Long QT and Brugada syndrome gene mutations in New Zealand

Long QT and Brugada syndrome gene mutations in New Zealand Seo-Kyung Chung, BSc (Hons),*† Judith M. MacCormick, MB ChB,†‡ Caroline H. McCulley, BSc (Hons),* Jackie Crawford, NZCS,†‡ Carey-Anne Eddy, BSc (Hons), MSc (Med),†§ Edwin A. Mitchell, FRACP, FRCPCH, DSc,储 Andrew N. Shelling, BPhEd, BSc (Hons), PhD,†§ John K. French, BMedSc, MB ChB, MSc, PhD,†¶ Jonathan R. Skinner, MB ChB, FRACP, FRCPCH, MD,†‡ Mark I. Rees, BSc (Hons), PhD*†# *From the Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand, † Cardiac Inherited Disease Group, Auckland City Hospital, Grafton, Auckland, New Zealand, ‡ Greenlane Paediatric and Congenital Cardiac Services, Starship Hospital, Grafton, Auckland, New Zealand, § Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand, 储 Department of Paediatrics, University of Auckland, Auckland, New Zealand, ¶ Department of Cardiology, and South West Sydney Clinical School (UNSW) Liverpool Hospital, Sydney, Australia, # Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom. BACKGROUND Genetic testing in long QT syndrome (LQTS) is moving from research into clinical practice. We have recently piloted a molecular genetics program in a New Zealand research laboratory with a view to establishing a clinical diagnostic service. OBJECTIVE This study sought to report the spectrum of LQTS and Brugada mutations identified by a pilot LQTS gene testing program in New Zealand. METHODS Eighty-four consecutive index cases referred for LQT gene testing, from New Zealand and Australia, were evaluated. The coding sequence and splice sites of 5 LQTS genes (KCNQ1, HERG, SCN5A, KCNE1, and KCNE2) were screened for genomic variants by transgenomics denaturing high-performance liquid chromatography (dHPLC) system and automated DNA sequencing. RESULTS Forty-five LQTS mutations were identified in 43 patients (52% of the cohort): 25 KCNQ1 mutations (9 novel), 13 HERG mutations (7 novel), and 7 SCN5A mutations (2 novel). Forty

Long QT syndrome (LQTS) represents a diverse range of disorders associated with prolonged ventricular repolarization.1 After initial description in 1957,2 LQTS was regarded as a rare disorder. It is now widely accepted that the incidence of LQTS previously was underestimated,3 with milder clinical phenotypes being increasingly recognized.4,5

Supported by Cure Kids (Child Health Research Foundation of New Zealand), the Lion Foundation, Greenlane Research and Education Fund, the University of Auckland Vice-Chancellor Fund, and the John Neutze Fund. Jackie Crawford, Clinical Service Coordinator, is funded by a bursary from Medtronic. Address reprint requests and correspondence: Dr. Jon Skinner, Paediatric and Congenital Cardiac Services, Auckland City Hospital, Level 3, Building 32, Private Bag 92 189, Auckland 1030, New Zealand. E-mail address: [email protected]. (Received April 20, 2007; accepted June 21, 2007)

patients had LQTS, and 3 had Brugada syndrome. Mutations were identified in 14 patients with resuscitated sudden cardiac death: 4 KCNQ1, 5 HERG, 5 SCN5A. In 17 cases there was a family history of sudden cardiac death in a first-degree relative: 8 KCNQ1, 6 HERG, 2 SCN5A, and 1 case with mutations in both KCNQ1 and HERG. CONCLUSION The spectrum of New Zealand LQTS and Brugada mutations is similar to previous studies. The high proportion of novel mutations (40%) dictates a need to confirm pathogenicity for locally prevalent mutations. Careful screening selection criteria, cellular functional analysis of novel mutations, and development of locally relevant control sample cohorts will all be essential to establishing regional diagnostic services. KEYWORDS Long QT; Mutations; Arrhythmia; Ion channels; Sudden cardiac death (Heart Rhythm 2007;4:1306 –1314) © 2007 Heart Rhythm Society. All rights reserved.

