Patients

The patients were 70 Danish LQTS probands from unrelated families where mutation screening in the five LQTS associated genes KCNQ1, KCNH2, KCNE1, KCNE2 and SCN5A had led to the identification of a disease-causing mutation. LQT diagnosis was based on the clinical examination of patients, which was performed according to guidelines issued by the Danish Cardiology Society [6] by specialists in cardiology from Danish cardiology departments at Rigshospitalet, Skejby Hospital, Aalborg Hospital, Gentofte Hospital, Haderslev Hospital and Odense Hospital. All patients had a QT_C interval > 440 ms for men and 450 ms for women. All patients were Caucasian. A clinical description of patients identified through a Danish nationwide survey comprising 59 families (all contributing to the current survey) has recently been published [12].

Mutation identification

Genomic DNA was extracted from EDTA-blood using the commercially available Maxwell™ 16 Blood DNA purification kit on the Maxwell^R 16 System (Promega Biotech AB, Nacka, Sweden). Genetic screening was performed by bi-directional sequencing of PCR amplified exons with associated flanking intronic regions. Primers sequences are available on request. A minor proportion of mutations were identified at genetic departments at Skejby University Hospital, Rigshospitalet as well as in Norway and the Netherlands using other, but similar, technologies. All probands had the coding regions of the five genes sequenced. All mutations were verified by sequencing a second amplified amplicon. A large proportion of patients were examined for large deletions in KCNQ1, KCNH2, KCNE1, KCNE2 and a small part of SCN5A by multiplex-ligation-dependent amplification (MLPA) using the SALSA MLPA P114 kit (MRC-Holland, Amsterdam, The Netherlands).

Mutation and protein nomenclature

Mutation nomenclature uses numbering with the A of the initiation codon ATG as +1 (http://www.hgvs.org/mutnomen), based on the following RefSeqs: NM_000218.2 (KCNQ1), NM_000238.2 (KCNH2), NM_000335.4 (SCN5A), NM_000219.3 (KCNE1) and NM_172201.1 (KCNE2). All mutations were checked using Mutalyzer. The protein nomenclature was that used in the recent mutation update on LQTS [2].

Evaluation of sequence changes

Deletions, frameshift-, splice- and nonsense mutations were considered disease-causing if not found in controls. Concerning missense mutations, familial segregation was ascertained if possible, but nuclear family size was in all cases so small that it precluded a proper linkage analysis [12]. Instead it was ascertained that the family did not contain affected members that did not carry the family mutation. Conservation of residues across several species was examined. All genetic variants were evaluated in 182 randomly and anonymously collected blood donor controls (364 alleles). The frequency of identified variants was assessed using the Exome Variant Server (EVS) v.0.0.21. (http://evs.gs.washington.edu). It was established whether genetic variants had previously been associated with LQTS and whether functional analysis had been performed. The potential functional effect of changes in amino acid composition was assessed in silico using the prediction servers Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) [13], SIFT (http://sift.bii.a-star.edu.sg) [14], and MutationAssessor (http://www.ngrl.org.uk/Manchester/page/mutation-assessor) [15]

Haplotyping KCNH2

Haplotyping was performed using the microsatellites D7S1824, D7S1826 up-stream of KCNH2 and D7S636, D7S3070, D7S483 and D7S1803 downstream of KCNH2 (Figure 1). CentiMorgan distances were obtained from the Map-O-Mat database for microsatellites (http://compgen.rutgers.edu/Mapomat/). PCR amplicons were generated using fluorescently end-labelled primers (available at NCBI UniSTS) at 0.4 μM per primer, per reaction. A loading mix of 0.5 μl amplicon, 9 μl HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 0.5 μl 600LIZ size standard (Applied Biosystems) was prepared, and DNA products were electrophoresed on an ABI PRISM® 3100 Genetic Analyser. Data were analysed using ABI GeneMapper software v4.0 (Applied Biosystems).

Mutation screening

KCNQ1mutations

The twenty-two mutations identified in KCNQ1 seemed evenly spread out over the gene (Table 1). Only three of the mutations (p.F157C, IVS6_IVS10del and p.R583G) had not previously been described in other populations. The IVS6_IVS10del results, theoretically, either in the excision of a considerable part of the protein rendering it non-functional or in haploinsufficiency due to mRNA surveillance mechanisms [62], and is thus considered pathogenic. The remaining missense mutations all affect highly conserved residues and many missense mutations have been described in the S2-S3 and C-terminal regions of the protein, respectively [2]. The p.R583G affects a codon, where another missense mutation, p.R583C, has been shown to have an electrophysiological phenotype similar to that expected for a LQTS-associated mutation [42]. Among the 18 mutations previously seen in other populations, there were two frameshift mutations, two deletions, one splice-site mutation and one nonsense mutation. All of these must be expected to result in haploinsufficiency due to mRNA surveillance mechanisms. Of the remaining missense mutations, four had been electrophysiologically characterized in vitro, i.e. p.V254M [21], p.L273I [21, 27, 28], p.Y315C [43] and p.R583C [42]. Thus, only four out of 15 LQTS-associated missense mutations (27%) had established electrophysiological characteristics at the molecular level.

