 12 1 this 2776 † del2906 † p968fs / 4 c - terminus 12 1 this del2959 - p986fs / c - terminus 12 1 this 2960 † 130 c3040t † r1014x c - terminus 13 2 this del3094 † g1031fs / c - terminus 13 1 this 24 insg3107 - g1036fs / c - terminus 13 1 berthet 3108 82 et al ., 1999 insc3303 - p1101fs c - terminus 14 1 this 3304 † [ 0066 ] table 4 summary of all scn5a mutations number of nucleotide change coding effect position exon rw 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 - del1505 - diii / div 26 4 wang et al ., 1995a ; wang et 4519 1507 al ., 1995b del4850 - delf1617 div / s3 / s4 28 1 this 4852 † 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 g536oa † s1787n c - terminus 28 1 this a5369g d1790g c - terminus 28 1 an et al ., 1998 instga insd1795 - c - terminus 28 1 bezzina et al ., 1999 5385 - 5386 1796 [ 0067 ] table 5 summary of all kcne1 mutations * number nucleotide coding 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 - l51h s1 3 1 jln bianchi et 152at 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 [ 0068 ] table 6 summary of all kcne2 mutations number nucleotide coding of change effect position exon families study c25g q9e n - 1 1 abbott et al ., 1999 terminus 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 [ 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 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 . ackerman m j , et al . 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( 1990 ). nucl . acids res . 18 : 2699 - 2705 . wattanasirichaigoon d , et al . ( 1999 ). am . j . med genet . 86 : 470 - 476 . atg gcc gcg gcc tcc tcc ccg ccc agg gcc gag agg aag cgc tgg ggt 48 tgg ggc cgc ctg cca ggc gcc cgg cgg ggc agc gcg ggc ctg gcc aag 96 aag tgc ccc ttc tcg ctg gag ctg gcg gag ggc ggc ccg gcg ggc ggc 144 gcg ctc tac gcg ccc atc gcg ccc ggc gcc cca ggt ccc gcg ccc cct 192 gcg tcc ccg gcc gcg ccc gcc gcg ccc cca gtt gcc tcc gac ctt ggc 240 ccg cgg ccg ccg gtg agc cta gac ccg cgc gtc tcc atc tac agc acg 288 cgc cgc ccg gtg ttg gcg cgc acc cac gtc cag ggc cgc gtc tac aac 336 arg arg pro val leu ala arg thr his val gln gly arg val tyr asn ttc ctc gag cgt ccc acc ggc tgg aaa tgc ttc gtt tac cac ttc gcc 384 phe leu glu arg pro thr gly trp lys cys phe val tyr his phe ala gtc ttc ctc atc gtc ctg gtc tgc ctc atc ttc agc gtg ctg tcc acc 432 atc gag cag tat gcc gcc ctg gcc acg ggg act ctc ttc tgg atg gag 480 ile glu gln tyr ala ala leu ala thr gly thr leu phe trp met glu atc gtg ctg gtg gtg ttc ttc ggg acg gag tac gtg gtc cgc ctc tgg 528 tcc gcc ggc tgc cgc agc aag tac gtg ggc ctc tgg ggg cgg ctg cgc 576 ttt gcc cgg aag ccc att tcc atc atc gac ctc atc gtg gtc gtg gcc 624 tcc atg gtg gtc ctc tgc gtg ggc tcc aag ggg cag gtg ttt gcc acg 672 ser met val val leu cys val gly ser lys gly gln val phe ala thr tcg gcc atc agg ggc atc cgc ttc ctg cag atc ctg agg atg cta cac 720 gtc gac cgc cag gga ggc acc tgg agg ctc ctg ggc tcc gtg gtc ttc 768 atc cac cgc cag gag ctg ata acc acc ctg tac atc ggc ttc ctg ggc 816 ctc atc ttc tcc tcg tac ttt gtg tac ctg gct gag