Current estimates are that the incidence of LQTS gene mutations is 1 in 1,000 to 5,000.6,7 To date, 9 LQTS genes have been identified and are associated with specific phenotypes of LQTS. It is clinically relevant to regard these different genes as triggers for individual disorders, not only in their mode of presentation, but also in their response to ␤-blockers and the relative risk of sudden death.8,9 Determining genotype is imperative in optimizing treatment. Nearly one third of LQTS gene carriers have a QTc ⬍ 460 ms on electrocardiogram.10,11 Thus, genetic screening is vital in identifying with certainty those at risk of adverse events and of transmitting this risk to future generations. Once a causative mutation has been identified in the index case, the family members can undergo

1547-5271/$ -see front matter © 2007 Heart Rhythm Society. All rights reserved.

doi:10.1016/j.hrthm.2007.06.022

Chung et al

Long QT and Brugada Syndrome

cascade screening, testing for presence or absence of the known mutation. KCNQ1 (LQT1) and HERG (LQT2) encode ␣-subunits of the voltage-gated K⫹ channel and are responsible for IKs and IKr, respectively. Loss of function mutations results in prolongation of the QT interval. SCN5A (LQT3) encodes the ␣-subunit of a voltage-gated Na⫹ channel. Gain of function mutations in SCN5A cause long QT type 3, whereas those mutations creating a loss of function result in Brugada syndrome. KCNE1 (LQT5) and KCNE2 (LQT6) code for the ␤-subunit of the K⫹ channel, affecting IKs and IKr, respectively. Brugada syndrome, like LQTS, is a hereditary cause of sudden cardiac death with a variable clinical phenotype. Mutations in SCN5A have also been associated with sudden unexplained nocturnal death syndrome,12 cardiac conduction disorder,13 sudden infant death syndrome,14 sick-sinus syndrome,15 and dilated cardiomyopathy.16 Several LQTS genetic screening studies have been published, identifying over 600 mutations.7,17,18 In response to the clear clinical importance of genetic screening for LQTS and Brugada syndrome, we piloted a grant-funded university-based research program in New Zealand in 2001. We wished to identify the local spectrum of mutations and examine factors relevant to establishing a clinical genetic diagnostic service in a relatively small population (4.2 million). This report describes the spectrum of mutations identified and our early experience of this national pilot program.

Methods Patients suspected of having LQTS or Brugada syndrome were referred to our cardiac service. The initial presentations to medical services included personal symptoms (syncope, seizures, or resuscitated SCD), SCD of a relative, and the incidental finding of a prolonged QTc interval. Those whose history and electrocardiogram findings supported a clinical diagnosis of LQTS or Brugada syndrome proceeded to molecular genetic analysis. Informed consent for genetic testing was obtained in all cases, following the protocols established in our multicenter ethical approval from the regional ethics committee. Eighty-four index cases were screened for mutations in five genes associated with LQTS: KCNQ1, HERG, SCN5A, KCNE1, and KCNE2. DNA was extracted from blood samples using standard phenol-chloroform extraction. The coding sequence and splice sites of the 5 LQTS genes were screened for genomic variants by transgenomics dHPLC system and automated DNA sequencing as described below. DNA was amplified using a polymerase chain reaction (PCR). The exons and flanking intron boundaries of KCNQ1 and HERG were amplified using primers from previous reports.19,20 Primers for SCN5A, KCNE1, and KCNE2 were designed using Primer 3.0 program Whitehead Institute, Cambridge, MA (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi). Before dHPLC, PCR products were denatured at 95°C for 5 minutes and slowly cooled to 4°C (0.1°C/sec) using an