KCNH2mutations

Twenty-eight mutations were identified in KCNH2. Fourteen of these were located in the N-terminus, and five of these were located in the Per-Arnt-Sim domain. Five were located in the C-terminus, only five mutations were located in the pore-region (Table 2). This is surprising, as the majority of previously identified LQTS associated mutations in KCNH2 have been located in the pore region [2]. Fifteen of the mutations have only been described in the Danish population. Eight of the Danish specific mutations were deletions, or frameshift mutations. The remaining eight Danish-specific mutations were missense mutations and all involved conserved amino acid residues. Among the mutations found in other populations, two were frameshift mutations, one was nonsense and 10 were missense mutations. Only one of these missense mutations, p.F29L, had been demonstrated to have an in vitro electrophysiological effect compatible with LQTS [43], so the remainder were considered disease-associated based on the evolutionary conservation of the involved amino acid residue and the absence of the mutation among 364 control alleles as well as a previously reported association with LQTS. The observation of the association of these mutations with LQTS in the Danish population corroborates their role in the causation of LQTS. Finally, a large number of mutations in the regions affected by the mutations described here, have been associated with LQTS [2].

All the remaining missense mutations in KCNH2, except p.A913V, were conserved to the level of zebrafish. The p.A913V mutation changed an alanine into a valine, where the valine was found at the same codon in rodents. However, the mutation had previously been associated with LQTS [16] and in the absence of deviations from familial disease segregation, and the absence of other mutations in the five genes as well as of the mutation in 364 control alleles, it was considered disease-causing.

SCN5Amutations

Nine mutations were identified in SCN5A (Table 3), five of which have only been described in the Danish population. Four mutations have not been reported before. Two mutations were intronic splice-site mutations and the remaining mutations were missense mutations located in the DI, DIV and C-terminal regions. This distribution is compatible with the one found when accumulating all known SCN5A LQTS-associated mutations [2]. The splice-site mutations were considered disease-causing because they are believed to result in aberrant mRNA splicing resulting in the synthesis of SCN5A ion channels with prolonged depolarisation contributing to delayed repolarisation. The splice mutations most likely do not result in haploinsufficiency as this would most likely give a Brugada syndrome phenotype [63]. One of the missense mutations, p.P2005A, has previously been associated with sudden infant death syndrome and shown to result in late persistent I _Na current, compatible with LQTS [53]. An in vitro electrophysiological analysis of the functional consequence of the p.L1785Q mutation has shown that it results in increased late persistent I _Na current, but also in a reduction of the total I _Na (Kanters et al., submitted), suggesting that the electrophysiological phenotype may be a combination of Brugada syndrome and LQTS, as previously described for SCN5A mutations [63], e.g. the p.E1784K mutation [64]. The remaining missense mutations interfere with conserved residues and have not been found in controls; further the mutations p.F1596I and p.V1597M, are located in the C-terminal part of SCN5A, where mutations causing LQTS are particularly frequent [2]. The rare polymorphism p.D1819N, known to be associated with increased QT-interval in normal individuals [65], was found in a single p.F29L family and not considered pathogenic, despite being previously reported as associated with LQTS [57] The C-terminally located mutations are likely to interfere with fast inactivation of the I _Na current [66]. Likewise, the remaining missense mutations, p.G319S, p.R340W and p.V411M, are located in the DI-S5-S6 region, a region with a high frequency of LQTS-associated mutations [2].

KCNE1mutations

Three mutations were found in KCNE1, two of which, p.G60D and p.A93T, have only been found in the Danish population and have not been reported before. The p.A93T was found associated with p.R583G in KCNQ1 in an isolated proband, where family data were not attainable. The p.G60D and p.D76N interfered with amino acid residues conserved in human, rat, mouse, cow and frog, whereas the alanine at residue 93 was only conserved in mouse, rat, cow and human. However, the N-terminus of frog minK is not conserved at all from amino acid residue 85 – 105 in the human sequence (corresponding to residue 80 – 95 in the frog minK sequence). However, the missense mutations p. Y81C, p.W87R, p.R98W, and p.P127T – as well as p.D76N, located in the cytoplasmic C-terminus of minK, have previously been associated with LQTS [2].

KCNE2 mutations

Two mutations were found, p.I57T and p.V65M, both previously described in other populations as associated with LQTS [57, 58] and shown to interfere with Kv7.1 function [59] in a way compatible with an association with LQTS. Both interfered with highly conserved residues and were not found in 384 control alleles.

Disease causation

The association between a mutation and disease is of paramount importance when the mutation findings are used for cascade screening and clinical management as is the case in Denmark [12]. This is a particular problem when family data are not sufficient to establish linkage. As none of the mutations are present in the 364 control alleles and all but three (SCN5A:p.P2005A, KCNE1:p.D76N and KCNE2:p.I57T) were absent from the EVS, moreover, the three variants reported here occurred at frequencies < 1:1000, it is therefore unlikely that the variants reported here are polymorphisms. Frame-shift, splice-site- nonsense- or indel mutations represents 31.2% of the mutations identified in this cohort (Tables 1, 2, 3). Such mutations have a direct effect on the integrity of the polypeptide chain and are considered explanatory for LQTS. However, the remaining 68.8% are missense mutations, that need to be differentiated from the naturally occurring functionally insignificant non-synonymous variants in the same proteins. A classical method of supporting disease causality in LQTS is electrophysiological examination of mutated channel proteins to disclose a reduction in repolarising K⁺- current (KCNQ1, KCNH2, KCNE1, KCNE2) or late persistence of depolarising Na⁺- current (SCN5A) [2]. Such information, however, was only available for 22.7% of the missense mutations (Tables 1, 2, 3).

The founder mutations in KCNH2

Two of the missense mutations, p.F29L [43] and p.K101E [44], were found in five and two “unrelated” families, respectively. Haplotype analysis, using six polymorphic microsatellite markers, spanning 24.6 cM, flanking the KCNH2 gene at distances ranging from 10.1 Mb 3’ to 2.9 Mb 5’ as shown in Figure 1, demonstrated that both mutations were founder mutations (Additional file 1: Table S1).

Population distribution of mutations