Haplotype-Sharing Analysis Implicates Chromosome 7q36 Harboring DPP6 in Familial Idiopathic Ventricular Fibrillation
2009; Elsevier BV; Volume: 84; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2009.02.009
ISSN1537-6605
AutoresMariëlle Alders, Tamara T. Koopmann, Imke Christiaans, Pieter G. Postema, Leander Beekman, Michael W.T. Tanck, Katja Zeppenfeld, Peter Loh, Karel T. Koch, Sophie Demolombe, Marcel M.A.M. Mannens, Connie R. Bezzina, Arthur A.M. Wilde,
Tópico(s)Viral Infections and Immunology Research
ResumoIdiopathic Ventricular Fibrillation (IVF) is defined as spontaneous VF without any known structural or electrical heart disease. A family history is present in up to 20% of probands with the disorder, suggesting that at least a subset of IVF is hereditary. A genome-wide haplotype-sharing analysis was performed for identification of the responsible gene in three distantly related families in which multiple individuals died suddenly or were successfully resuscitated at young age. We identified a haplotype, on chromosome 7q36, that was conserved in these three families and was also shared by 7 of 42 independent IVF patients. The shared chromosomal segment harbors part of the DPP6 gene, which encodes a putative component of the transient outward current in the heart. We demonstrated a 20-fold increase in DPP6 mRNA levels in the myocardium of carriers as compared to controls. Clinical evaluation of 84 risk-haplotype carriers and 71 noncarriers revealed no ECG or structural parameters indicative of cardiac disease. Penetrance of IVF was high; 50% of risk-haplotype carriers experienced (aborted) sudden cardiac death before the age of 58 years. We propose DPP6 as a gene for IVF and increased DPP6 expression as the likely pathogenetic mechanism. Idiopathic Ventricular Fibrillation (IVF) is defined as spontaneous VF without any known structural or electrical heart disease. A family history is present in up to 20% of probands with the disorder, suggesting that at least a subset of IVF is hereditary. A genome-wide haplotype-sharing analysis was performed for identification of the responsible gene in three distantly related families in which multiple individuals died suddenly or were successfully resuscitated at young age. We identified a haplotype, on chromosome 7q36, that was conserved in these three families and was also shared by 7 of 42 independent IVF patients. The shared chromosomal segment harbors part of the DPP6 gene, which encodes a putative component of the transient outward current in the heart. We demonstrated a 20-fold increase in DPP6 mRNA levels in the myocardium of carriers as compared to controls. Clinical evaluation of 84 risk-haplotype carriers and 71 noncarriers revealed no ECG or structural parameters indicative of cardiac disease. Penetrance of IVF was high; 50% of risk-haplotype carriers experienced (aborted) sudden cardiac death before the age of 58 years. We propose DPP6 as a gene for IVF and increased DPP6 expression as the likely pathogenetic mechanism. Sudden cardiac death (SCD) is the major cause of mortality in developed countries, and the majority of SCD is caused by ventricular fibrillation (VF).1Zipes D.P. Camm A.J. Borggrefe M. Buxton A.E. Chaitman B. Fromer M. Gregoratos G. Klein G. Moss A.J. Myerburg R.J. et al.ACC/AHA/ESC 2006 Guidelines for Management of Patients With Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death: a report of the American College of Cardiology/American Heart Association Task Force and the European Society of Cardiology Committee for Practice Guidelines (writing committee to develop Guidelines for Management of Patients With Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death): developed in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society.Circulation. 2006; 114: e385-e484Crossref PubMed Scopus (894) Google Scholar, 2Consensus Statement of the Joint Steering Committees of the Unexplained Cardiac Arrest Registry of Europe and of the Idiopathic Ventricular Fibrillation Registry of the United StatesSurvivors of out-of-hospital cardiac arrest with apparently normal heart. Need for definition and standardized clinical evaluation.Circulation. 