1307 automated program on the PTC-200 PCR machine (MJ Research Watertown, MA) to generate either homoduplex or heteroduplex molecules if a mismatch of base pairs was present. To identify single-nucleotide polymorphisms and mutations in LQTS genes, sequence variation was detected by dHPLC analysis in a mixture of DNA from index cases and normal control samples.21 dHPLC was performed on the Transgenomic 2100 Waver DNA fragment analysis system (Transgenomic, Omaha, NE) using a DNASep HT cartridge and Navigator version 1.5.1 software (2003, Transgenomic, Omaha, NE). For each amplimer assay, the optimal partial denaturing temperatures were determined using interpretation of the DNA melting properties by the Navigator version 1.5.1 software. PCR products of DNA samples with variant dHPLC profiles underwent electrophoresis on 1.5% agarose gels and were purified with the Qiaquick PCR purification kit (Qiagen, Inc, Hilden, Germany). The purified DNA fragments were sequenced using Big Dye Terminator kits and an ABI 3100 automated sequencer (Applied Biosystems, Foster City, CA) at the Center for Gene Technology, University of Auckland, New Zealand. The population frequency of single-nucleotide polymorphisms and suspected functional variants were assessed in 100 control chromosomes using restriction fragment length polymorphisms analysis if a suitable restriction enzyme was available. Candidate mutations that failed to generate restriction fragment length polymorphism changes were assessed by dHPLC of controls vs mutation positive profiles to test the frequency of the abnormal profile within the control population. Variants were designated as mutations by excluding them from normal population controls and aligning them against other GeneBank databases to assess the degree of evolutionary/phylogenetic conservation.

Results Of the 43 patients, 31 (72%) were female and 12 (28%) were male. The median age was 21 years, with a range of 0 to 60 years. Ethnicity data were as follows: 31 (78%) European, 5 (12%) Pacific, 4 (9%) New Zealand Maori, 2 (5%) Chinese, 1 (2%) Middle Eastern. The median corrected QT interval was 500 ms, with a range from 450 to 660 ms. Forty-five mutations were found in 43 patients, reflecting a detection rate of 52%: 25 KCNQ1, 13 HERG, and 7 SCN5A. Forty-two of these mutations, in 40 patients, were consistent with LQTS, whereas 3 SCN5A mutations were associated with Brugada syndrome. Five mutations were detected in more than 1 unrelated index case. In the KCNQ1 group, there were 3 patients with the mutation L266P, 2 with R360G, and 2 with R518X. A further 3 patients had a D774Y mutation in HERG, and 2 were positive for R1193Q in SCN5A. In total there were 12

1308 unrelated index cases sharing a mutation with at least 1 other individual. Of the 38 different mutations identified in this study, 17 (45%) were novel (Table 1, Figure 1), whereas 21 (55%) were previously reported (Table 2). Novel mutations were found in all 3 genes: 8 in KCNQ1, 7 in HERG, and 2 in SCN5A. Phylogenetic alignments for all novel missense variants are shown in Figure 2. Two cases of Jervell and Lange-Neilsen syndrome, associated with sensorineural deafness, segregated with KCNQ1 compound heterozygosity (R518X/G189fs94X) or consanguineous homozygosity (maternal and paternal del998-999[GT] ⫻ 2). One case of LQTS was associated with 2 frameshift mutations in KCNQ1 (IVS10-1G¡T) and HERG (Ins513[CTGCTG]). The majority of LQT1 mutations were missense mutations in regions encoding transmembrane domains, particularly S5 and S6 regions, and the intracellular C-terminal (Figure 3). Most of the HERG mutations were localized to within intracellular N- and C-terminal domains, with 3 HERG missense mutations detected in the S4 and S5 transmembrane domains (Figure 4). The majority of SCN5A mutations were identified in regions encoding intracellular domains (Figure 5). A SCN5A mutation in the DII-DIII linker (R1193Q) is represented in 2 Brugada cases and was previously identified as a determinant in a sudden unexplained nocturnal death syndrome case.12 This mutation was considered a polymorphism in certain populations22,23; however, functional analysis of R1193Q confirms a persistent sodium current that can explain QTc prolongation.24 No mutations were identified in KCNE1 and KCNE2. There was a personal history of syncope in 35 (81%) cases (Tables 3 and 4). Of these, 14 had required resuscitation, including 7 who had received DC cardioversion. Family history was positive for sudden unexpected death in a first-degree relative at age ⬍35 years in 17 (40%) cases. In total, of the 43 gene-positive patients, 28 (65%) had either a personal history of resuscitated sudden cardiac death, a family history of sudden death, or both. In these 28 patients, there were 29 mutations, of which 13 were in KCNQ1, 10 were in HERG, and 6 were in SCN5A. These mutations that were associated with severe clinical events made up 52% of the LQT1 mutations, 78% of the LQT2 mutations, 100% of the LQT3 mutations, and 67% of the mutations causing Brugada syndrome. Clinical management is not determined by the Cardiac Inherited Disease Group and is dependent on local cardiologist and patient preference. We are aware of 16 patients from our gene-positive cohort who were offered insertion of an implantable cardioverter defibrillator (ICD). Of these, 14 patients proceeded with ICD insertion and 2 declined. Patients offered ICDs included most of those who had suffered events requiring resuscitation and those with mutations in SCN5A. One patient, who had been resuscitated after near-drowning, suffered severe hypoxic ischemic encephalopathy and was not offered an ICD.