aag gac gcg gtg 864 aac gag tca ggc cgc gtg gag ttc ggc agc tac gca gat gcg ctg tgg 912 asn glu ser gly arg val glu phe gly ser tyr ala asp ala leu trp tgg ggg gtg gtc aca gtc acc acc atc ggc tat ggg gac aag gtg ccc 960 cag acg tgg gtc ggg aag acc atc gcc tcc tgc ttc tct gtc ttt gcc 1008 gln thr trp val gly lys thr ile ala ser cys phe ser val phe ala atc tcc ttc ttt gcg ctc cca gcg ggg att ctt ggc tcg ggg ttt gcc 1056 ctg aag gtg cag cag aag cag agg cag aag cac ttc aac cgg cag atc 1104 ccg gcg gca gcc tca ctc att cag acc gca tgg agg tgc tat gct gcc 1152 pro ala ala ala ser leu ile gln thr ala trp arg cys tyr ala ala gag aac ccc gac tcc tcc acc tgg aag atc tac atc cgg aag gcc ccc 1200 glu asn pro asp ser ser thr trp lys ile tyr ile arg lys ala pro cgg agc cac act ctg ctg tca ccc agc ccc aaa ccc aag aag tct gtg 1248 gtg gta aag aaa aaa aag ttc aag ctg gac aaa gac aat ggg gtg act 1296 cct gga gag aag atg ctc aca gtc ccc cat atc acg tgc gac ccc cca 1344 pro gly glu lys met leu thr val pro his ile thr cys asp pro pro gaa gag cgg cgg ctg gac cac ttc tct gtc gac ggc tat gac agt tct 1392 gta agg aag agc cca aca ctg ctg gaa gtg agc atg ccc cat ttc atg 1440 val arg lys ser pro thr leu leu glu val ser met pro his phe met aga acc aac agc ttc gcc gag gac ctg gac ctg gaa ggg gag act ctg 1488 ctg aca ccc atc acc cac atc tca cag ctg cgg gaa cac cat cgg gcc 1536 acc att aag gtc att cga cgc atg cag tac ttt gtg gcc aag aag aaa 1584 ttc cag caa gcg cgg aag cct tac gat gtg cgg gac gtc att gag cag 1632 phe gln gln ala arg lys pro tyr asp val arg asp val ile glu gln tac tcg cag ggc cac ctc aac ctc atg gtg cgc atc aag gag ctg cag 1680 tyr ser gln gly his leu asn leu met val arg ile lys glu leu gln agg agg ctg gac cag tcc att ggg aag ccc tca ctg ttc atc tcc gtc 1728 arg arg leu asp gln ser ile gly lys pro ser leu phe ile ser val tca gaa aag agc aag gat cgc ggc agc aac acg atc ggc gcc cgc ctg 1776 ser glu lys ser lys asp arg gly ser asn thr ile gly ala arg leu aac cga gta gaa gac aag gtg acg cag ctg gac cag agg ctg gca ctc 1824 atc acc gac atg ctt cac cag ctg ctc tcc ttg cac ggt ggc agc acc 1872 ccc ggc agc ggc ggc ccc ccc aga gag ggc ggg gcc cac atc acc cag 1920 ccc tgc ggc agt ggc ggc tcc gtc gac cct gag ctc ttc ctg ccc agc 1968 aac acc ctg ccc acc tac gag cag ctg acc gtg ccc agg agg ggc ccc 2016 arg arg pro val leu ala arg thr his val gln gly arg val tyr asn phe leu glu arg pro thr gly trp lys cys phe val tyr his phe ala ile glu gln tyr ala ala leu ala thr gly thr leu phe trp met glu ser met val val leu cys val gly ser lys gly gln val phe ala thr asn glu ser gly arg val glu phe gly ser tyr ala asp ala leu trp gln thr trp val gly lys thr ile ala ser cys phe ser val phe ala pro ala ala ala ser leu ile gln thr ala trp arg cys tyr ala ala glu asn pro asp ser ser thr trp lys ile tyr ile arg lys ala pro pro gly glu lys met leu thr val pro his ile thr cys asp pro pro val arg lys ser pro thr leu leu glu val ser met pro his phe met phe gln gln ala arg lys pro tyr asp val arg asp val ile glu gln tyr ser gln gly