Patent Publication Number: US-2009220949-A1

Title: Mutations Associated with the Long QT Syndrome and Diagnostic Use Thereof

Description:
FIELD OF THE INVENTION 
     The invention relates to new genetic mutations in KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2 genes and to diagnostic tests for their identification. 
     STATE OF THE ART 
     The Long QT Syndrome is an inherited disease predisposing to cardiac arrhythmias and sudden death at a young age. It is characterized by a prolonged QT interval on the electrocardiogram. 
     Two phenotypic variants have been recognized: an autosomal dominant variant known as Romano Ward Syndrome (RWS) and an autosomal recessive variant termed Jervell Lange Nielsen Syndrome (JLNS) (Romano C et al. Clin Ped 1963; 45:656-657; Ward D C. J Irish Med As 1964; 54:103; Jervell A &amp; Lange-Nielsen F. Am. Heart J 1957; 54:59-61). More recently, two other forms with extra-cardiac involvement have been reported (Splawski I et al. Cell 2004; 119:19-31; Plaster N M et al. Cell 2001; 105:511-519). 
     The genetic loci associated with the first type of pathology have been located on chromosomes 3, 4, 7, 11 and 21 and the respective genes have been identified. The gene associated with the LQT 1 locus is KCNQ1 (formerly termed KvLQT1), the gene associated with the LQT2 locus is KCNH2 (formerly termed HERG), the gene associated with the LQT3 locus is SCN5A, the gene associated with the LQT4 locus is ANK2, the gene associated with the LQT5 locus is KCNE1 (formerly termed minK) and finally the gene associated with the LQT6 locus is KCNE2 (formerly termed MIRP). All the genes involved encode ion channels (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) except for the gene encoding the cardiac form of “ankyrin”, a structural protein which anchors ion channels to the cell membrane (ANK2): however there are very few patients (4-5 families world-wide) showing an involvement of this protein, therefore it is not possible to perform genotype—phenotype relation studies in such a small population. 
     So far, several mutations in the genes encoding the five ion channels have been reported: for instance US2005/003445 describes mutations in KCNQ1 (or KvLQT1), KCNE1 (or Min K), KCNE2 (or MIRP), KCNH2 (or HERG) and SCN5A genes. 
     Currently, the diagnosis of Long QT Syndrome is primarily based on identification in a surface electrocardiogram of a heart-rate corrected QT prolongation (QTc≧440 msec for males and QTc≧460 for females). Prolongation of the QT interval may or may not be associated with symptoms linked to the presence of arrhythmias, such as syncopal episodes, however the finding of a prolonged QT interval remains the basic diagnostic element in the disease. However, epidemiological data have shown that only 70% of the subjects affected by this Syndrome displays a prolonged QT interval, therefore it can be deduced that genetic diagnosis is a fundamental tool for the diagnosis of disease at the pre-symptomatic stage. Moreover, since the type of underlying genetic defect affects the seriousness of the Long QT Syndrome and the response to the therapy, it is also evident the importance of molecular diagnosis for assessment of the arrhythmic risk and the following therapeutic choice. 
     However, a limit to the diffusion of molecular diagnosis is represented by the high number of mutations (for a list see http://pc4.fsm.it:81/cardmoc/) that can cause the disease, many of which are “private” mutations found in a single patient or family. So far, this has made necessary to screen the entire coding region (ORF) of all genes involved in the Syndrome. Such an approach is expensive and requires a very log time to formulate the report. 
     SUMMARY OF THE INVENTION 
     The present invention relates to novel nucleotide mutations in KCNQ1, KCNH2, SCN5, KCNE1, KCNE2 genes that are associated with the full-blown Long QT Syndrome or are associated with the predisposition to said syndrome or with the susceptibility to develop arrhythmias during exposure to trigger-events (food, drugs), and to a method to identify such mutations. Said mutations affect the coding region of the above defined genes and always result in corresponding amino acid changes, leading to the expression of variant proteins with altered functionality compared to wild type proteins. 
     According to a further primary aspect, the invention relates to a method for identification of about 40% of the carriers of the Long QT Syndrome or of carriers of a predisposition to said Syndrome. Such method involves the detection of a group of about 70 non-private mutations. According to a further aspect, the invention relates to a method for identification of about 20% of the carriers of the Long QT Syndrome or of carriers of a predisposition to said syndrome, comprising the detection of a further selection of about 20 non-private mutations. The detection is performed by well known techniques for identification of point mutations, insertions, deletions, duplications. 
     Nucleic acids containing previously unreported mutations, vectors containing said nucleic acids and cells transformed with said vectors are also included in the invention. 
     According to a further aspect, the invention relates to the detection, at the amino acid sequence level, of novel mutations or of different groups of non-private mutations. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1  shows the distribution of QT intervals in subjects not affected by LQTS and, in red, in subjects affected by LQTS. A wide overlap of the QT interval duration curve between affected and non affected subjects is noticed, therefore only genetic analysis can allow a correct diagnosis in affected subjects with a QT within normal limits. 
         FIG. 2 . Nucleotide and amino acid sequences of wild type KCNQ1 cDNA. 
         FIG. 3 . Nucleotide and amino acid sequences of wild type KCNH2 cDNA. 
         FIG. 4 . Nucleotide and amino acid sequences of wild type SCN5 cDNA. 
         FIG. 5 . Nucleotide and amino acid sequences of wild type KCNE1 cDNA. 
         FIG. 6 . Nucleotide and amino acid sequences of wild type KCNE2 cDNA. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     For the purpose of the present invention, the following definitions have been used: 
     Mutation: for the purpose of the present invention, it is meant by mutation, unless otherwise indicated, any change of the nucleotide sequence, involving one or more nucleotides, hence including a permutation, insertion or deletion that is absent in the DNA from control individuals or in wild type DNA corresponding to the cDNA sequences deposited in the GenBank with accession numbers: AF00571 (cDNA KvLQT1; gene: KCNQ1: AJ006345), NM005136 (cDNA: MiRP; gene: KCNE2, AB009071), NM000335 (cDNA: Nav1.5; gene: SCN5A NT — 022517.17), NM000238 (cDNA: HERG; gene: KCNH2: NT — 011512.10), NM00219 (KCNE1; gene: AP000324), resulting also in an amino acid sequence change in the encoded protein. 
     In the present invention, unless otherwise indicated, the positions of a single mutated nucleotide or of several mutated nucleotides are indicated with the number of the mutated nucleotide (like, for instance, in table 1 where the reference of the identifying sequence can be found, with the mutated nucleotide underlined) or with the respective codon or with the amino acid affected by the mutation: therefore, the name of the P345 mutation refers to the amino acid change, which is proline in wild type, as well as to the mutation of one of the nucleotides of the proline codon at position 345 of the amino acid sequence: in this case, the specific mutation of the nucleotide sequence is also reported. 
     In the present invention, the definitions “locus” and “gene” are used interchangeably and they correspond to, respectively: LQT1 locus—KCNQ1 (o KvLQT1) gene, LQT2 locus—KCNH2 (o HERG) gene, LQT3 locus—SCN5A gene, LQT5 locus—KCNE1 (o minK) gene, LQT6 locus—KCNE2 (o MiRP) gene. 
     QTc by QTc it is meant the QT interval corrected for the heart rate expressed as RR interval according to the formula QTc=QT/v RR.
 
IQR: Interquartile range. Values corresponding to the 75th percentile and the 25th percentile of a variable are reported under this definition.
 
Long QT Syndrome (QTS) Two phenotypic variants of the Long QT Syndrome have been recognized: an autosomal dominant variant known as Romano Ward Syndrome (RWS) and an autosomal recessive variant termed Jervell Lange Nielsen Syndrome (JLNS). In addition, two other forms with extra-cardiac involvement have been reported.
 
ORF: Open Reading Frame=the coding portion of a gene.
 