Heart Rhythm, Vol 4, No 10, October 2007 KCNQ1 T265I

*

KCNQ1 F296S

KCNQ1 del CT [998-999]

KCNQ1 F339S

KCNQ1 R360G

KCNQ1 H455Y

KCNQ1 IVS10-1 G>T

HERG I42N

*

*

KCNQ1 P630A

*

HERG ins513 [6bp]

HERG ins983 [13bp]

HERG dup 1871-1881

HERG N629T

HERG del 3094-3107

HERG P1075L

SCN5A R190Q

SCN5A insG 4295-4299

Figure 1 Panel of novel sequences: sequence panel of novel mutations identified in this study. The position of mutations are indicated by an arrow, and the sequence is displayed in forward direction unless marked otherwise (*).

Discussion Genetic testing in LQTS is moving from research laboratories into clinical practice. This study identifies and highlights several issues to be considered when developing a clinical diagnostic service. Substantial allelic heterogeneity in LQTS genes means molecular testing is complex, and consideration needs to be given to the best practice for genetic screening. Our study illustrates the frequent finding of previously unclassified variants when screening LQTS genes. To aid in interpretation of these abnormalities, it is essential to establish appropriate selection criteria for screening and to undertake careful correlation with clinical information. In addition, there is a need to develop large locally relevant control sample cohorts and to perform cellular functional studies to confirm pathogenicity of mutations. There is clear evidence to support the need for LQTS gene screening as a clinical test. Knowledge of genotype assists in offering appropriate therapeutic intervention to

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Long QT and Brugada Syndrome

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Figure 2 Phylogenetic alignments in A: amino acid sequence alignment of KCNQ1 novel mutations, B: amino acid sequence alignment of HERG novel mutations, C: amino acid sequence alignment of SCN5A novel mutations.

individuals at risk of life-threatening arrhythmia. There is no evidence showing that subjects with LQT 3 respond to ␤-blocker therapy (unlike LQT 1 and LQT2), meaning ICDs are required for symptomatic individuals. Appropriate targeting of therapies according to genotype is the major factor in making LQTS genetic testing cost-effective in the clinical setting.25 In addition, identifying the mutation in the index case enables cascade screening of family members. After detection of the LQTS mutations reported in this study, we have conducted genotype testing in over 170 at-risk family members. Of the first 84 LQTS and Brugada index patients screened in New Zealand for mutations in the coding sequence of 5 LQTS genes (KCNQ1, HERG, SCN5A, KCNE1, and KCNE2), there were 42 LQTS mutations and 3 Brugada mutations detected in 43 unrelated index cases.