his leu asn leu met val arg ile lys glu leu gln arg arg leu asp gln ser ile gly lys pro ser leu phe ile ser val ser glu lys ser lys asp arg gly ser asn thr ile gly ala arg leu atg gca aac ttc cta tta cct cgg ggc acc agc agc ttc cgc agg ttc 48 aca cgg gag tcc ctg gca gcc atc gag aag cgc atg gcg gag aag caa 96 gcc cgc ggc tca acc acc ttg cag gag agc cga gag ggg ctg ccc gag 144 gag gag gct ccc cgg ccc cag ctg gac ctg cag gcc tcc aaa aag ctg 192 cca gat ctc tat ggc aat cca ccc caa gag ctc atc gga gag ccc ctg 240 gag gac ctg gac ccc ttc tat agc acc caa aag act ttc atc gta ctg 288 glu asp leu asp pro phe tyr ser thr gln lys thr phe ile val leu aat aaa ggc aag acc atc ttc cgg ttc agt gcc acc aac gcc ttg tat 336 asn lys gly lys thr ile phe arg phe ser ala thr asn ala leu tyr gtc ctc agt ccc ttc cac cca gtt cgg aga gcg gct gtg aag att ctg 384 gtt cac tcg ctc ttc aac atg ctc atc atg tgc acc atc ctc acc aac 432 tgc gtg ttc atg gcc cag cac gac cct cca ccc tgg acc aag tat gtc 480 cys val phe met ala gln his asp pro pro pro trp thr lys tyr val gag tac acc ttc acc gcc att tac acc ttt gag tct ctg gtc aag att 528 ctg gct cga gct ttc tgc ctg cac gcg ttc act ttc ctt cgg gac cca 576 tgg aac tgg ctg gac ttt agt gtg att atc atg gca tac aca act gaa 624 trp asn trp leu asp phe ser val ile ile met ala tyr thr thr glu ttt gtg gac ctg ggc aat gtc tca gcc tta cgc acc ttc cga gtc ctc 672 cgg gcc ctg aaa act ata tca gtc att tca ggg ctg aag acc atc gtg 720 ggg gcc ctg atc cag tct gtg aag aag ctg gct gat gtg atg gtc ctc 768 aca gtc ttc tgc ctc agc gtc ttt gcc ctc atc ggc ctg cag ctc ttc 816 atg ggc aac cta agg cac aag tgt gtg cgc aac ttc aca gcg ctc aac 864 met gly asn leu arg his lys cys val arg asn phe thr ala leu asn ggc acc aac ggc tcc gtg gag gcc gac ggc ttg gtc tgg gaa tcc ctg 912 gac ctt tac ctc agt gat cca gaa aat tac ctg ctc aag aac ggc acc 960 tct gat gtg tta ctg tgt ggg aac agc tct gac gct ggg aca tgt ccg 1008 gag ggc tac cgg tgc cta aag gca ggc gag aac ccc gac cac ggc tac 1056 glu gly tyr arg cys leu lys ala gly glu asn pro asp his gly tyr acc agc ttc gat tcc ttt gcc tgg gcc ttt ctt gca ctc ttc cgc ctg 1104 atg acg cag gac tgc tgg gag cgc ctc tat cag cag acc ctc agg tcc 1152 met thr gln asp cys trp glu arg leu tyr gln gln thr leu arg ser gca ggg aag atc tac atg atc ttc ttc atg ctt gtc atc ttc ctg ggg 1200 tcc ttc tac ctg gtg aac ctg atc ctg gcc gtg gtc gca atg gcc tat 1248 gag gag caa aac caa gcc acc atc gct gag acc gag gag aag gaa aag 1296 cgc ttc cag gag gcc atg gaa atg ctc aag aaa gaa cac gag gcc ctc 1344 acc atc agg ggt gtg gat acc gtg tcc cgt agc tcc ttg gag atg tcc 1392 cct ttg gcc cca gta aac agc cat gag aga aga agc aag agg aga aaa 1440 cgg atg tct tca gga act gag gag tgt ggg gag gac agg ctc ccc aag 1488 arg met ser ser gly thr glu glu cys gly glu asp arg leu pro lys tct gac tca gaa gat ggt ccc aga gca atg aat cat ctc agc ctc acc 1536 ser asp ser glu asp gly pro arg ala met asn his leu ser leu thr cgt ggc ctc agc agg act tct atg aag cca cgt tcc agc cgc ggg agc 1584 att ttc acc ttt cgc agg cga gac