     The present invention is based on the identification of new mutations in KCNQ1 (also termed KvLQT1), KCNH2 (also termed HERG), SCN5A, KCNE1 (also termed minK), KCNE2 (also termed MiRP) genes encoding ion channels involved in the control of cardiac electrical activity and particularly in generation of the cardiac action potential. A genetically based dysfunction of the proteins encoded by these genes can cause the Long QT Syndrome. 
     Therefore, according to a first aspect, the invention relates to 139 new mutations of the coding region in the genomic DNA corresponding to KCNQ1, KCNH2 SCN5A, KCNE1, KCNE2 genes, enlisted in table 1, to nucleic acids, either RNA or DNA, and preferably cDNA and genomic DNA comprising the specific mutations and encoding the entire protein in variant forms with altered function compared to the wild type protein and with the potential to cause the Long QT Syndrome. Moreover, the invention relates to nucleic acid fragments and their encoded proteins, characterized in that they comprise at least one of the amino acid changes reported in Table 1 (SEQ ID NO: 11-149) and are useful for diagnostic or research purposes. 
     The invention also refers to fragments, polynucleotides and oligonucleotides, comprising at least the 9-nucleotide sequence reported in Table 1 (SEQ ID NO: 11-149) including the mutation and, alternatively or optionally, depending on their use, the adjacent nucleotides that can be derived from the sequences of KCNQ1, KCNH2 SCN5A, KCNE1, KCNE2 genes or cDNAs as described for wild type. 
     In a preferred embodiment, the length of the oligonucleotides of the invention, which are preferably used for diagnostic purposes, is shorter than or equal to 50 nucleotides, preferably between 40 and 15 nucleotides, even more preferably between 30 and 20 nucleotides. These oligonucleotides comprise the nonanucleotides defined in Table 1 or oligonucleotides suitable for detection of position, structure and type of mutations defined therein. Suitable oligonucleotides can be designed by an expert in the field based on the mutated sequences of in the present invention, on the published sequence of each wild type gene, whose accession number is herein reported, and on his/her own knowledge of the field. The oligonucleotides of the invention, and/or their complementary sequences, are chemically synthesized and can comprise chemically modified nucleotides (for instance phosphorothioated nucleotides) or a fluorochrome or chromophore label, preferably at the 5′ and/or 3′ terminus. 
     Such oligos can be used for gene amplification reactions or for hybridization in homogeneous or heterogeneous phase: they can be used as such or in a form bound to a solid matrix or a two-dimensional or three-dimensional support, for instance a membrane, or to the bottom of a well in a plate or to a microchip. 
     The nucleic acids of the invention are double or single stranded: wherein single stranded molecules include also oligonucleotides and complementary DNA (cDNA) or antisense DNA. 
     The nucleic acids of the invention, particularly the cDNA and its fragments, comprising at least one mutation according to the invention, can be cloned into vectors, for instance expression vectors for the production of high amounts of recombinant protein useful for functional characterization of different variants or to set up immunoassays. 
     Therefore, vectors containing the nucleotide sequences and cells transformed with such vectors, and expressing the mutant proteins, are also comprised in the invention. 
     The nucleic acids and proteins of the invention are claimed for diagnostic use in the Long QT Syndrome, particularly in the Romano Ward Syndrome and/or the Jervell Lange-Nielsen Syndrome in in vitro methods. Moreover, the recombinant proteins and their fragments comprising the mutation are useful for production of specific antibodies against the mutant protein, which are able to specifically bind the mutated but not the wild type protein. Together with the proteins, said antibodies are used to set up diagnostic immunoassays in vitro. 
     Therefore, according to a preferred embodiment, the invention relates to a method for identification in a sample of at least 1 of the mutations in Table 1, where such identification has both prognostic and diagnostic value for the Long QT Syndrome, in particular for the Romano Ward and/or Jervell Lange types for example by hybridization or by PCR. The identification of at least one of the mutations reported in Table 1 is carried out according to molecular methods well known in the art. The presence of said mutations in the nucleic acids of the sample correlates with a predisposition to develop such disease or with the full-blown disease. 
     The sample is preferably represented by nucleic acids purified from a biological sample, such as cells obtained from biological fluids, as for instance blood or other tissues. The nucleic acids are preferably genomic DNA or mRNA. In the latter case, the sample can be retrotranscribed into cDNA prior to sequence analysis. 
     A further aspect of the invention relates to a diagnostic method based on the identification of a group of about 70 non-private mutations in KCNQ1, KCNH2 and SCN5A genes, selected among new mutations shown in Table 1, and mutations well known in the art, selected among those detected by the authors of the present invention, which occur at high rate in a statistically significant sample of probands and which are able to identify about 40% of the probands. 
     According to a further aspect, the method to diagnose the Long QT Syndrome of RW and/or Jervell Lange type, or the genetic predisposition to said syndrome which represents the genetic cause of QT interval alterations found in an electrocardiogram, comprises at least the identification of hot spot mutations as defined below. Hot spot mutations are the most commonly found according to the population studied and occur in at least 3 or more clinically affected individuals of different families. In the KCNQ1 gene, such hot spot mutations affect the codons encoding for: R190, preferably R190W, where even more preferably W is encoded by the corresponding codon in sequence SEQ ID NO: 17, R231C and more preferably R231H, where even more preferably H is encoded by the corresponding codon in sequence SEQ ID NO: 24, V254 more preferably V254M and V254L, where even more preferably L is encoded by the corresponding codon in sequence SEQ ID NO: 26, and so on according to the preferred embodiments enlisted in Table 2 for the following mutations in the KCNQ1 gene: G269, S277, G314, A341, A344; in the KCNH2 gene in codons: A561, G572 (preferably identified by sequence SEQ ID NO: 97), G628; in the SCN5A gene in codons P1332 and E1784. 
     Hot spot mutations characterize 24% of the probands with electrocardiographic alterations; therefore the present invention comprises a method for identification of hot spot mutations as defined in the present invention which make use of methods well known in the art. In the KCNQ1 gene, the rate of said hot spot mutations in the sample is the following for each indicated codon: 190 (n=12), 231 (n=4) 254 (n=4), 269 (n=4), 277 (n=5), 314 (n=4), 341 (n=6), 344 (n=9), in the KCNH2 gene it is for codon 561 (n=7), 572 (n=4) and 628 (n=7); in the SCN5A gene is the following for each indicated codon: 1332 (n=5) and 1784 (n=3). 
     It should be noted that mutations in KCNQ1 and KCNH2 (LQT1 and LQT2) genes are more common in patients with Long QT Syndrome; they account for the genetic cause in 90% of these pathologies. 
     The preferential search identification of mutations in one of the hot spot codons, or of the mutations listed in Table 2, allows a rapid and highly cost-effective diagnosis of the Long QT Syndrome, with remarkable reduction of costs and expansion of the diagnostic potential to the general population. 
     In a particularly preferred aspect, the invention relates to a method for the molecular diagnosis (carried out on the nucleic acids of the patient) of the Long QT Syndrome, particularly the inherited forms Romano-Ward and/or Jervell Lange, comprising the identification of a group of non-private mutations (i.e. found in at least two individuals belonging to different families) affecting the following codons or groups of codons:
         in the KCNQ1 gene: L137 (exon 2), R174, G179, R190 (exon 3), I204 (exon 4), R231, D242, V254, H258, R259 (exon 5), L262, G269, S277, V280, A300, W305, (exon 6), G314, Y315, T322, G325, A341, P343, A344 (exon 7), R360 (exon 8), R518 (exon 12), R539 (exon 13), I567 (exon 14), R591, R594 (exon 15), according to the numbering of codons or amino acids, and according to the numbering of nucleotides with mutation 1514 +1G&gt;A, identified by oligonucleotide SEQ ID NO: 52, 1513-1514delCA, identified by oligonucleotide SEQ ID NO: 53, with mutation 921+1 G&gt;A and with mutation 921+2 T&gt;C;   in the KCNH2 gene: Y43, E58, IAQ82-84 (exon 2), W412, S428 (exon 6), R534, L552, A561, G572, R582, G604, D609, T613, A614, T623, G628 (exon 7), S660 (exon 8), R752 (exon 9), S818, R823 (exon 10) according to the numbering of codons or amino acids, and according to the numbering of nucleotides with mutation 453delC, 453-454insCC, 576delG identified by the oligonucleotide with sequence SEQ ID NO: 79, 578-582deICCGTG identified by the oligonucleotide with sequence SEQ ID NO: 80, G2398+3A&gt;G identified by the oligonucleotide with sequence SEQ ID NO: 110, G2398+3A&gt;T identified by the oligonucleotide with sequence SEQ ID NO: 111, 3093-3106del identified by the oligonucleotide with sequence SEQ ID NO: 125, 3093-3099del/insTTCGC identified by the oligonucleotide with sequence SEQ ID NO: 126, and 3100delC identified by the oligonucleotide with sequence SEQ ID NO: 128;   In the SCN5A gene: A413 (exon 10), T1304, P1332 (exon 21), 1505-1507del (exon 26), R1623 (exon 10), R1644, Y1767, E1784 (exon 28),
 
where the presence of a mutation in at least one of the above-mentioned codons or positions indicates the presence of a functional abnormality of the ion channel and that the subject carrying such mutation is affected by or predisposed to the Long QT Syndrome, preferably of the Romano-Ward and/or Jervell Lange type. Table 2 outlines the group of most representative mutations used for the diagnostic methods described above.
       