1997; 95: 265-272Crossref PubMed Scopus (180) Google Scholar, 3Zipes D.P. Wellens H.J. Sudden cardiac death.Circulation. 1998; 98: 2334-2351Crossref PubMed Scopus (1560) Google Scholar In the absence of identifiable structural heart disease or known repolarization abnormalities, it is referred to as idiopathic ventricular fibrillation (IVF [MIM 603829]). IVF accounts for as many as 10% of sudden deaths, mainly in the young.2Consensus Statement of the Joint Steering Committees of the Unexplained Cardiac Arrest Registry of Europe and of the Idiopathic Ventricular Fibrillation Registry of the United StatesSurvivors of out-of-hospital cardiac arrest with apparently normal heart. Need for definition and standardized clinical evaluation.Circulation. 1997; 95: 265-272Crossref PubMed Scopus (180) Google Scholar The recurrence of VF in patients with IVF is about 30%, and the only effective therapy is implantation of an implantable cardioverter defibrillator (ICD).2Consensus Statement of the Joint Steering Committees of the Unexplained Cardiac Arrest Registry of Europe and of the Idiopathic Ventricular Fibrillation Registry of the United StatesSurvivors of out-of-hospital cardiac arrest with apparently normal heart. Need for definition and standardized clinical evaluation.Circulation. 1997; 95: 265-272Crossref PubMed Scopus (180) Google Scholar, 4Champagne J. Geelen P. Philippon F. Brugada P. Recurrent cardiac events in patients with idiopathic ventricular fibrillation, excluding patients with the Brugada syndrome.BMC Med. 2005; 3: 1Crossref PubMed Scopus (28) Google Scholar In up to 20% of IVF cases, a family history of sudden cardiac death or IVF is present, suggesting that at least a subset of IVF is hereditary.5Haissaguerre M. Shoda M. Jaïs P. Nogami A. Shah D.C. Kautzner J. Arentz T. Kalushe D. Lamaison D. Griffith M. et al.Mapping and ablation of idiopathic ventricular fibrillation.Circulation. 2002; 106: 962-967Crossref PubMed Scopus (510) Google Scholar, 6Noda T. Shimizu W. Taguchi A. Aiba T. Satomi K. Suyama K. Kurita T. Aihara N. Kamakura S. Malignant entity of idiopathic ventricular fibrillation and polymorphic ventricular tachycardia initiated by premature extrasystoles originating from the right ventricular outflow tract.J. Am. Coll. Cardiol. 2005; 46: 1288-1294Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7Viskin S. Belhassen B. Idiopathic ventricular fibrillation.Am. Heart J. 1990; 120: 661-671Abstract Full Text PDF PubMed Scopus (178) Google Scholar Because no cardiac abnormalities are observed in IVF patients, family members that may be at risk cannot be identified. Elucidation of underlying genetic defects will provide more insight into the pathogenesis of the disorder and, crucially, will allow presymptomatic identification of individuals at risk. The identification of genes involved in IVF is very difficult. Classical linkage analysis is hampered for several reasons. Unlike that of other monogenic arrhythmia syndromes, the diagnosis of the disorder cannot be made on the basis of ECG abnormalities; it can be made only after the occurrence of (aborted) sudden cardiac death. Many affected patients die young, thus leaving only small numbers of patients and material available for analysis. We set out to identify the culprit gene in three families (families A, B, and C; Figure 1) in which multiple individuals died suddenly or were successfully resuscitated from VF at young age. These families originate from the same area in The Netherlands and are genealogically linked through multiple lines. This unique situation of three distantly related IVF families enabled a gene-identification strategy consisting of searching for haplotypes shared in affected patients. This method identifies chromosomal segments that are identical by descent (IBD) and are likely to harbor the disease-causing gene.8He B. Giedraitis V. Ligers A. Binzer M. Andersen P.M. Forsgren L. Sandkuijl L.A. Hillert J. Sharing of a conserved haplotype suggests a susceptibility gene for multiple sclerosis at chromosome 17p11.Eur. J. Hum. Genet. 