ctg ggt tct gaa gca gat ttt gca 1632 gat gat gaa aac agc aca gcg cgg gag agc gag agc cac cac aca tca 1680 ctg ctg gtg ccc tgg ccc ctg cgc cgg acc agt gcc cag gga cag ccc 1728 agt ccc gga acc tcg gct cct ggc cac gcc ctc cat ggc aaa aag aac 1776 agc act gtg gac tgc aat ggg gtg gtc tca tta ctg ggg gca ggc gac 1824 cca gag gcc aca tcc cca gga agc cac ctc ctc cgc cct gtg atg cta 1872 pro glu ala thr ser pro gly ser his leu leu arg pro val met leu gag cac ccg cca gac acg acc acg cca tcg gag gag cca ggc ggc ccc 1920 cag atg ctg acc tcc cag gct ccg tgt gta gat ggc ttc gag gag cca 1968 gln met leu thr ser gln ala pro cys val asp gly phe glu glu pro gga gca cgg cag cgg gcc ctc agc gca gtc agc gtc ctc aca agc gca 2016 ctg gaa gag tta gag gag tct cgc cac aag tgt cca cca tgc tgg aac 2064 cgt ctc gcc cag cgc tac ctg atc tgg gag tgc tgc ccg ctg tgg atg 2112 arg leu ala gln arg tyr leu ile trp glu cys cys pro leu trp met tcc atc aag cag gga gtg aag ttg gtg gtc atg gac ccg ttt act gac 2160 ser ile lys gln gly val lys leu val val met asp pro phe thr asp ctc acc atc act atg tgc atc gta ctc aac aca ctc ttc atg gcg ctg 2208 gag cac tac aac atg aca agt gaa ttc gag gag atg ctg cag gtc gga 2256 glu his tyr asn met thr ser glu phe glu glu met leu gln val gly aac ctg gtc ttc aca ggg att ttc aca gca gag atg acc ttc aag atc 2304 asn leu val phe thr gly ile phe thr ala glu met thr phe lys ile att gcc ctc gac ccc tac tac tac ttc caa cag ggc tgg aac atc ttc 2352 ile ala leu asp pro tyr tyr tyr phe gln gln gly trp asn ile phe gac agc atc atc gtc atc ctt agc ctc atg gag ctg ggc ctg tcc cgc 2400 atg agc aac ttg tcg gtg ctg cgc tcc ttc cgc ctg ctg cgg gtc ttc 2448 aag ctg gcc aaa tca tgg ccc acc ctg aac aca ctc atc aag atc atc 2496 ggg aac tca gtg ggg gca ctg ggg aac ctg aca ctg gtg cta gcc atc 2544 atc gtg ttc atc ttt gct gtg gtg ggc atg cag ctc ttt ggc aag aac 2592 tac tcg gag ctg agg gac agc gac tca ggc ctg ctg cct cgc tgg cac 2640 atg atg gac ttc ttt cat gcc ttc cta atc atc ttc cgc atc ctc tgt 2688 gga gag tgg atc gag acc atg tgg gac tgc atg gag gtg tcg ggg cag 2736 gly glu trp ile glu thr met trp asp cys met glu val ser gly gln tca tta tgc ctg ctg gtc ttc ttg ctt gtt atg gtc att ggc aac ctt 2784 gtg gtc ctg aat ctc ttc ctg gcc ttg ctg ctc agc tcc ttc agt gca 2832 gac aac ctc aca gcc cct gat gag gac aga gag atg aac aac ctc cag 2880 ctg gcc ctg gcc cgc atc cag agg ggc ctg cgc ttt gtc aag cgg acc 2928 acc tgg gat ttc tgc tgt ggt ctc ctg cgg cac cgg cct cag aag ccc 2976 thr trp asp phe cys cys gly leu leu arg his arg pro gln lys pro gca gcc ctt gcc gcc cag ggc cag ctg ccc agc tgc att gcc acc ccc 3024 tac tcc ccg cca ccc cca gag acg gag aag gtg cct ccc acc cgc aag 3072 gaa aca cag ttt gag gaa ggc gag caa cca ggc cag ggc acc ccc ggg 3120 gat cca gag ccc gtg tgt gtg ccc atc gct gtg gcc gag tca gac aca 3168 gat gac caa gaa gag gat gag gag aac agc ctg ggc acg gag gag gag 3216 tcc agc aag cag cag gaa tcc cag cct gtg tcc ggc tgg ccc aga ggc 3264 cct ccg gat tcc agg acc tgg agc cag gtg tca gcg act gcc tcc tct 3312 gag gcc