     In a further embodiment the invention also relates to a two-dimensional or three-dimensional support comprising oligonucleotides capable of selectively detecting the mutations defined in Table 2. According to an even more preferred embodiment, said support comprises polynucleotides or oligonucleotides comprising at least one of the preferred nonanucleotides chosen from those that are most commonly found in the sequences of the probands and reported in Table 1: SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 140, SEQ ID NO: 142, or their complementary sequences. Alternatively, an expert in the field can design the sequence of oligonucleotides capable of detecting the mutations defined in the present invention, for example by software tools. 
     According to a further embodiment, the invention comprises a support wherein polynucleotides and/or oligonucleotides include at least one of the nonanucleotides with sequence from SEQ ID NO: 11 to SEQ ID NO: 149 or at least one of their complementary oligonucleotides. 
     Table 2 shows both the position of the mutated nucleotide and the position of the codon which, as result of the mutation, is different from the wild type. In Tables 1 and 2 it is also possible to identify, in patients affected by the Long QT Syndrome, one or more amino acids preferably found as result of mutations in the codons of a gene. 
     According to a further aspect the invention comprises the use of a oligonucleotide comprising anyone of the nonamer with wild type sequence corresponding to those identified in SEQ ID NO: 11-149 and in Table 2 for the diagnosis of a full-blown Long QT Syndrome or for the diagnosis of a genetic predisposition to the Long QT Syndrome in in vitro methods. 
     For the purpose of the present invention it is intended to be comprised within the disclosure of the present invention any nucleotidic mutation leading to any amino acid change different from wild type, in the same position, as herein disclosed, with the exclusion of the mutations already well-known (see references of Table 2) regardless of the codons that are preferably generated as the result of the specific mutation. 
     Thus, just as an example, the present invention comprises all the mutations affecting, in the case of the KCNQ1 gene, the histidine codon at position 258 (wild type) which, according to the invention, changes from a histidine codon to a different codon that, according to a preferred embodiment, is an arginine (Arg or R) codon, if the mutation affects the second nucleotide of the CAC codon (His), thus changing from A into G (A→G) and producing a CGC codon which encodes for Arg; in the same starting codon a different change, C→A produces a AAC codon which enclodes for Asp as result of a mutation of the first nucleotide of the codon. In this case the mutation according to the invention identifies any amino acid at position 258 that is different from histidine, and preferably identifies arginine or asparagine. Table 2 shows the preferred embodiments for each mutation. 
     All nucleotide mutations (generally in the third-nucleotide of the codon) which, due to the degeneracy of the genetic code, change the codon giving rise to the same amino acid, identical to the amino acid preferred in the protein sequence, are also intended to be comprised in the present invention. Just as an example, in the case of the mutation of codon 258 in the KCNQ1 gene, already used in the previous example, all the permutations, due to genetic code degeneracy, that change the CAC codon into any of the codons encoding Arginine, or, in the second case, into any of the codons encoding Asp, are intended to be comprised in the invention. Each preferred embodiment of the mutations identified as being related to the Long QT Syndrome found and used in the method of the invention enlisted in Table 1 which reports the mutated nucleotide and/or amino acid according to the nucleotide or amino acid numbering of the corresponding wild type gene sequence which is reported as annex in the Sequence List from SEQ ID NO: 1 to 10. 
     The identification, according to the invention, of the most representative mutations in subjects at risk for the Long QT Syndrome can be carried out also on the protein product, corresponding to the various amino acid variants, using methods well known in the art, as for instance specific antibodies or differences in the electrophoretic migration pattern. 
     Based on the findings of the authors of the present invention, which have been also confirmed in an independent sample, at least 40% of mutation carriers (or probands) carries a mutation of one of the codons or one of the mutations reported in Table 2. Therefore, this molecular method represents the first level of molecular screening for the Long QT Syndrome. 
     The molecular method according to the invention involves also a second level of investigation carried out preferentially on subjects that are found to be negative at the first level screening. Such second level of investigation consists in the characterization of sequences of the Open Reading Frames in KCNQ1 and KCNH2 genes. Finally, the molecular method according to the invention involves a third level for subjects that turned out to be negative in the second level, comprising the analysis of the genes responsible for the less prevalent genetic variants of LQTS, that is for SCN5A, KCNE1 and KCNE2 genes. Said third level can include a confirmation of the sequences of SCN5A, KCNE1 and KCNE2 gene ORFs, for instance by direct sequencing following gene amplification with primer oligonucleotides which can be derived, by methods well known in art, from the published sequence. According to a preferred embodiment, the primers used for direct sequencing of exons are listed in Table 4. 
     The identification performed with molecular methods according to the present invention can be associated with other measurements or other clinical diagnostic/prognostic methods. 
     In this respect, the authors of the present invention have evaluated in parallel the sensitivity and specificity of several QTc cut-off values: a cut-off value of 440 ms turned out to have 81% specificity, 89% sensitivity and 91% positive predictive accuracy for the diagnosis of Long QT Syndrome. Instead, specific cut-off values for gender (=440 ms for males and =460 ms for females) proved to be very specific (96%) but not very sensitive (72%). 
     QTc duration correlates with the presence of genetic mutations: it is in fact decreasingly long for probands, family members carrying the disease at the genetic level and healthy family members (p&lt;0.0001; Table 5). However the distribution of QTc values is very similar among individuals affected and unaffected by genetic mutations, even though the epidemiological data have shown that only 70% of subjects affected by the Syndrome has a prolonged QT interval (the overlap between the two populations is shown in  FIG. 1 ). Therefore, it is demonstrated that the genetic diagnosis is an important tool for diagnosis of the disease at the presymptomatic stage. Moreover, since the type of underlying genetic defect affects the severity of the long QT Syndrome and the response to therapy, it is also evident the importance of molecular diagnosis for assessment of the arrhythmic risk and the following therapeutic choice (drugs, implantable defibrillator). 
     The data provided in the Table 3 and shown in  FIG. 1 , highlight the penetrance values of the disease (i.e. the percentage of carriers showing a prolongation of the QT interval in the surface electrocardiogram): from these data it is deduced that 30% of the carriers of at least one mutation according to the invention have a QT interval that is not different from normal. Therefore the effectiveness of a molecular screening for genotyping, like the one proposed here, is clear. Where necessary, such screening can be also coupled to a further investigation at the level of population screening, in order to identify genetic defects at birth, and/or identify iatrogenic long QT susceptibility, and/or screen competitive athletes and other populations in which the identification of a subclinical form of congenital long QT can prevent arrhythmic events, cardiac arrest and sudden cardiac death. This is made possible by the identification of a susceptibility to develop arrhythmias during exposure to trigger events (food, drugs etc) and the avoidance of conditions known to entail a higher risk, as for instance harmful life style habits, use of drugs and food/drinks contraindicated in subjects carrying such genetic defects. 
     The three levels of investigation in the method of the invention allow a significant saving of time and reagents: the first level of investigation is limited to the screening of about 70 mutations identified in Tables 2 A and 2B which are present in at least 40% of the patients that can be genotyped on the basis of mutations found so far. This way, a quick diagnosis is obtained that has limited costs and is nevertheless significant. Such analysis can be easily performed also in the forms of genetic screening at birth, screening for competitive athletes or screening for patients that need to be treated with drugs that can cause a prolongation of then QT interval. In fact, in all these categories of patients and sports persons, the analysis of the whole codifying portion of all disease genes is possible but is not used on a routine basis due to costs and excessive time length required. 
     The molecular method in its various embodiments makes use of well known methods for identification of mutations. Any method for detection of nucleotide mutations in a nucleic acid sequence can be used on the basis of the sequence information herein provided. 
     Among well known methods, the following are reported, although the list is not exhaustive: a) recognition of an enzymatic digestion pattern based for instance on the use of restriction enzymes by which the DNA fragment derived from the sample is selectively cut generating alternative patterns for mutated and “wild type” sequences, b) use of direct sequencing of nucleic acids, c) use of methods based on hybridization (or base pairing) with homologous or highly homologous sequences, d) use of the selective removal of specific sequences by methods of chemical or enzymatic breakage. The above techniques may or may not be used in association with steps of gene amplifications and may comprise, according to a particularly preferred embodiment, the use of solid platforms (microchip) with high/medium or low density binding of oligonucleotide probes (microarrays). Among hybridization-based methods beside the Southern Blotting technique, the following methods can also be used: Single Strand Conformation Polymorphism (SSCP Orita et al. 1989), clamped denaturing gel electrophoresis (CDGE, Sheffield et al. 1991), Denaturing Gradient Gel Electrophoresis (DGGE), heteroduplex analysis (HA, White et al. 1992), Chemical Mismatch Cleavage (CMC, Grompe et al., 1989), ASO (Allele Specific Oligonucleotides), RNase protection method (D B Thompson and J Sommercorn J. Biol. Chem., March 1992; 267: 5921-5926) and TCGE temporal gradient capillary electrophoresis Integrated platform for detection of DNA sequence variants using capillary array electrophoresis (Qingbo Li et al., Electrophoresis 2002, vol. 23, 1499-1511. 
     In the case of DNA direct sequencing (genes and ORFs) the primers used according to a preferred embodiment are those reported in Table 4. Insertions and deletions can be identified by methods well known in the art, such as RFLP (Restriction Fragment Length Polymorphism). Other methods are reported, for instance, in Sambrook et al. Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press NY. USA, 1989, or in Human Molecular Genetics, ed Strachan T. and Read A. P., 2 nd  ed. 1999, BIOS Scientific Publisher. 
     In a preferred embodiment the method of the invention, when based on nucleic acid hybridization, comprises the use of oligonucleotide probes derived from KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2 genes, and comprising the nonanucleotides with sequence from SEQ ID NO: 11 to SEQ ID NO: 149 in the Sequence List or their complementary sequences, or oligonucleotides suitable to identify the mutations disclosed in the present invention. 
     Alternatively, the invention relates to oligonucleotides with wild type sequence, which are however suitable to distinguish, in a biological sample under appropriate conditions of hybridization stringency, the mutations or groups of mutations according to the invention because they cannot perfectly match with the mutated sequence present in the sample. 
     Considering that about 10-15% of subjects with iatrogenic (i.e. caused by drugs) torsions of the tip and sudden death show a Long QT Syndrome mutation as genetic substratum (Yang P et al Circulation. 2002 Apr. 23; 105(16):1943-8, Napolitano et al J Cardiovasc Electrophysiol. 2000 June; 11(6):691-6), one embodiment of the proposed method is definitively the identification of subjects with contraindication for drugs that prolong the QT interval by blocking the Ikr current (the so-called” pre-prescription genotyping”). These include antibiotics, prokinetic drugs, antipsychotic drugs, antidepressants, antiarrhythmic drugs and drugs belonging to other therapeutic classes. 
     Therefore, the method of the invention allows to avoid the risks associated with drug administration to subjects carrying the subclinical form of the Long QT Syndrome and to assess the sensitivity of a subject to the following drugs: 
     Albuterol, Alfuzosin, Haloperidol, Amantadine, Amiodarone, Amitriptyline, Amoxapine, Amphetamine/dextroamphetamine, AmpicillinArsenic trioxide, Atomoxetine, Azithromycin, Bepridil, Quinidine, Chloral hydrate, Chloroquine, Chlorpromazine, Ciprofloxacin, Cisapride, Citalopram, Clarithromycin, Clomipramine, Cocaine, Desipramine, Dextroamphetamine, Disopiramide, Dobutamine, Dofetilide, Dolasetron, Domperidone, Dopamine, Doxepin, Droperidol, Ephedrine, Epinephrine, Erythromycin, Felbamate, Fenfluramine, Phentermine, Phenylephrine, Phenylpropanolamine, Flecainide, Fluconazole, Fluoxetine, Foscarnet, Fosphenytoin, Galantamine, Gatifloxacin, Gemifloxacin, Granisetron, Halofantrine, Ibutilide, Imipramine, Indapamide, Isoproterenol, Isradipine, Itraconazole, Ketoconazole, Levalbuterol, Levofloxacin, Levomethadyl, Lithium, Mesoridazine, Metaproterenol, Methadone, Methylphenidate, Mexiletine, Midodrine, Moexipril/HCTZ, Moxifloxacin, Nicardipine, Norepinephrine, Nortriptyline, Octreotide, Ofloxacin, Ondansetron, Paroxetine, Pentamidine, Pimozide, Procainamide, Procainamide, Protriptyline, Pseudoephedrine, Quetiapine, Risperidone, Ritodrine, Roxithromycin, Salmeterol, Sertraline, Sibutramine, Solifenacin, Sotalol, Sparfloxacin, Tacrolimus, Tamoxifen, Telithromycin, Terbutaline, Thioridazine, Tizanidine, Trimethoprim-Sulfamethoxazole, Trimipramine, Vardenafil, Venlafaxine, Voriconazole, Ziprasidone, where the presence of at least one of the mutations identified in Table 1 or of one of the mutations identified in Table 2 indicates a sensitivity to one of the drugs mentioned above. 
     The assay can also be used to identify a possible susceptibility to develop arrhythmias during exposure to trigger events other than drugs (e.g. food). According to a preferred embodiment, the invention comprises kits for the realization of the diagnostic or prognostic methods according to each of the aspects described above. Therefore, said kits are obtained according to a preferred embodiment comprising at least one of the oligonucleotides in Table 1, having sequence from SEQ ID NO: 11 to SEQ ID NO: 149 (mutant sequences), and/or their complementary sequences, and optionally other reagents such as buffers, enzymes, etc. necessary to carry out the method of the invention. According to a preferred embodiment, the kit comprises a set of at least 2 oligonucleotides each including at least one of the following nonanucleotides (identified by the SEQ ID NO) or of their complementary sequences, where said nonanucleotides are chosen from:
         KCNQ1 gene or locus: SEQ ID NO: 13 (L137), SEQ ID NO: 16 (R174P), SEQ ID NO: 17 (R190W), SEQ ID NO: 21 (I204M), SEQ ID NO: 24 (R231H), SEQ ID NO: 26 (V254L), SEQ ID NO: 27 (H258N), SEQ ID NO: 28 (H258R), SEQ ID NO: 29 (L262V), SEQ ID NO: 34 (V280E), SEQ ID NO: 39 (T322M), SEQ ID NO: 40 (P343L), SEQ ID NO: 41 (P343R), SEQ ID NO: 46 (R360T), SEQ ID NO: 55 (R518G), SEQ ID NO: 56 (R518P), SEQ ID NO: 59 (I567T), SEQ ID NO: 52, SEQ ID NO: 53;   KCNH2 gene or locus: SEQ ID NO: 67 (Y43C), SEQ ID NO: 69 (E58A), SEQ ID NO: 70 (E58G), SEQ ID NO: 71 (E58D), SEQ ID NO: 75 (delIAQ), SEQ ID NO: 85 (W412stop), SEQ ID NO: 88 (S428L), SEQ ID NO: 97 (G572D), SEQ ID NO: 98 (R852L), SEQ ID NO: 99 (D609H), SEQ ID NO: 106 (S660L), SEQ ID NO: 113 (S818P), SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 128;   SCN5A gene or locus: SEQ ID NO: 133 (A413E), SEQ ID NO: 134 (A413T), SEQ ID NO: 140 (R1644C), SEQ ID NO: 142 (Y1767C).       