2002; 10: 271-275Crossref PubMed Scopus (14) Google Scholar, 9Houwen R.H. Baharloo S. Blankenship K. Raeymaekers P. Juyn J. Sandkuijl L.A. Freimer N.B. Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis.Nat. Genet. 1994; 8: 380-386Crossref PubMed Scopus (266) Google Scholar Informed consent was obtained from all individuals studied. The proband of family A (A-1) visited our cardiogenetics outpatient clinic because of the SCD of her younger brother and sister. Her brother died suddenly at age 31, in the early morning, and postmortem examination did not reveal a cause of death or cardiac abnormalities. Her sister died suddenly at age 31, at night. She underwent complete cardiac examination, which was unremarkable, a month before her death. Cardiac evaluation of the proband A-1 did not reveal any abnormalities. Six months after this examination, she suffered VF at age 44 and was successfully resuscitated. Implantation of an ICD followed, and it has never discharged during a follow-up period of 2.5 years. Just a few months later, her nephew (A-2) was also resuscitated from VF. He had an ICD implanted, and it discharged appropriately several times in the next two years after his first event. The proband of family B (B-1) and his children contacted our cardiogenetics outpatient clinic because of the SCD of his two sons, who died at age 37 in rest and at age 32 during sleep, respectively. Postmortem examination revealed no cause of death, and cardiac abnormalities were absent in both. Although B-1 had no history of arrhythmias, he was believed to be an obligate carrier of the disease, because three siblings of his mother also died suddenly at young age. Cardiac examination ([exercise-]ECG, echocardiogram) of the proband and his children revealed no cardiac abnormalities. The proband of family C (C-1) was admitted to our hospital after resuscitation from VF at age 33. His cardiac examination revealed no abnormalities, and an ICD was implanted. During a follow-up of four years, he had several appropriate ICD discharges. Two sons of his great grandmother had died suddenly at age 30. Eight individuals from these three families, indicated by arrows in Figure 1, were genotyped by genome-wide SNP analysis with the use of Illumina HumanHap 300 BeadChips. The obtained genotypes were analyzed in Microsoft Office Access. Haplotypes of individuals A-1 and B-1 were constructed by defining which allele was and which allele was not transmitted to the offspring. Each haplotype of A-1 was compared with each haplotype of B-1 and with the genotypes of patients A-2 and C-1. Shared segments of >5 contiguous SNPs were plotted (Figure 2A). One large haplotype of 301 contiguous SNPs on chromosome 7q36 was shared between the three families (Figure 2A), and this was confirmed by additional genotyping of microsatellite markers within this segment (D7S483, D7S798, D7S1491, D7S2462, D7S2546, D7S2447, D7S1823). Identity by descent of four smaller shared segments of >100 SNPs on chromosomes 2, 5, 6, and 12 was excluded by microsatellite analysis (chr12: D12S88, D12S365; chr2: D2S2196, D2S1334; chr5: D5S2086, D5S2024; chr6: D6S2986). Primer sequences of all markers were obtained from the UniSTS database (see Web Resources). Genomic DNA was amplified in the presence of Cy3 dCTP, and fragments were separated on an ABI310 genetic analyzer (Applied Biosystems). Results were processed with Genemapper software (Applied Biosystems). The shared haplotype on chromosome 7q36 was actually 350 SNPs in length when patients B-1, A-1, and C-1 were compared. A recombination occurred when the haplotype was transmitted to A-2, decreasing the size of the shared haplotype to 301 SNPs. This haplotype was bordered by SNPs rs940261 and rs4960710. It was 2.5Mb in length and contained only four validated genes: HTR5A [MIM 601305], PAX-IP1 [MIM 608254], DPP6 [MIM 126141], and exons 10–12 of ACTR3B (Figure 2B). DPP6 encoding dipeptidyl-peptidase 6 was considered particularly interesting, because it encodes a putative subunit of potassium-channel complexes. It is predominantly expressed in brain, where it is associated with Kv4 potassium channels and Kv channel-interacting protein (KChIP) and underlies the transient subthreshold-activating, somatodendritic A-type potassium current (ISA).10Nadal M.S. Ozaita A. Amarillo Y. Vega-Saenz de Miera E. Ma Y. Mo W. Goldberg E.M. Misumi Y. Ikehara Y. Neubert T.A. et al.The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels.Neuron. 