This is a 52% detection rate, which is consistent with a recent study finding,7 but is slightly lower than previously published PCR-based studies.17,18 The detection rate is likely to depend on the screening criteria and on the degree of pretest clinical suspicion.7 In 17 families from our gene-positive group, a sudden death occurred. It was often many years before another family member presented with or was investigated for the diagnosis of LQTS. LQTS gene mutation analysis, together with clinical review of the family, needs to be integrated into the forensic investigation of young sudden death. Ensuring that there are pathways for this to occur is part of establishing a clinical service for LQTS testing. The relative preponderance of KCNQ1 mutations in our study, 60% (95% confidence interval 45% to 74%) of those causing LQTS may be due to chance or may be truly

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Figure 3 Mutations in KCNQ1. A: Schematic diagram of KCNQ1 genomic structure and approximate location of pathological mutations. B: Twodimensional schematic representation of predicted KCNQ1 polypeptide with locations of KCNQ1 mutations.

representative of the local population. Previous studies have reported the percentage of KCNQ1 mutations as 42% (95% confidence interval 37% to 47%).7,18 The recurrence of 3 KCNQ1 mutations in 7 unrelated index cases might well suggest a common ancestor effect. The results could, however, also reflect a referral bias: an event occurring with exercise (typical of LQT1) may be a more easily recognizable clinical presentation of LQTS than an episode during sleep, for example. In addition, both New Zealand and Australia have a youth culture with a strong emphasis on sporting activity, including water-based sports. Substantial allelic heterogeneity seems to be the common feature of global LQTS data sets,6,7,17,18,26 with the exception of Finland, where 4 KCNQ1 and HERG mutations account for 73% LQTS cases.27 This allelic heterogeneity makes LQTS gene mutation screening an expensive and challenging process. Alternative methods, such as hierarchical gene screening and genotype-phenotype analysis, could be considered to increase the efficiency of LQTS screening.28 Hierarchical mutational analysis is based on published data of gene prevalence and sequentially targets the most

likely sites of mutation. This approach might be less expensive and excessive in terms of the number of assays required. However, it is apparent from this and other studies that allelic and locus compound heterozygosity exists in LQTS cohorts.29,30 Without complete coding region coverage of at least LQT1-3 (and LQT5, 6), alternative screening approaches may miss those with 2 mutations. This will lead to underreporting of severely affected individuals. In addition, during family cascade testing, some individuals will be falsely reassured by the absence of a mutation within 1 LQTS gene, and yet remain at risk of sudden cardiac death from a mutation in another known LQTS gene. In contrast to hierarchical screening, the genotype-phenotype approach uses targeted screening based on the relationship between the phenotype and the genotype. It is known that the majority of LQT1 patients have events precipitated by physical exercise, whereas LQT2 patients are more likely to develop arrhythmia after emotion and LQT3 patients tend to be symptomatic at rest or during sleep.8 This approach would be an attractive option for rationalization of gene-testing if the genotype-phenotype relationship in LQTS was robust. However, it is known that

Figure 4 Mutations in HERG. A: Schematic diagram of HERG genomic structure and approximate location of pathogenic mutations. B: Two-dimensional schematic representation of predicted HERG polypeptide with locations of HERG mutations.

Chung et al Table 1

Long QT and Brugada Syndrome

1311

Novel LQTS mutations identified in this study

Sequence changes Mutations- KCNQ1 C794T T887C del [998-999] ⫻ 2 T1016C A1078G C1363T IVS10-1 G⬎T C1888G Mutations-HERG T125A ins513[6bp] ins983[13bp] dup [1871-1881] A1886C del [3094-3107] C3224T Mutations-SCN5A G569A insG[4295-4299]

Exon

Classification of mutation

Predicted consequence

Protein position

Patient number (refers to Table 3)

6 6 7 7 8 10 11 16

Missense Missense Homozygous deletion Missense Missense Missense Splice site alteration Missense