     Alternatively the kit comprises oligonucleotides suitable to detect a group of at least 20 of the mutations reported in Table 2, where said oligonucleotides are designed with methods or software well known in the art. According to a preferred embodiment, the kit to realize the diagnostic method of the invention comprises oligonucleotides suitable to detect the following mutations, identified, according to the numbering of codons or amino acids in the KCNQ1 gene: 
     L137F, where F is preferably identified with the corresponding codon in SEQ ID NO: 13; R174C and R174P, where P is preferably identified with the corresponding codon in SEQ ID NO: 16; G179S, R190W where W is preferably identified with the corresponding codon in SEQ ID NO: 17; and R190Q, I204M where M is preferably identified with the corresponding codon in SEQ ID NO: 20; R231C and R231H where H is preferably identified with the corresponding codon in SEQ ID NO: 24; D242N, V254L where L is preferably identified with the corresponding codon in SEQ ID NO: 26; and V254M, H258N where N is preferably identified with the corresponding codon in SEQ ID NO: 27; and H258R where R is preferably identified with the corresponding codon in SEQ ID NO: 28; R259C, L262V where V is preferably identified with the corresponding codon in SEQ ID NO: 29; G269D and G269S, S277L, V280E where E is preferably identified with the corresponding codon in SEQ ID NO: 34; A300T, W305S and W305stop, G314D and G314S, Y315C, T322M where M is preferably identified with the corresponding codon in SEQ ID NO: 39; G325R, A341E and A341V, P343C where C is preferably identified with the corresponding codon in SEQ ID NO: 40; and P343R where R is preferably identified with the corresponding codon in SEQ ID NO: 41; A344E, R360T where T is preferably identified with the corresponding codon in SEQ ID NO: 46; R518G where G is preferably identified with the corresponding codon in SEQ ID NO: 55; and R518P where P is preferably identified with the corresponding codon in SEQ ID NO: 56; and R518stop, R539W, 1567T where T is preferably identified with the corresponding codon in SEQ ID NO: 59; R591H, R594Q and, according to nucleotide numbering, with the mutations: 1514+1G&gt;A, (SEQ ID NO: 52), 1513-1514delCA corresponding to SEQ ID NO: 53, with the mutation 921+1 G&gt;A and with the mutation 921+2 T&gt;C;
         in the KCNH2 gene, according to the numbering of codons or amino acids: Y43C where C is preferably identified with the corresponding codon in SEQ ID NO: 67; E58A where A is preferably identified with the corresponding codon in SEQ ID NO: 69; and E58G where G is preferably identified with the corresponding codon in SEQ ID NO: 70; and E58D where D is preferably identified with the corresponding codon in SEQ ID NO: 71; and E58K, del82-84IAQ preferably identified with SEQ ID NO: 75; W412stop, S428L where L is preferably identified with the corresponding codon in SEQ ID NO: 88; R534C and R534L where L is preferably identified with the corresponding codon in SEQ ID NO: 91; L552S, A561T and A561V, G572C and G572D where D is preferably identified with the corresponding codon in SEQ ID NO: 97; R582C and R582L where L is preferably identified with the corresponding codon in SEQ ID NO: 98; G604S, D609H where H is preferably identified with the corresponding codon in SEQ ID NO: 99; and D609G, T613M, A614V, T6231, G628S, S660L where L is preferably identified with the corresponding codon in SEQ ID NO: 106; R752W, S818L, R823W and, according to nucleotide numbering, with the mutations: 453delC, 453-454insCC, 576delG (SEQ ID NO: 79), 578-582deICCGTG (SEQ ID NO: 80), G2398+3A&gt;G (SEQ ID NO: 110), G2398+3A&gt;T (SEQ ID NO: 111), 3093-3106del (SEQ ID NO: 125), 3093-3099del/insTTCGC (SEQ ID NO: 126), and 3100delC (SEQ ID NO: 128);   in the SCN5A gene: A413E where E is preferably identified with the corresponding codon in SEQ ID NO: 133; and A413T where T is preferably identified with the corresponding codon in SEQ ID NO: 134; T1304M, P1332L, 1505-1507delKPQ, R1623Q, R1644C where C is preferably identified with the corresponding codon in SEQ ID NO: 140; Y1767C where C is preferably identified with the corresponding codon in SEQ ID NO: 142, E1784K.       

     According to a different embodiment, the kit comprises a set of at least 20 mutations among those defined in Table 2 and optionally other reagents such as buffers, enzymes, etc. necessary to carry out the method of the invention. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 New mutations identified in probands. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Gene 
                   
                   
                   
                   
                   
               
               
                 Oligo IDN 
                   
                 Nucleotide 
                 Coding Effect (by aa* number) 
                 Region 
                 Type of mutation 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 KCNQ1 
                   
                   
                   
                   
               
               
                 11 
                 C CCG  A CG GG 
                 G136A 
                 A46T 
                 N-TERM 
                 Missense 
               
               
                 12 
                 CGCTC T TAC 
                 151-152insT 
                 L50fs + 233x 
                 N-TERM 
                 Insertion 
               
               
                 13 
                 C TGC  T TC AT 
                 C409T 
                 L137F 
                 S1 
                 Missense 
               
               
                 14 
                 C ATC  A AG CA 
                 G436A 
                 E146K 
                 S1-S2 
                 Missense 
               
               
                 15 
                 GTG G A C CGC 
                 T518A 
                 V173D 
                 S2-S3 
                 Missense 
               
               
                 16 
                 GTC C C C CTC 
                 G521C 
                 R174P 
                 S2-S3 
                 Missense 
               
               
                 17 
                 G GGG  T GG CT 
                 C568T 
                 R190W 
                 S2-S3 
                 Missense 
               
               
                 18 
                 CTG C C C TTT 
                 G575C 
                 R192P 
                 S2-S3 
                 Missense 
               
               
                 19 
                 GCCCGAAGC 
                 584del 
                 R195fs + 41X 
                 S2-S3 
                 Deletion 
               
               
                 20 
                 C ATC  C GTGA 
                 G604C 
                 D202H 
                 S3 
                 Missense 
               
               
                 21 
                 TC AT G  GTG G 
                 C612G 
                 I204M 
                 S3 
                 Missense 
               
               
                 22 
                 GCC T T C ATG 
                 C626T 
                 S209F 
                 S3 
                 Missense 
               
               
                 23 
                 C TGC  A TG GG 
                 G643A 
                 V215M 
                 S3 
                 Missense 
               
               
                 24 
                 ATC C A C TTC 
                 G692A 
                 R231H 
                 S4 
                 Missense 
               
               
                 25 
                 ATG C C A CAC 
                 T716C 
                 L239P 
                 S4 
                 Missense 
               
               
                 26 
                 C TCC  T TG GT 
                 G760T 
                 V254L 
                 S4-S5 
                 Missense 
               
               
                 27 
                 C ATC  A AC CG 
                 C772A 
                 H258N 
                 S4-S5 
                 Missense 
               
               
                 28 
                 ATC C G C CGC 
                 A773G 
                 H258R 
                 S4-S5 
                 Missense 
               
               
                 29 
                 AG GAG  G TG AT 
                 C784G 
                 L262V 
                 S4-S5 
                 Missense 
               
               
                 30 
                 CACCTGTAC 
                 796del 
                 T265fs + 22X 
                 S5 
                 Deletion 
               
               
                 31 
                 CTG G A C CTC 
                 G815A 
                 G272D 
                 S5 
                 Missense 
               
               
                 32 
                 TCTCGTACT 
                 828-830del 
                 S277del 
                 S5 
                 Deletion 
               
               
                 33 
                 TCC T G G TAC 
                 C830G 
                 S277W 
                 S5 
                 Missense 
               
               
                 34 
                 TTT G A G TAC 
                 T839A 
                 V280E 
                 S5 
                 Missense 
               
               
                 35 
                 GAC G A G GTG 
                 C860A 
                 A287E 
                 S5-PORE 
                 Missense 
               
               
                 36 
                 A GAT  A CG CT 
                 G904A 
                 A302T 
                 PORE 
                 Missense 
               
               
                 37 
                 CAG G A C ACA 
                 T923A 
                 V308D 
                 PORE 
                 Missense 
               
               
                 38 
                 TAT G A G GAC 
                 G947A 
                 G316E 
                 PORE 
                 Missense 
               
               
                 39 
                 CAG A T G TGG 
                 C965T 
                 T322M 
                 PORE-S6 
                 Missense 
               
               
                 40 
                 CTC C T A GCG 
                 C1028T 
                 P343L 
                 S6 
                 Missense 
               
               
                 41 
                 CTC C G A GCG 
                 C1028G 
                 P343R 
                 S6 
                 Missense 
               
               
                 42 
                 T GGC  C CG GG 
                 T1045C 
                 S349P 
                 S6 
                 Missense 
               
               
                 43 
                 C TCG  C GG TT 
                 G1048C 
                 G350R 
                 S6 
                 Missense 
               
               
                 44 
                 GGG T C T GCC 
                 T1052C 
                 F351S 
                 S6 
                 Missense 
               
               
                 45 
                 GTGCAGCAG 
                 1067-1072del 
                 QT 356-357del 
                 C-TERM 
                 Deletion 
               
               
                 46 
                 CAG A C G CAG 
                 G1079C 
                 R360T 
                 C-TERM 
                 Missense 
               
               
                 47 
                 GCA G A C TCA 
                 C1115A 
                 A372D 
                 C-TERM 
                 Missense 
               
               
                 48 
                 TGG A T G ATC 
                 A1178T 
                 K393M 
                 C-TERM 
                 Missense 
               
               
                 49 
                 GGGG G TGAC 
                 1291-1292insG 
                 G430fs + 31x 
                 C-TERM 
                 Missense 
               
               
                 50 
                 GGGT G GACT 
                 1292-1293insG 
                 V431fs + 31X 
                 C-TERM 
                 Insertion 
               
               
                 51 
                 AGACTGCTG 
                 1486-1487del 
                 T495fs + 18X 
                 C-TERM 
                 Deletion 
               
               
                 52 
                 CACA A TGAG 
                 1514 + 1 G &gt; A 
                 S504sp 
                 C-TERM 
                 Splice Error 
               
               
                 53 
                 CTCAGTGAG 
                 1513-1514del 
                 S504fs + 9X 
                 C-TERM 
                 Deletion 
               
               
                 54 
                 GGCCACATT 
                 1538delC 
                 T513fs + 78X 
                 C-TERM 
                 Deletion 
               
               
                 55 
                 C ATT  G GA CG 
                 C1552G 
                 R518G 
                 C-TERM 
                 Missense 
               
               
                 56 
                 ATT C C A CGC 
                 G1553C 
                 R518P 
                 C-TERM 
                 Missense 
               