2003; 37: 449-461Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar Studies by Radicke et al.11Radicke S. Cotella D. Graf E.M. Ravens U. Wettwer E. Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative beta-subunit of human cardiac transient outward current encoded by Kv4.3.J. Physiol. 2005; 565: 751-756Crossref PubMed Scopus (106) Google Scholar showed expression in heart and are suggestive for a role of DPP6 in cardiac transient outward current (Ito). Three human isoforms are known, and they differ at the N terminus (GenBank, NIH). Direct sequencing of all 28 coding exons in patients B-1, A-1, and C-1 revealed no mutations in the coding sequences of DPP6. However, a C→T transition 340 bases upstream of the ATG initiation codon of isoform 2 of the DPP6 gene ( NM_001936 ) was identified in all three patients. It was not present in a control group of 350 Dutch individuals of European descent (700 alleles) and is specific for this particular haplotype. For examination of the involvement of DPP6 in other IVF families, 42 probands of additional, independent families with one or more cases of IVF were screened for mutations in DPP6. These probands contacted our cardiogenetics outpatient clinic or were admitted to our hospital within the last 12 years (1996–2008). IVF was diagnosed if spontaneous VF occurred in the absence of electrolyte abnormalities, antiarrhythmic drugs, and any known cardiac disease as based on routine cardiac investigation, including ECG, exercise-ECG, Holter monitoring, echocardiography, coronary angiography, and MRI. In the case of unexplained sudden cardiac death, IVF was diagnosed in the absence of an identifiable cause of death upon autopsy and/or no cardiac abnormalities in relatives upon cardiac investigation. No mutations were detected in the coding region of DPP6 in these 42 probands. However, the same variant c.1–340C→T in isoform 2 of DPP6 was identified in seven additional families. Additional haplotyping with microsatellite markers revealed that the extended haplotype was also shared between these families. However, in four of these families (families E, G, H, and I; Figure 3), the shared chromosomal region did not span the full length of the original haplotype. Recombinations must have occurred between D7S2546 and rs3807218 in families G and E, between the DPP6 variant c.1–340C→T and D7S2546 in intron 3 of DPP6 in family H, and between D7S1491 and D7S798 in families E and I (Figure 3, recombinations indicated by arrows), reducing the size of the risk haplotype to approximately 1.5 Mb and, importantly, excluding ACTR3B, PAXIP1, and HTR5A from the shared region (Figure 2B). The identification of the variant in a substantial proportion of this independent patient group (7/42; 16.6%) and its absence in 350 controls strongly implies that this haplotype contains the disease-causing mutation (p = 1.2 × 10−7, Fisher's exact test). It also implies that along with families A, B, and C, the seven additional families are also descended from the same ancestor. Genealogical research identified links between most families >5 generations ago. The probands of families H and G, however, were linked more closely and were found to be third cousins. Multipoint parametric linkage analysis in the families with multiple affected individuals (families A, B, G+H, and I) was performed with the use of the easyLinkage software package12Lindner T.H. Hoffmann K. easyLINKAGE: a PERL script for easy and automated two-/multi-point linkage analyses.Bioinformatics. 