T265I F296S S333fs128X F339S R360G H455Y Altered exon splicing P630A

S5 S5/pore S6 S6 C-terminal C-terminal C-terminal C terminal

1 2 3 4 5,6 7 8* 9

2 4 5 7 7 13 14

Missense Insertion Insertion Duplication Missense Deletion Missense

I42N ins172 LL R328fs24X F627fs89X N629T G1031fs27X P1075L

N-terminal N-terminal N-terminal pore pore C-terminal C-terminal

10 8* 11 12 13 14 15

5 24

Missense Insertion

R190Q G1434fs1X

D1/S2-S3 DIII S6

16 17†

Numbering of all mutations is based on the full-length cDNA sequence (GenBank accession numbers AF000571 (KCNQ1), U04270 (HERG), AAK74065 (SCN5A)), with nucleotide 1 assigned to the first base of the start cordon (ATG) in accordance with current mutation nomenclature recommendations.31 *Novel locus heterozygosity (IVS10-1 G⬎T) in KCNQ1 and (ins513[CTGCTG]) in HERG was found in a severe case. †Identified in Brugada syndrome.

Table 2

Previously reported LQTS mutations detected in this study

Sequence Mutations- KCNQ1 G136A G502A insG567 T797C G805A C905T G947A G973A IVS7 -2A¡G C1066T C1552T C1637T G1781A Mutations-HERG C1600T G1704C G1714A G2320T Mutations-SCN5A C2074A G3578A A3974G A5302G

Exon

Classification of mutation

Predicted consequence

Protein position

Reference

Patient number (refers to Table 4)

1 3 3 6 6 6 7 7 8 8 12 13 15

Missense Missense Insertion Missense Missense Missense Missense Missense Splice site Nonsense Nonsense Missense Missense

A46T G168R G189fs94X L266P G269S A302V G316E G325R Frameshift Q356X R518X S546L R594Q

N terminal S2 S2-S3 S5 S5 Pore Pore S6 C-terminal C-terminal C-terminal C-terminal C-terminal

17 7, 26, 30, 32 7, 33 7, 18 7, 34-36 7, 36 17 18, 32, 37, 38 27 18 7, 18, 39-41 7, 36 7, 18, 26

18 19 20* 21,22,23 24 25 26 27 28 29 20*,30 31 32

7 7 7 9

Missense Missense Missense Missense

R534C W568C G572S D774Y

S4 S5 S5/pore C-terminal

7, 42, 43 44 7 7

33 34 35 36,37,38

14 20 23 28

Missense Missense Missense Missense

Q692K R1193Q N1325S I1768V

DI-DII DII-DIII DIII S4-S5 DIV S6

28 12, 45 7, 46 28, 47

39 40B,41† 42 43

All principles are identical to those in Table 1, except a list of publications are included that describe the detection of the recurrent mutations. *Heterozygous compound mutations (InsG567/R518X) were identified in a Jervell-Lange-Neilson syndrome proband. †Identified in Brugada syndrome.

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Table 3

Clinical information for patients in this study with novel mutations

Patient no.

Sequence changes

Diagnosis

Age (yrs)

Gender

Ethnicity

Syncope

RSCD

Identified trigger/s

QTc (ms)

C794T T887C del [998-999] ⫻ 2 T1016C A1078G A1078G C1363T IVS10-1/G⬎T/ins513 [6bp] C1888G T125A ins983[13bp] dup [1871-1881] A1886C del [3094-3107] C3224T G569A insG [4295-4299] (n)

LQT1 LQT1 JLNS LQT1 LQT1 LQT1 LQT1 LQT1/LQT2

40 14 ⬍1 44 9 15 38 60

F F M F M F F F

European European Middle East European European European European European

Y

Y

Rest/sibutramine

660 490 500 500 480 600 470 520

13 32 38 37 3 9 35 24 1

F F F F F M F M F

Pacific Maori Pacific Chinese European European European European European

Y Y Y Y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

LQT1 LQT2 LQT2 LQT2 LQT2 LQT2 LQT2 LQT3 Brugada

Y Y

Exercise Exercise (water)

Y Y Y* Y Y

Y Y Y Y

Y Y*

Postpartum Stress Rest/stress Intercurrent illness

560 540 480 480 500 460 500 480 450

SD of 1° relative

Y Y Y

Y Y Y

JLNS ⫽ Jervell and Lange-Neilson syndrome; RSCD ⫽ resuscitated sudden cardiac death; SD of 1° relative ⫽ Sudden death of first-degree relative at age ⬍35 years; Y* ⫽ required DC cardioversion.