               
                 57 
                 CAG G A C CAC 
                 G1643A 
                 G548D 
                 C-TERM 
                 Missense 
               
               
                 58 
                 ATG G C G CGC 
                 T1661C 
                 V554A 
                 C-TERM 
                 Missense 
               
               
                 59 
                 TCC A C T GGG 
                 T1700C 
                 I567T 
                 C-TERM 
                 Missense 
               
               
                 60 
                 AAGCCTCAC 
                 1710delC 
                 P570fs + 22X 
                 C-TERM 
                 Deletion 
               
               
                 61 
                 TG TT A  ATC T 
                 C1719A 
                 F573L 
                 C-TERM 
                 Missense 
               
               
                 62 
                 ATCTCTCAG 
                 1725-1728del 
                 S575fs + 16X 
                 C-TERM 
                 Missense 
               
               
                 63 
                 GAT C A C GGC 
                 G1748A 
                 R583H 
                 C-TERM 
                 Insertion 
               
               
                 64 
                 C AGC  G AC AC 
                 A1756G 
                 N586D 
                 C-TERM 
                 Missense 
               
               
                 65 
                 CCCCCAGAG 
                 1893delC 
                 P631fs + 33X 
                 C-TERM 
                 Deletion 
               
               
                 66 
                 GGGCCACAT 
                 1909delC 
                 H637fs + 29X 
                 C-TERM 
                 Deletion 
               
               
                   
                 KCNH2 
               
               
                 67 
                 ATC T G C TGC 
                 A128G 
                 Y43C 
                 N-TERM 
                 Missense 
               
               
                 68 
                 TTC T G C GAG 
                 G146A 
                 C49Y 
                 N-TERM 
                 Missense 
               
               
                 69 
                 GCC G C G GTG 
                 A173C 
                 E58A 
                 N-TERM 
                 Missense 
               
               
                 70 
                 GCC G G G GTG 
                 A173G 
                 E58G 
                 N-TERM 
                 Missense 
               
               
                 71 
                 CC GA C  GTG A 
                 G174C 
                 E58D 
                 N-TERM 
                 Missense 
               
               
                 72 
                 C GAC  C TC CT 
                 T202C 
                 F68L 
                 N-TERM 
                 Missense 
               
               
                 73 
                 G CAC GGG CCG 
                 G211C 
                 G71R 
                 N-TERM 
                 Missense 
               
               
                 74 
                 CGC A C G CAG 
                 C221T 
                 T74M 
                 N-TERM 
                 Missense 
               
               
                 75 
                 GCAGGCAC 
                 244-252del 
                 IAQ82-84del 
                 N-TERM 
                 Deletion 
               
               
                 76 
                 GATGATGGT 
                 308-310ins ATG 
                 103InsD 
                 N-TERM 
                 Insertion 
               
               
                 77 
                 TGTGCCCGT 
                 337-339del 
                 V113del 
                 N-TERM 
                 Deletion 
               
               
                 78 
                 CGG TTCGC CG 
                 557- 
                 A185fs + 143X 
                 N-TERM 
                 Deletion/Insertion 
               
               
                   
                   
                 566del/Ins + TTCGC 
               
               
                 79 
                 CCGGGGCC 
                 576delG 
                 G192fs + 7X 
                 N-TERM 
                 Deletion 
               
               
                 80 
                 GGGGGTGGT 
                 578-582del 
                 G192fs + 135X 
                 N-TERM 
                 Deletion 
               
               
                 81 
                 GCC CC CGGC 
                 735-6InsCC 
                 P245fs + 114X 
                 N-TERM 
                 Insertion 
               
               
                 82 
                 ATCG T CCCG 
                 C751T 
                 P251S 
                 N-TERM 
                 Missense 
               
               
                 83 
                 G CTG  T AG G 
                 C1171T 
                 Q391X 
                 N-TERM 
                 Nonsense 
               
               
                 84 
                 C GTG T C G GAC 
                 G1229C 
                 W410S 
                 S1 
                 Missense 
               
               
                 85 
                 G GAC TG A  CTC 
                 G1235A 
                 W412X 
                 S1 
                 Nonsense 
               
               
                 86 
                 C ACA C A C TAC 
                 C1277A 
                 P426H 
                 S1-S2 
                 Missense 
               
               
                 87 
                 A CCC  C AC TCG 
                 T1279C 
                 Y427H 
                 S1-S2 
                 Missense 
               
               
                 88 
                 C TAC T T G GCT 
                 C1283T 
                 S428L 
                 S1-S2 
                 Missense 
               
               
                 89 
                 C GTG  T AC ATC 
                 G1378T 
                 D460Y 
                 S2 
                 Missense 
               
               
                 90 
                 C ATC  C AC ATG 
                 G1501C 
                 D501H 
                 S3 
                 Missense 
               
               
                 91 
                 G GTG C T C GTG 
                 G1601T 
                 R534L 
                 S4 
                 Missense 
               
               
                 92 
                 CGGATCGCT 
                 1613-1619del 
                 R537fs + 24X 
                 S4 
                 Deletion 
               
               
                 93 
                 GCC T C C ATC 
                 G1697C 
                 C566S 
                 S5 
                 Missense 
               
               
                 94 
                 TGCATTGGT 
                 1701delC 
                 I567fs + 26X 
                 S5 
                 Deletion 
               
               
                 95 
                 C ATC  C GG TAC 
                 T1702C 
                 W568R 
                 S5 
                 Missense 
               
               
                 96 
                 C GCC  G TC GC 
                 A1711G 
                 I571V 
                 S5 
                 Missense 
               
               
                 97 
                 C ATC G A C AA 
                 G1715A 
                 G572D 
                 S5 
                 Missense 
               
               
                 98 
                 TCA C T C ATC 
                 G1745T 
                 R582L 
                 S5-PORE 
                 Missense 
               
               
                 99 
                 AAG  C AC AAG 
                 G1825C 
                 D609H 
                 S5-PORE 
                 Missense 
               
               
                 100 
                 GCG  T TC TAC 
                 C1843T 
                 L615F 
                 PORE 
                 Missense 
               
               
                 101 
                 GC AG G  CTC A 
                 C1863G 
                 S621R 
                 PORE 
                 Missense 
               
               
                 102 
                 CCC G C C AGC 
                 G1877C 
                 G626A 
                 PORE 
                 Missense 
               
               
                 103 
                 TCA GA C  AAG 
                 G1911C 
                 E637D 
                 PORE-S6 
                 Missense 
               
               
                 104 
                 C TGC  T TC AT 
                 G1930T 
                 V644F 
                 S6 
                 Missense 
               
               
                 105 
                 ATC T G C GGC 
                 T1967G 
                 F656C 
                 S6 
                 Missense 
               
               
                 106 
                 TG T T G GCC A 
                 C1979T 
                 S660L 
                 S6 
                 Missense 
               
               
                 107 
                 CAG C C C CTC 
                 G2087C 
                 R696P 
                 S6-CNBD 
                 Missense 
               
               
                 108 
                 TGACGAGTG 
                 2164-2181dup 
                 E722-D727 
                 S6-CNBD 
                 Duplication 
               
               
                 109 
                 CTTCCAGGG 
                 2231delG 
                 F743fs + 12X 
                 S6-CNBD 
                 Deletion 
               
               
                 110 
                 GGGT G TGGG 
                 G2398 + 3A &gt; G 
                 L799sp 
                 CNBD 
                 Splice Error 
               
               
                 111 
                 GGGT T TGGG 
                 G2398 + 3A &gt; T 
                 L799sp 
                 CNBD 
                 Splice Error 
               
               
                 112 
                 CCTG T GTAT 
                 G2398T 
                 G800W 
                 CNBD 
                 Missense 
               
               
                 113 
                 AAG C C G AAC 
                 T2452C 
                 S818P 
                 CNBD 
                 Missense 
               
               
                 114 
                 T GGC  T AG TC 
                 A2494T 
                 K832X 
                 CNBD 
                 Nonsense 
               
               
                 115 
                 TTC  C AC CTG 
                 A2581C 
                 N861H 
                 C-TERM 
                 Missense 
               
               
                 116 
                 GGGTGCAAC 
                 2638-2648del 
                 G879fs + 35X 
                 C-TERM 
                 Deletion 
               
               
                 117 
                 TCCGACGGA 
                 2676-2682del 
                 R892fs + 79X 
                 C-TERM 
                 Deletion 
               
               
                 118 
                 CCGGCCGGG 
                 2732-2766del 
                 P910 + 16X 
                 C-TERM 
                 Deletion 
               
               
                 119 
                 GGCGGGCCG 
                 2738-2739insCGGGC 
                 A913fs + 62X 
                 C-TERM 
                 Insertion 
               
               
                 120 
                 GGGGCCGTG 
                 2775delG 
                 G925fs + 47X 
                 C-TERM 
                 Deletion 
               
               
                 121 
                 CG TG A  GGG G 
                 G2781A 
                 W927X 
                 C-TERM 
                 Nonsense 
               
               
                 122 
                 CCCGGGTGG 
                 2895-2905del 
                 G965fs + 148X 
                 C-TERM 
                 Deletion 
               
               
                 123 
                 CCG C T G GGT 
                 C2903T 
                 P968L 
                 C-TERM 
                 Missense 
               
               
                 124 
                 CGATG A CCCGC 
                 C3045A 
                 C1015X 
                 C-TERM 
                 Nonsense 
               
               
                 125 
                 CCCGGGGCG 
                 3093-3106del 
                 G1031fs + 86X 
                 C-TERM 
                 Deletion 
               
               
                 126 
                 GGG TTCGC C 
                 3093- 
                 G1031fs + 20X 
                 C-TERM 
                 Deletion/Insertion 
               
               
                   
                   
                 3099del/insTTCGC 
               
               
                 127 
                 GGCGCCCCG 
                 3099delG 
                 R1033 + 22X 
                 C-TERM 
                 Missense 
               
               
                 128 
                 GCGGCCCGG 
                 3100delC 
                 P1034 fs + 63X 
                 C-TERM 
                 Deletion 
               
               
                 129 
                 CAGGTGGAG 
                 3154delC 
                 R1051fs + 4X 
                 C-TERM 
                 Deletion 
               
               
                 130 
                 CCC C ACCCT 
                 3304InsC 
                 P1101fs + 16X 
                 C-TERM 
                 Insertion 
               
               
                 131 
                 CCCACGACG 
                 3397-3398del 
                 T1133fs + 135X 
                 C-TERM 
                 Deletion 
               
               
                   
                 SCN5A 
               
               
                 132 
                 CTG  T AC AGA 
                 C3457T 
                 H1153Y 
                 C-TERM 
                 Missense 
               
               
                 133 
                 GGTCG A A ATG 
                 C1238A 
                 A413E 
                 IS6 
                 Missense 
               