2005; 21: 405-407Crossref PubMed Scopus (149) Google Scholar running the GENEHUNTER v2.1 program,13Kruglyak L. Daly M.J. Reeve-Daly M.P. Lander E.S. Parametric and nonparametric linkage analysis: a unified multipoint approach.Am. J. Hum. Genet. 1996; 58: 1347-1363PubMed Google Scholar, 14Kruglyak L. Lander E.S. Faster multipoint linkage analysis using Fourier transforms.J. Comput. Biol. 1998; 5: 1-7Crossref PubMed Scopus (185) Google Scholar with the assumption of an autosomal-dominant pattern of inheritance, a disease-allele frequency of 0.0001, and a disease penetrance of 0.70. This yielded a maximum additive LOD score of 5.9. Despite the lack of mutations in the coding sequence of DPP6, it remains the only gene in the shared region. Because mutations outside coding sequences are also known to affect gene expression, we analyzed expression of DPP6 in heart biopsies of five risk-haplotype carriers (A-2, C-1, H-2, I-1, and J-1; Figure 3). The cardiac biopsies from these patients were taken from the right interventricular septum (IVS) and were immediately frozen in liquid nitrogen. Control heart biopsies (n = 13) were taken from the IVS of hearts of controls (n = 3; Academic Medical Center Amsterdam) or from explanted nondiseased human hearts (n = 10, University of Szeged) that were technically unusable for transplantation and previously described by Gaborit et al.15Gaborit N. Le Bouter S. Szuts V. Varro A. Escande D. Nattel S. Demolombe S. Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart.J. Physiol. 2007; 582: 675-693Crossref PubMed Scopus (353) Google Scholar All experimental protocols were approved by the Ethical Review Committees of the Academic Medical Center Amsterdam and the Medical Center of the University of Szeged (No. 51-57/1997 OEJ). Total RNA was extracted from pulverized heart tissue with RNA-Bee reagent (Tel-Test) or RNeasy Fibrous Tissue Mini Kit (QIAGEN) and reverse transcribed with Superscript II or Superscript III (Invitrogen) and random hexamers, in accordance with the manufacturer's protocol. All five patients were heterozygous for SNP rs3807218 in exon 7 of DPP6. Amplification of DPP6 cDNA, with the use of primers in exons 6 and 8 (present in all isoforms), and subsequent sequencing showed an imbalance in expression between the two alleles (Figure 4A). The A allele of rs3807218, representing the DPP6 risk haplotype (see Figure 3), was overrepresented relative to the G allele, suggesting increased expression of DPP6 from the risk-haplotype allele. To confirm upregulation of DPP6 expression in these heart biopsies, we performed real-time quantitative PCR on a Roche LightCycler 480 Real-Time PCR System, using primers that recognize all DPP6 transcripts and primers specific for transcript isoforms 2 and 3 (primer sequences available upon request). Isoform 1 was not tested, because no expression of this isoform could be detected in heart. Values were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression levels, and measurements were done in triplicate. Data were analyzed with LinRegPCR software and were corrected for between-session variation as described previously.16Ruijter J.M. Thygesen H.H. Schoneveld O.J.L.M. Das A.T. Berkhout B. Lamers W.H. Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology.Retrovirology. 2006; 3: 2Crossref PubMed Scopus (134) Google Scholar Average overall DPP6 expression was increased 22-fold in patients compared to controls (p = 0.01, Mann-Whitney U test) (Figure 4B). Both isoforms 2 and 3 exhibited increased expression, although increased expression was more pronounced for isoform 2 (21-fold; p = 0.03, Mann-Whitney U test) than for isoform 3 (8-fold; p = 0.03, Mann-Whitney U test). Expression of the neighboring genes ACTRB3 and PAXIP1 was not elevated (data not shown), suggesting that the upregulation of DPP6 is specific rather than due to a general increase of expression of genes in the region. For evaluation of the clinical consequences of carrying the risk haplotype, a total of 155 relatives from the ten IVF families sharing the identical haplotype on chromosome 7q36 were genotyped and risk-haplotype carrier status was determined. Individuals