gag gcc agt gca tct cag gcc gac tgg cgg cag cag tgg aaa 3360 gcg gaa ccc cag gcc cca ggg tgc ggt gag acc cca gag gac agt tgc 3408 tcc gag ggc agc aca gca gac atg acc aac acc gct gag ctc ctg gag 3456 cag atc cct gac ctc ggc cag gat gtc aag gac cca gag gac tgc ttc 3504 gln ile pro asp leu gly gln asp val lys asp pro glu asp cys phe act gaa ggc tgt gtc cgg cgc tgt ccc tgc tgt gcg gtg gac acc aca 3552 cag gcc cca ggg aag gtc tgg tgg cgg ttg cgc aag acc tgc tac cac 3600 gln ala pro gly lys val trp trp arg leu arg lys thr cys tyr his atc gtg gag cac agc tgg ttc gag aca ttc atc atc ttc atg atc cta 3648 ctc agc agt gga gcg ctg gcc ttc gag gac atc tac cta gag gag cgg 3696 aag acc atc aag gtt ctg ctt gag tat gcc gac aag atg ttc aca tat 3744 lys thr ile lys val leu leu glu tyr ala asp lys met phe thr tyr gtc ttc gtg ctg gag atg ctg ctc aag tgg gtg gcc tac ggc ttc aag 3792 aag tac ttc acc aat gcc tgg tgc tgg ctc gac ttc ctc atc gta gac 3840 lys tyr phe thr asn ala trp cys trp leu asp phe leu ile val asp gtc tct ctg gtc agc ctg gtg gcc aac acc ctg ggc ttt gcc gag atg 3888 ggc ccc atc aag tca ctg cgg acg ctg cgt gca ctc cgt cct ctg aga 3936 gct ctg tca cga ttt gag ggc atg agg gtg gtg gtc aat gcc ctg gtg 3984 ggc gcc atc ccg tcc atc atg aac gtc ctc ctc gtc tgc ctc atc ttc 4032 gly ala ile pro ser ile met asn val leu leu val cys leu ile phe tgg ctc atc ttc agc atc atg ggc gtg aac ctc ttt gcg ggg aag ttt 4080 trp leu ile phe ser ile met gly val asn leu phe ala gly lys phe ggg agg tgc atc aac cag aca gag gga gac ttg cct ttg aac tac acc 4128 gly arg cys ile asn gln thr glu gly asp leu pro leu asn tyr thr atc gtg aac aac aag agc cag tgt gag tcc ttg aac ttg acc gga gaa 4176 ile val asn asn lys ser gln cys glu ser leu asn leu thr gly glu ttg tac tgg acc aag gtg aaa gtc aac ttt gac aac gtg ggg gcc ggg 4224 leu tyr trp thr lys val lys val asn phe asp asn val gly ala gly tac ctg gcc ctt ctg cag gtg gca aca ttt aaa ggc tgg atg gac att 4272 tyr leu ala leu leu gln val ala thr phe lys gly trp met asp ile atg tat gca gct gtg gac tcc agg ggg tat gaa gag cag cct cag tgg 4320 met tyr ala ala val asp ser arg gly tyr glu glu gln pro gln trp gaa tac aac ctc tac atg tac atc tat ttt gtc att ttc atc atc ttt 4368 ggg tct ttc ttc acc ctg aac ctc ttt att ggt gtc atc att gac aac 4416 ttc aac caa cag aag aaa aag tta ggg ggc cag gac atc ttc atg aca 4464 gag gag cag aag aag tac tac aat gcc atg aag aag ctg ggc tcc aag 4512 aag ccc cag aag ccc atc cca cgg ccc ctg aac aag tac cag ggc ttc 4560 ata ttc gac att gtg acc aag cag gcc ttt gac gtc acc atc atg ttt 4608 ctg atc tgc ttg aat atg gtg acc atg atg gtg gag aca gat gac caa 4656 agt cct gag aaa atc aac atc ttg gcc aag atc aac ctg ctc ttt gtg 4704 gcc atc ttc aca ggc gag tgt att gtc aag ctg gct gcc ctg cgc cac 4752 ala ile phe thr gly glu cys ile val lys leu ala ala leu arg his tac tac ttc acc aac agc tgg aat atc ttc gac ttc gtg gtt gtc atc 4800 ctc tcc atc gtg ggc act gtg ctc tcg gac atc atc cag aag tac ttc 4848 leu ser ile val gly thr val leu ser asp ile ile gln lys tyr phe ttc