               
                 134 
                 GGTC A CAAT 
                 G1237A 
                 A413T 
                 IS6 
                 Missense 
               
               
                 135 
                 TGCC  G AG GG 
                 C1717G 
                 Q573E 
                 I-II 
                 Missense 
               
               
                 136 
                 TCCC  A GA AC 
                 G1735A 
                 G579R 
                 I-II 
                 Missense 
               
               
                 137 
                 AAC C A T CTC 
                 G2066A 
                 R689H 
                 I-II 
                 Missense 
               
               
                 138 
                 T GCC A C G AAG 
                 T4493C 
                 M1498T 
                 III-IV 
                 Missense 
               
               
                 139 
                 TC TTC C C A GTC 
                 G4877C 
                 R1626P 
                 IV-S4 
                 Missense 
               
               
                 140 
                 GATC  T GC ACG 
                 C4930T 
                 R1644C 
                 IV-S4 
                 Missense 
               
               
                 141 
                 C AAC  G TC GG 
                 A4978G 
                 I1660V 
                 IV-S5 
                 Missense 
               
               
                 142 
                 ATG T G C ATT 
                 A5300G 
                 Y1767C 
                 IV-S6 
                 Missense 
               
               
                 143 
                 CACC  A AG CC 
                 G5360A 
                 S1787N 
                 C-TERM 
                 Missense 
               
               
                 144 
                 GAG G G C GAC 
                 A5369G 
                 D1790G 
                 C-TERM 
                 Missense 
               
               
                 145 
                 TTC C A C AGG 
                 G5738A 
                 R1913H 
                 C-TERM 
                 Missense 
               
               
                   
                 KCNE1 
               
               
                 146 
                 CCC C A C AGC 
                 G107A 
                 R36H 
                 S1 
                 Missense 
               
               
                 147 
                 GGA T G C TTC 
                 T158G 
                 F53C 
                 S1 
                 Missense 
               
               
                   
                 KCNE2 
               
               
                 148 
                 GTG G TG  ATG   
                 A166G + 169InsATG 
                 M56V + M57ins 
                 S1 
                 Missense/Insertion 
               
               
                 149 
                 CTGTAGGTG 
                 156-161del 
                 52-54del.YLM &gt; X 
                 S1 
                 Deletion 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2A 
               
             
            
               
                   
               
               
                 Mutations found in at least 40% of probands. 
               
               
                 MISSENSE MUTATIONS AND IN-FRAME INSERTIONS OR DELETIONS 
               
               
                 NUMBERING REFERS TO THE AMINO ACID SEQUENCE 
               
            
           
           
               
               
               
            
               
                 KCNQ1 
                 KCNH2 
                 SCN5A 
               
               
                   
               
               
                 L137 → (Z-L) F (SEQ ID NO: 13) 
                 Y43 → (Z -Y) C (SEQ ID NO: 67) 
                 A413 → (Z -A) E (SEQ ID NO: 133), T 
               
               
                   
                   
                 (SEQ ID NO: 134) 
               
               
                 R174 → (Z-R) C 1 , P (SEQ ID NO: 16) 
                 E58 → (Z -E) A, G, D (SEQ ID NO: 
                 T1304 → (Z -T) M 3   
               
               
                   
                 69, 70, 71), K 2   
               
               
                 G179 → (Z-G) S 4   
                 del 82-84: IAQ (SEQ ID NO: 75) 
                 P1332 V (Z -P) L 5   
               
               
                 R190 → (Z-R) W (SEQ ID NO: 17), Q 1   
                 W412 → (Z -W); X (SEQ ID NO: 85) 
                 del 1505-1507: KPQ 6   
               
               
                 I204 → (Z -I) M (SEQ ID NO: 21) 
                 S428 → (Z -S) L (SEQ ID NO: 88), X 7   
                 R1623 → (Z -R) Q 8   
               
               
                 R231 → (Z -R) C 9 , H (SEQ ID NO: 24) 
                 R534 → (Z -R) C 10   
                 R1644 → (Z -R) C (SEQ ID NO: 140), H 4   
               
               
                 D242 → (Z -D) N 10   
                 L552 → (Z -L) S 4   
                 Y1767 → (Z -Y) C (SEQ ID NO: 142) 
               
               
                 V254 → (Z -V) L (SEQ ID NO: 26), M 1   
                 A561 → (Z -A) T 4 , V 4   
                 E1784 → (Z -E) K 4   
               
               
                 H258 → (Z-H) N (SEQ ID NO: 27), R (SEQ 
                 G572 → (Z -G) C 4 , D (SEQ ID NO: 97) 
               
               
                 ID NO: 28) 
               
               
                 R259 → (Z -R) C 11   
                 R582 → (Z -R) C 12 , L (SEQ ID NO: 98) 
               
               
                 L262 → (Z -L) V (SEQ ID NO: 29) 
                 G604 → (Z -G) S 4   
               
               
                 G269 → (Z -G) D 1 , S 13   
                 D609 → (Z -D) H (SEQ ID NO: 99), G 16   
               
               
                 S277 → (Z -S) L 14   
                 T613 → (Z -T) M 4   
               
               
                 V280 → (Z -V) E (SEQ ID NO: 34) 
                 A614 → (Z -A) V 7   
               
               
                 A300 → (Z -A) T 15   
                 T623 → (Z -T) I 16   
               
               
                 W305 → (Z -W) S 17 , X 18   
                 G628 → (Z -G) S 4   
               
               
                 G314 → (Z -G) D 19 , S 1   
                 S660 → (Z -S) L (SEQ ID NO: 106) 
               
               
                 Y315 → (Z -Y) C 20   
                 R752 → (Z -R) W 4   
               
               
                 T322 → (Z -T) M (SEQ ID NO: 39) 
                 S818 → (Z -S) L 21 , P (SEQ ID NO: 113) 
               
               
                 G325 → (Z -G) R 1   
                 R823 → (Z -R) W 4   
               
               
                 A341 → (Z -A) E 4 , V 4   
               
               
                 P343 → (Z -P) L, R (SEQ ID NO: 40, 
               
               
                 41) 
               
               
                 A344 → (Z -A) E 16   
               
               
                 R360 → (Z -R) T (SEQ ID NO: 46) 
               
               
                 R518 → (Z -R) G, P (SEQ ID NO: 55, 
               
               
                 56), X 4   
               
               
                 R539 → (Z -F) W 22   
               
               
                 I567 → (Z -I) T (SEQ ID NO: 59) 
               
               
                 R591 → (Z -R) H 23   
               
               
                 R594 → (Z -R) Q 4   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2B 
               
             
            
               
                   
               
               
                 SPLICING AND FRAMESHIFTS MUTATIONS (NUMBERING 
               
               
                 REFERS TO THE NUCLEOTIDE SEQUENCE) 
               
            
           
           
               
               
            
               
                 KCNQ1 
                 KCNH2 
               
               
                   
               
               
                 1514 + 1 G &gt; A (SEQ ID NO: 52) 
                 453delC 24 ; 453-454insCC 16   
               
               
                 1513-1514delCA (SEQ ID NO: 53) 
                 576delG (SEQ ID NO: 79) 
               
               
                 921 + 1 G &gt; A 4   
                 578-582del CCGTG (SEQ ID NO: 80) 
               
               
                 921 + 2 T &gt; C 4   
                 G2398 + 3A &gt; G (SEQ ID NO: 110) 
               
               
                   
                 G2398 + 3A &gt; T (SEQ ID NO: 111) 
               
               
                   
                 3093-3106del (SEQ ID NO: 125) 
               
               
                   
                 3093-3099del/insTTCGC (SEQ ID NO: 126) 
               
               
                   
                 3100delC (SEQ ID NO: 128) 
               
               
                   
               
            
           
         
       
     
     Legend to Tables 1 and 2. 
     Table 1. 
     Mutation Abbreviations: 
     del: deletion. 796del indicates that the nucleotide at position 796 is deleted. When more than one nucleotide is deleted, as for instance in 828-830 (SEQ ID NO: 32), all the intervening nucleotides and the extreme nucleotides are deleted (e.g. 828, 829, 830). The deleted nucleotide/nucleotides can be also specified (e.g. in SEQ ID NO: 129, 3154delC indicates that the cytosine at position 3154 is deleted).
 
ins: insertion. The explanation is similar to del.
 
     Generally, the nucleotide or amino acid number refers to the residue (nucleotide or amino acid) affected by the mutation. However, for frame shift mutations (fs), the amino acid residue indicated with the one-letter code and the number corresponding to its position in the protein sequence, corresponds to the last residue identical to wild type. 
     Moreover, for frame-shift mutations, that easily generate a stop codon upstream of the natural stop codon, it is also indicated the number of amino acids out of frame (different from the natural protein sequence) that follow the last wild type amino acid (for instance, R195fs+41X, SEQ ID NO: 19, indicates that the nucleotide mutation generates a frame-shift due to which Arg at position 195 of the KCNQ1 gene is the last wild type amino acid, followed by a tail of 41 amino acids different from wild type). 
     Coding for splicing (sp) errors: the number indicates the last nucleotide of the exon; the number after the +sign indicates the position, relative to this nucleotide, of the intronic base that is substituted. For instance, 921+2 T&gt;C indicates a T to C substitution of the second intronic base after the last exonic nucleotide at position 921. 
     Table 2A 
     Z: any amino acid; (Z-W): any amino acid but W. Therefore, the detection of the mutation refers to the codon identified by the number: Detection may refer to any codon different from wild type (e.g. for KCNQ1, the detection of R518 refers to any codon not encoding the wild type amino acid isoleucine, and preferably refers to any codon encoding glycine or proline, even more preferably refers to glycine and proline codons as identified by SEQ ID NO: 55 and SEQ ID NO: 56). 
     Table 2A and 2B: 
     X.: STOP codon. Other abbreviations and symbols have the same meaning as in Table 1.
 
Numbering refers to the number of the wild type amino acid (Table 2A) or nucleotide (Table 2B) as reported in the Sequence List.
 
Superscript numbers refer to references for mutations already described. The mutations identified in the present invention are characterized by the corresponding identification n° (SEQ ID NO) of Table 1.
 
Additional references for the symbols used are reported in: Antonarakis S E. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working group. Hum. Mutat., 1998; 11:1-3.
 