Table 4

Clinical information for patients in this study with previously reported mutations

Patient no.

Sequence changes

Diagnosis

Age (yrs)

Gender

Ethnicity

Syncope

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

G136A G502A insG567/C1552T T797C T797C T797C G805A C905T G947A G973A IVS7 -2A¡G C1066T C1552T C1637T G1781A C1600T G1704C G1713A G2320T G2320T G2320T C2074A G3575A G3575A A3974G A5302G

LQT1 LQT1 JLNS LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT1 LQT2 LQT2 LQT2 LQT2 LQT2 LQT2 LQT3 Brugada Brugada LQT3 LQT3

57 39 26 35 19 12 42 12 8 53 35 42 3 9 9 12 50 9 31 21 29 10 ⬍1 9 52 16

F F F F F M M F M F F F F M M F F F F F F M M F M F

European European European European European European European Maori European European Maori European European European European Chinese European Pacific European European Maori European Pacific Pacific European European

Y Y Y Y Y Y Y Y

RSCD

Identified trigger/s

Y*

Stress Stress Exercise/stress General anesthetic

Y*

Exercise/stress Exercise (water)

Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Intercurrent illness Exercise Exercise/stress Exercise (water) Exercise (water) Exercise Postpartum Exercise Y

Stress

Y* Y* Y*

Rest Intercurrent illness Rest

Y

Y

QTc (ms)

SD of 1° relative

620 500 520 590 480 520 470 490 560 520 590 520 470 480 470 500 450 510 470 510 470 540 600 530 460 630

Y Y Y Y Y

Y Y Y

Y Y Y

JLNS ⫽ Jervell and Lange-Neilson syndrome; RSCD ⫽ resuscitated sudden cardiac death; SD of 1° relative ⫽ Sudden death of first-degree relative at age ⬍35 years; Y* ⫽ required DC cardioversion.

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Figure 5 Mutations in SCN5A. A: Schematic diagram of SCN5A genomic structure and approximate location of pathogenic mutations. B: Twodimensional schematic representation of predicted SCN5A polypeptide with locations of SCN5A mutations.

clinical manifestations of LQTS are highly variable even among the carriers of the same mutation; patients with LQT1 still may die in their sleep.

Conclusions Our study identified 17 unique novel mutations and 21 different previously reported mutations causing LQTS. The latter have been described in the literature with varying levels of support for mutation pathogenicity. To date, 648 independent mutations in LQTS genes have been reported (http://www.fsm.it/cardmoc/), and our data expands this repertoire to 665 LQTS mutations. There has been a sizeable amount of previous sequencing across LQTS coding regions in other larger LQTS cohorts as well as this study. This provides additional confirmation of the unique nature of our novel mutations, despite our relatively small and underpowered control-sample cohort. The importance of the development of large locally relevant and ethnically matched control samples is clear. In addition, we are currently involved in comprehensive collaborative studies to characterize the biophysical properties of the novel mutations, as well as those recurrent mutations without previous functional characterization. Thus, broadly speaking, the findings of this modest-sized cohort are consistent with those from larger cohorts. We confirm that any such diagnostic program must use full screening in index cases of at least the 3 most common genes associated with LQTS; KCNQ1, HERG, and SCN5A. The high proportion of novel mutations (40%) dictates a need to confirm pathogenicity for locally prevalent mutations. Careful screening selection criteria, cellular functional analysis of novel mutations, and development of locally relevant control sample cohorts all will be essential to establishing regional diagnostic services for LQTS.

Acknowledgements The authors thank Drs. Joanne Dixon, Hugh McAlister, and Ian Crozier for allowing us to include data on their patients.

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