tcc ccg acg ctc ttc cga gtc atc cgc ctg gcc cga ata ggc cgc 4896 atc ctc aga ctg atc cga ggg gcc aag ggg atc cgc acg ctg ctc ttt 4944 gcc ctc atg atg tcc ctg cct gcc ctc ttc aac atc ggg ctg ctg ctc 4992 ttc ctc gtc atg ttc atc tac tcc atc ttt ggc atg gcc aac ttc gct 5040 tat gtc aag tgg gag gct ggc atc gac gac atg ttc aac ttc cag acc 5088 tyr val lys trp glu ala gly ile asp asp met phe asn phe gln thr ttc gcc aac agc atg ctg tgc ctc ttc cag atc acc acg tcg gcc ggc 5136 phe ala asn ser met leu cys leu phe gln ile thr thr ser ala gly tgg gat ggc ctc ctc agc ccc atc ctc aac act ggg ccg ccc tac tgc 5184 trp asp gly leu leu ser pro ile leu asn thr gly pro pro tyr cys gac ccc act ctg ccc aac agc aat ggc tct cgg ggg gac tgc ggg agc 5232 cca gcc gtg ggc atc ctc ttc ttc acc acc tac atc atc atc tcc ttc 5280 ctc atc gtg gtc aac atg tac att gcc atc atc ctg gag aac ttc agc 5328 gtg gcc acg gag gag agc acc gag ccc ctg agt gag gac gac ttc gat 5376 atg ttc tat gag atc tgg gag aaa ttt gac cca gag gcc act cag ttt 5424 met phe tyr glu ile trp glu lys phe asp pro glu ala thr gln phe att gag tat tcg gtc ctg tct gac ttt gcc gac gcc ctg tct gag cca 5472 ctc cgt atc gcc aag ccc aac cag ata agc ctc atc aac atg gac ctg 5520 leu arg ile ala lys pro asn gln ile ser leu ile asn met asp leu ccc atg gtg agt ggg gac cgc atc cat tgc atg gac att ctc ttt gcc 5568 pro met val ser gly asp arg ile his cys met asp ile leu phe ala ttc acc aaa agg gtc ctg ggg gag tct ggg gag atg gac gcc ctg aag 5616 phe thr lys arg val leu gly glu ser gly glu met asp ala leu lys atc cag atg gag gag aag ttc atg gca gcc aac cca tcc aag atc tcc 5664 tac gag ccc atc acc acc aca ctc cgg cgc aag cac gaa gag gtg tcg 5712 tyr glu pro ile thr thr thr leu arg arg lys his glu glu val ser gcc atg gtt atc cag aga gcc ttc cgc agg cac ctg ctg caa cgc tct 5760 ttg aag cat gcc tcc ttc ctc ttc cgt cag cag gcg ggc agc ggc ctc 5808 tcc gaa gag gat gcc cct gag cga gag ggc ctc atc gcc tac gtg atg 5856 ser glu glu asp ala pro glu arg glu gly leu ile ala tyr val met agt gag aac ttc tcc cga ccc ctt ggc cca ccc tcc agc tcc tcc atc 5904 tcc tcc act tcc ttc cca ccc tcc tat gac agt gtc act aga gcc acc 5952 agc gat aac ctc cag gtg cgg ggg tct gac tac agc cac agt gaa gat 6000 ser asp asn leu gln val arg gly ser asp tyr ser his ser glu asp ctc gcc gac ttc ccc cct tct ccg gac agg gac cgt gag 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tyr val lys trp glu ala gly ile asp asp met phe asn phe gln thr phe ala asn ser met leu cys leu phe gln ile thr thr ser ala gly trp asp gly leu leu ser pro ile leu asn thr gly pro pro tyr cys met phe tyr glu ile trp glu lys phe asp pro glu ala thr gln phe leu arg ile ala lys pro asn gln ile ser leu ile asn met asp leu pro met val ser gly asp arg ile his cys met asp ile leu phe ala phe thr lys arg val leu gly glu ser gly glu met asp ala leu lys tyr glu pro ile thr thr thr leu arg arg lys his glu glu val ser ser glu glu asp ala pro glu arg glu gly leu ile ala tyr val met ser asp asn leu gln val arg gly ser asp tyr ser his ser glu asp