     REFERENCES FOR TABLE 2 
     
         
         1. Donger, C, et al. Circulation. 1997; 96:2778-2781. 
         2. Lupoglazoff, J M, et al. Circulation. 2001; 103:1095-1101. 
         3. Wattanasirichaigoon, D, et al. Am J Med Genet. 1999; 86:470-476. 
         4. Splawski, I, et al. Circulation. 2000; 102:1178-1185. 
         5. Kehl, H G, et al. Circulation. 2004; 109:e205-e206. 
         6. Wang, Q, et al. Cell. 1995; 80:805-811. 
         7. Priori, S G, et al. Circulation. 1999; 99:529-533. 
         8. Kambouris, N G, et al. Circulation. 1998; 97:640-644. 
         9. Lupoglazoff, J M et al. J Am Coll Cardiol. 2004; 43:826-830. 
         10. Itoh, T, et al. Hum. Genet. 1998; 102:435-439. 
         11. Kubota, T, et al. J Cardiovasc Electrophysiol. 2000; 11:1048-1054. 
         12. Jongbloed, R J, et al. Hum. Mutat. 1999; 13:301-310. 
         13. Ackerman, M J, et al N. Engl. J. Med. 1999; 341:1121-1125. 
         14. Liu, W, et al. Hum. Mutat. 2002; 20:475-476. 
         15. Priori, S G, et al. Circulation. 1998; 97:2420-2425. 
         16. Tester, D J, et al. Heart Rhythm. 2005; 2:507-517. 
         17. Neyroud, N, et al. Eur. J. Hum. Genet. 1998; 6:129-133. 
         18. Chen, S et al. Clin Genet. 2003; 63:273-282. 
         19. Choi, G, et al. Circulation. 2004; 110:2119-2124. 
         20. Napolitano, C et al. J. Cardiovasc. Electrophysiol. 2000; 11:691-696. 
         21. Berthet, M, et al. Circulation. 1999; 99:1464-1470. 
         22. Chouabe, C, et al. Cardiovasc Res. 2000; 45:971-980. 
         23. Neyroud, N, et al. Circ. Res. 1999; 84:290-297. 
         24. Swan, H, et al J. Am. Coll. Cardiol. 1999; 34:823-829. 
       
    
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 N 
                 QTc (ms) 
                 Mean 
                 IQR 
                 % penetrance 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 LQT1 
                 450 
                 465 ± 41 
                 461 
                 440-488 
                  64** 
               
               
                 LQT2 
                 279 
                  486 ± 48* 
                 477 
                 455-511 
                  81# 
               
               
                 LQT3 
                 63 
                  489 ± 49* 
                 481 
                 460-515 
                  83# 
               
               
                 LQT5 
                 20 
                 447 ± 36 
                 439 
                 424-467 
                 40 
               
               
                 LQT6 
                 5 
                 434 ± 24 
                 425 
                 414-459 
                 40 
               
               
                   
               
               
                 QT values in ms. 
               
               
                 IQR = Inter Quartile Range in ms; 
               
               
                 *p &lt; 0.005 vs. KCNQ1/KCNE1; 
               
               
                 **p &lt; 0.04 vs. KCNE1; 
               
               
                 #p &lt; 0.001 vs. KCNQ1/KCNE1/KCNE2 
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Sequencing primers (SEQ ID NO: 150-212) 
                   
               
            
           
           
               
               
               
               
            
               
                 GENE 
                 PRIMER ID 
                 SEQUENCE 
                 Length 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 KCNQI 
                 KV11.1 
                 cactcaaggccgagcctgcct 
                 21 
                   
               
               
                   
               
               
                 ″ 
                 KV5NA 
                 gccccacaccatctccttcg 
                 20 
               
               
                   
               
               
                 ″ 
                 KV6NI 
                 taccctaacccgggccac 
                 18 
               
               
                   
               
               
                 ″ 
                 KVDFn 
                 gaggagaagtgatgcgtgtc 
                 20 
               
               
                   
               
               
                 ″ 
                 KVDRn 
                 ggcaggacctgggcaccctc 
                 20 
               
               
                   
               
               
                 ″ 
                 KV1A1F 
                 cttcgctgcagctcccggtg 
                 20 
               
               
                   
               
               
                 ″ 
                 KV1A2R 
                 acgcgcgggtctaggctcac 
                 20 
               
               
                   
               
               
                 ″ 
                 KK2F 
                 gactgccgtgtccctgtcttg 
                 21 
               
               
                   
               
               
                 ″ 
                 KK2R 
                 gccatgccttcagatgctacg 
                 21 
               
               
                   
               
               
                 ″ 
                 KK12Rn 
                 ctgagggcaggaaggctcag 
                 20 
               
               
                   
               
               
                 ″ 
                 KK14F1 
                 ctgtctgtcccacagacgac 
                 20 
               
               
                   
               
               
                 ″ 
                 KK14Rn 
                 ctgggcccagagtaactgac 
                 20 
               
               
                   
               
               
                 ″ 
                 KK15Fn 
                 cggcccaccccagcacttggc 
                 21 
               
               
                   
               
               
                 ″ 
                 KK15Rn 
                 gaaccaccgcaggccggcgcg 
                 21 
               
               
                   
               
               
                 ″ 
                 KK16Fn 
                 cgtctgcctttgtccccg 
                 18 
               
               
                   
               
               
                 ″ 
                 KK16Rn 
                 cactcttggcctcccctc 
                 18 
               
               
                   
               
               
                 KCNE1 
                 MINK F 
                 ctgcagcagtggaccctta 
                 19 
               
               
                   
               
               
                 ″ 
                 MINK 1R 
                 agcttcttggagcggatgta 
                 20 
               
               
                   
               
               
                 ″ 
                 MINK 2F 
                 gtcctcatggtactgggatt 
                 20 
               
               
                   
               
               
                 ″ 
                 MINK R 
                 tttagccagtggtggggtt 
                 19 
               
               
                   
               
               
                 KCNE2 
                 MINK2-F2 
                 ccgttttcctaaccttgttcgcct 
                 24 
               
               
                   
               
               
                 ″ 
                 MINK2-R2 
                 gccacgatgatgaaagagaacattcc 
                 26 
               
               
                   
               
               
                 ″ 
                 MINK2-F3 
                 gtcatcctgtacctcatggtgat 
                 23 
               
               
                   
               
               
                 ″ 
                 MINK2-R3 
                 tggacgtcagatgttagcttggtg 
                 24 
               
               
                   
               
               
                 SCN5A 
                 SCN2.1Fnew 
                 ccc tgc tct ctg tcc ctg 
                 21 
               
               
                   
                   
                 ggc 
               
               
                   
               
               
                 ″ 
                 SCN2.1Rnew 
                 gca gcc cct ctc ggc tct 
                 20 
               
               
                   
                   
                 cc 
               
               
                   
               
               
                 ″ 
                 SCN2.2Fnew 
                 cat ggc aga gaa gca agc 
                 21 
               
               
                   
                   
                 ccg 
               
               
                   
               
               
                 ″ 
                 SCN6Fnew 
                 cct cct ctg act gtg tgt 
                 22 
               
               
                   
                   
                 ctc c 
               
               
                   
               
               
                 ″ 
                 SCN10Fnew 
                 cca gtg agg gtg acc tct 
                 21 
               
               
                   
                   
                 gcc 
               
               
                   
               
               
                 ″ 
                 SCN10Rnew 
                 ggc tta gag gct cct cgg 
                 21 
               
               
                   
                   
                 tgg 
               
               
                   
               
               
                 ″ 
                 SCN16F 
                 gag cca gag acc ttc aca 
                 21 
               
               
                   
                   
                 agg 
               
               
                   
               
               
                 ″ 
                 SCN17.1Fnew 
                 gct tgg cat ggt gca gtg 
                 24 
               
               
                   
                   
                 cct tgg 
               
               
                   
               
               
                 ″ 
                 SCN17.1Rnew 
                 gag gca cct tct ccg tct 
                 22 
               
               
                   
                   
                 ctg g 
               
               
                   
               
               
                 SCN5A 
                 SCN20Fnew 
                 cat tag atg tgg gca ttc 
                 24 
               
               
                   
                   
                 aca ggc 
               
               
                   
               
               
                 ″ 
                 SCN20Rnew 
                 cca gcc gtc cct gcc aca 
                 21 
               
               
                   
                   
                 acc 
               
               
                   
               
               
                 ″ 
                 SCN21Fnew 
                 ggt cca ggc ttc atg tcc 
                 21 
               
               
                   
                   
                 acc 
               
               
                   
               
               
                 ″ 
                 SCN21Rnew 
                 ggc aat ggg ttt ctc ctt 
                 22 
               
               
                   
                   
                 cct g 
               
               
                   
               
               
                 ″ 
                 SCN22Fnew 
                 ggg gag ctg ttc cca tcc 
                 22 
               
               
                   
                   
                 tcc c 
               
               
                   
               
               
                 ″ 
                 SCN22Rnew 
                 cgc ctc cca ctc cct ggt 
                 21 
               
               
                   
                   
                 ggg 
               
               
                   
               
               
                 ″ 
                 SCN23.1F 
                 ttg aaa agg aaa tgt gct 
                 23 
               
               
                   
                   
                 ctg gg 
               
               
                   
               
               
                 ″ 
                 SCN23.1R 
                 ttg ttc acg atg gtg tag 
                 22 
               
               
                   
                   
                 ttc a 
               
               
                   
               
               
                 ″ 
                 SCN23.2F 
                 cca gac aga ggg aga ctt 
                 21 
               
               
                   
                   
                 gcc 
               
               
                   
               
               
                 ″ 
                 SCN25Fnew 
                 ccc agc ctg tct gat ctc 
                 22 
               
               
                   
                   
                 cct g 
               
               
                   
               
               
                 ″ 
                 SCN25Rnew 
                 cca ccc tac cca gcc cag 
                 21 
               
               
                   
                   
                 tgg 
               
               
                   
               
               
                 KCNH2 
                 HM1F 
                 catgggctcaggatgccggt 
                 20 
               
               
                   
               
               
                 ″ 
                 KCNH2-1R 
                 cattgactcgcacttgccgacg 
                 22 
               
               
                   
               
               
                 ″ 
                 H2F 
                 cgctcacgcgcactctcctc 
                 20 
               
               
                   
               
               
                 ″ 
                 KH2R 
                 ttgaccccgcccctggtcgt 
                 20 
               
               
                   
               
               
                 ″ 
                 H3F 
                 ccactgagtgggtgccaaggg 
                 21 
               
               
                   
               
               
                 ″ 
                 H3R 
                 gagaccacgaacccctgagcc 
                 21 
               
               
                   
               
               
                 ″ 
                 H4.1F 
                 cccacgaccacgtgcctctcc 
                 21 
               
               
                   
               
               
                 ″ 
                 H8R 
                 gcctgccacccactggcc 
                 18 
               
               
                   
               
               
                 ″ 
                 KH9F 
                 atggtggagtagagtgtgggtt 
                 22 
               
               
                   
               
               
                 ″ 
                 KH9R 
                 agaaggctcgcacctcttgag 
                 21 
               
               
                   
               
               
                 ″ 
                 KH10F 
                 gagaaggtgcctgctgcctgg 
                 21 
               
               
                   
               
               
                 ″ 
                 KH10R 
                 acagctggaagcaggaggatg 
                 21 
               
               
                   
               
               
                 ″ 
                 H11F 
                 ggcaggagagcactgaaagggc 
                 22 
               
               
                   
               
               
                 ″ 
                 H11R 
                 ggtaaagcagacacggcccacc 
                 22 
               
               
                   
               
               
                 ″ 
                 H12A F 
                 gttctcctcccctctctgaggc 
                 22 
               
               
                   
               
               
                 ″ 
                 H12B R 
                 gggtagacgcaccaccgctgc 
                 21 
               
               
                   
               
               
                 ″ 
                 H13F 
                 gcagcggtggtgcgtctaccc 
                 21 
               
               
                   
               
               
                 ″ 
                 H13R 
                 gacctggaccagactccagggc 
                 22 
               
               
                   
               
               
                 ″ 
                 H14R 
                 gggtacatcgaggaagcagg 
                 20 
               
               
                   
               
            
           
         
       
     
     EXPERIMENTAL PART 
     Methods 
     Probands and their relatives were subjected to genetic analysis by molecular screening of the coding regions of genes associated with LQTS (Romano Ward variant). The diagnosis of probands was based on conventional clinical criteria (personal clinical history, evaluation of the QT interval by standard 12-lead ECG, Holter recording, with a cycloergometer exercise test). 
     Relatives were evaluated by genetic analysis independently from the diagnosis based on the clinical phenotype. 
     Methods for sample analysis: the entire coding regions of KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 genes were examined in a sample of 1621 subjects belonging to 430 families, using primers designed from intronic regions close to splicing sites (Table 4). 
     The population study comprised 430 LQTS probands with RWS and 1115 members of their families, and their data were collected by the Molecular Cardiology Laboratories of the Maugeri Foundation. 
     An informed consent was obtained from all subjects undergoing molecular analysis and, in the case of minors, from their legal tutors. 
     A group of 75 genotyped probands were used to verify the non-familial mutations identified in the previously examined population. 
     The study was approved by the Institutional Review Board of the Maugeri Foundation according to IRB regulations for all study subjects. 
     Statistical Analysis 
     Statistical analyses, when performed, were carried out with the SPSS Statistical Package (v. 12.01). The Kolmogorov-Smirnov test was used to determine the normal distribution of variables. Parametric tests (unpaired t-test and ANOVA with Bonferroni multiple-comparison correction) were used to compare normally distributed variables; instead the Kruskal-Wallis and the Mann-Whitney tests were used for not normally distributed variables. Chi-square test or Fisher exact test has been used for categorical variables. The data obtained are the mean±standard deviation (SD). For data that do not follow a normal distribution, the median and the interquartile range (25% and 75%) are reported. 
     Genotyping 
     Molecular analysis was performed on genomic DNA extracted from peripheral lymphocytes using methods well known in the art. The entire “open reading frames” of KCNQ1, KCNH2, SCN5A, KCNE1 and KCE2 genes were amplified by PCR with primers designed from intronic sequences flanking the exons (“exon-flanking intronic primer”). Primers are well known in the art or listed in Table 4. Amplicons were analysed by Denaturing High Performance Liquid Chromatography (DHPLC, Wave® Transgenomics) Each amplicon was subjected to DHPLC at least at two different temperatures, depending on the “melting” profile of the amplicon. When an abnormal chromatogram was detected, the amplicon was sequenced by a 310 Automated Genetic Analyzer® (Applied Biosystems) and/or cloned by PCR in a plasmid vector (Topo® cloning, Invitrogen). All DNA sequence variations that were absent in 400 control subjects (corresponding to 800 chromosomes) were defined as mutations. 
     Example 1 
     Clinical Characterization of the Sample 
     ECGs and clinical data were collected and analyzed in blind relative to the genetic status of the samples. QTc (QTc=QT/vRR) data were measured using the ECG recorded during the first visit. 
     The demographic characteristics of the sample are shown in the following table. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 5 
               
             
            
               
                   
                   
               
               
                   
                 Family members 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Genetically 
                 Genetically not 
                   
               
               
                   
                 Probands 
                 affected 
                 affected 
                 P-value 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 N 
                 310 
                 521 
                 594 
                   
               
               
                 Males (%) 
                 147 (47) 
                 231 (44) 
                 281 (47) 
               
               
                 Age, years (mean) 
                  21 ± 20 (16) 
                  33 ± 20 (35) 
                  29 ± 19 
                 &lt;0.002** 
               
               
                 QTc* (mean) 
                 495 ± 49 (490) 
                 461 ± 40 (458) 
                 406 ± 27 (408) 
                 &lt;0.001** 
               
               
                   
               
               
                 *In ms. 
               
               
                 **Post-hoc analysis between groups. 
               
               
                 QTc duration and occurrence by genotype. 
               
            
           
         
       
     
     The QTc value in the various populations examined has been reported in Table 3. As can be deduced from the table, in the mutation carriers the QTc was 474+46 msec (median value: 467 msec, IQR: 444-495 msec) while, among healthy family members, it was 406±27 msec (median value: 409 msec, IQR 390-425 msec). Incomplete penetrance was defined as the percentage of mutation carriers having a QTc longer than normal (e.g., QTc=440 ms for males and =460 ms for females). 
     The mean penetrance of the disease in the study population turned out to be 70%, but decreased to 57% among family members carrying the mutation. Patients with LQT2 and LQT3 showed a higher penetrance compared to patients with LQT1 and LQT5, whereas penetrance for patients with LQT6 could not be determined due to the low number (see Table 5). 
     QTc duration was decreasingly long for probands, family members carrying the disease at the genetic level and healthy family members (p&lt;0.0001; Table 5). However the distribution of QTc values was very similar between subjects affected by genetic mutations and subjects without mutations. (Instead, the QTc and the penetrance of LQTS were significantly different between carriers of multiple mutations (n=26) and family members with only one mutation (n=49): QTc 495±58 msec, IQR 450-523 msec versus 434+31 msec, IQR: 411-452 msec, p&lt;0.001; 30%, 15/49 versus 77% 20/26; p&lt;0.0001). 
     The analysis of 1411 subjects (296 probands, 521 genetically affected and 594 genetically unaffected family members) to determine the sensitivity and the specificity of different QTc cut-off intervals in individuals carrying genetic mutations, revealed that the best cut-off value for sensitivity and specificity is a QTc value=440 ms (81% specificity, 89% sensitivity and 91% positive predictive accuracy for diagnosis). Specific cut-off values for gender (=440 ms for males and =460 ms for females) proved to be very specific (96%) but poorly sensitive (72%). 
     Example 2 
     Genetic Characterization of the Sample 
     Genetic analysis of the sample revealed that 310/430 (72%) of the probands and 521 family members were carriers of 235 different mutations (139 of which were novel) that can determine the LQTS Syndrome. 
     From these 310 probands, the genetic analysis was extended to a total of 1115 family members: a mutation was found in 521 of them, while 594 were found to be healthy. In total, the study revealed 831 carriers of a mutation predisposing to LQTS symptoms and 594 healthy family members. 
     The mutation rate in the different genes was distributed as follows: 49% KCNQ1; 39% KCNH2; 10% SCN5A; 1.7% KCNE1; 0.7% KCNE2. About 90% of genotyped patients had mutations in KCNQ1 and KCNH2 genes, while 44% of the probands carried common mutations. These statistics were verified again with an independent set of 75 genotyped probands ( FIG. 1 ). 
     All the mutations identified and characterized for the first time in the present invention are reported in Table 1. 
     Two-hundred-ninety-six probands carried heterozygous mutations and 14 (4.5%) carried more than one genetic defect. Twelve probands turned out to be heterozygous for 2 (n=11) or 3 (n=1) combinations of mutations, 2 turned out to be homozygous RWS patients. In the group of 296 probands with a single genetic defect, KCNQ1mutations were most represented (n=144; 49%), followed by KCNH2 mutations (n=115; 39%), SCN5A mutations (n=30; 10%), KCNE1 mutations (n=5; 1.7%) and at last KCNE2 mutations (n=2; 0.7%). 
     In conclusion, 98% of the genotypic mutations detected in LQTS probands were identified in KCNQ1, KCNH2 and SCN5A genes. Twenty-nine of 247 probands, whose parents were both available for genetic analysis, turned out to be carriers of sporadic mutations (12%). 
     Overall, 235 different mutations were identified, of which 139 (KCNQ1 n=56, KCNH2 n=67, SCN5A n=13, KCNE1 n=2, KCNE2 n=2) were identified in this study for the first time. Missense mutations accounted for 72% (170/235) of the genetic defects. The remaining 28% comprised small intragenic deletions (n=33; 14.1%), splice errors (n=6; 2.7%), non-sense mutations (n=12; 5.1%), insertions (n=11; 4.7%), duplications or insertions/deletions (n=3; 1.4%). The most frequently mutated codons (hot-spots) were: in KCNQ1: codon 190 (n=12), 231 (n=4) 254 (n=4), 269 (n=4), 277 (n=5), 314 (n=4), 341 (n=6), 344 (n=9) and codons 561 (n=7), 572 (n=4) and 628 (n=7) in KCNH2, 1332 (n=5) and 1784 (n=3) in SCN5A. In total, 74/296 (25%) of the probands were genotyped based on hot-spot mutations and 129/296 of the probands (44%) carried one of the non-private mutations reported in Table 2 (i.e. mutations identified in more than one family). 
     Intra-Locus Variability 
     The distribution of mutations in the protein coding regions of the various LQTS genes, using the subdivision in regions already reported in other studies (e.g. Splawski I. et al. Circulation, 2000 102:1178-1185), was the following: mutations in the “pore region” and in the transmembrane region were identified in 61% of the patients, while in 32% of the patients the mutation was in a C-terminal region; only 7% of the patients carried a N-terminal mutation. 
     Annex 1 
     cDNA Sequence List:
         SEQ ID NO: 1: KvLQT1 cDNA (GenBank Acc. N o  AF000571); SEQ ID NO: 2: KvLQT1 protein (see  FIG. 2 )   SEQ ID NO: 3: KCNH2 cDNA (GenBank Acc. N o  NM000238); SEQ ID NO: 4: KCNH2 protein (see  FIG. 3 )   SEQ ID NO: 5: SCN5A cDNA (GenBank Acc. N o  NM000335); SEQ ID NO: 6: SCN5A protein (see  FIG. 4 )   SEQ ID NO: 7: KCNE1 cDNA (GenBank Acc. N o  NM000219); SEQ ID NO: 8: KCNE1 protein (see  FIG. 5 )   SEQ ID NO: 9 KCNE2 cDNA (GenBank Acc. N o  NM000335); SEQ ID NO: 10: KCNE2 protein (see  FIG. 6 ).       

     The oligonucleotides of the invention, comprising the mutations identified and characterized in the present invention, are numbered from 11 to 149 and are reported in Table 1.