with otherwise unexplained SCD at an age of 15 years of age were not significantly different between carriers and noncarriers of the risk haplotype (Table 1). ECG characteristics of early repolarization17Haïssaguerre M. Derval N. Sacher F. Jesel L. Deisenhofer I. de Roy L. Pasquié J.L. Nogami A. Babuty D. Yli-Mayry S. et al.Sudden cardiac arrest associated with early repolarization.N. Engl. J. Med. 2008; 358: 2016-2023Crossref PubMed Scopus (1040) Google Scholar were found in 3 of 47 risk-haplotype carriers (6%) and in none of the noncarriers (not significant). Ajmaline provocation tests in six high-risk individuals (three of which had had an event) did not reveal any sign of Brugada syndrome.Table 1Demographic Data, Method of Identification, Events, and ECG Characteristics in 155 Individuals from Ten Families with IVF Linked to 7q36: Defined by HaplotypeRisk-Haplotype Carrier (n = 84)Risk-HaplotypeNoncarrier (n = 71)p ValueDemographicsMean age (years)41 ± 2032 ± 210.01Age < 50 years of age58 (69)54 (76)0.37Male (%)51 (61)30 (24)0.02Method of IdentificationDNA haplotype70 (83)70 (99)Obligate∗6 (7)1 (1)SCD 15 years of age.Differences between high- and low-risk patients were analyzed with SOLAR.23Almasy L. Blangero J. Multipoint quantitative trait linkage analysis in general pedigrees.Am. J. Hum. Genet. 1998; 62: 1198-1211Abstract Full Text Full Text PDF PubMed Scopus (2512) Google Scholar p values < 0.05 were considered significant. Open table in a new tab Data are mean ± SD or number of patients (%). SCD denotes sudden cardiac death, asterisk indicates haplotype inferred. ECG characteristics are obtained from 47 haplotype carriers and 43 noncarriers, all >15 years of age. Differences between high- and low-risk patients were analyzed with SOLAR.23Almasy L. Blangero J. Multipoint quantitative trait linkage analysis in general pedigrees.Am. J. Hum. Genet. 1998; 62: 1198-1211Abstract Full Text Full Text PDF PubMed Scopus (2512) Google Scholar p values < 0.05 were considered significant. In three individuals who were resuscitated from VF, mode of spontaneous VF onset was recorded from their ICD (n = 3) or during an invasive electrophysiological study (EPS; n = 1). This showed relatively short-coupled isolated monomorphic extrasystoles, occasionally eliciting immediate VF (Figure 5). No specific electrocardiographic abnormalities (e.g., early repolarization, right precordial ST elevation, QT prolongation) preceded the VF episode. A part of spontaneous VF episodes was preceded by several single monomorphic extrasystoles (such as in Figure 5), whereas other episodes were initiated from regular sinus rhythm by one single extrasystole. During EPS, earliest activation of the extrasystoles was mapped to the right ventricular lower free anterior wall, compatible with the morphology on the 12-lead ECG (Figure 5). Because no abnormalities can be demonstrated using cardiac diagnostic methods in IVF, the only phenotype of the disease that can be studied is (aborted) SCD. Median survival (50%) in risk-haplotype carriers was 58 years (95% confidence interval 46–69 years; Figure 6). For the noncarriers, median age of survival could not be calculated because of the lack of events. Only one noncarrier died (no SCD); his haplotype could be reconstructed by haplotyping of his children. All other noncarriers are alive; this corresponds with survival being necessary for the obtaining of a blood sample for DNA tests. Thirty risk-haplotype carriers experienced a clinical event: 19, unexplained SCD; 11, resuscitated VF. The youngest individual experiencing an event was 16 years of age, and the oldest was 77 years of age. The mean age at event was 36 ± 13 years. Males were overrepresented in this group: they had 23 events, whereas there were seven events in females. Unlike other arrhythmia syndromes, such as Brugada syndrome or Long-QT syndrome, penetrance of IVF cannot be assessed on the basis of an electrocardiographic phenotype. In these syndromes, penetrance of ECG characteristics is variable but relatively high compared to the low penetrance of arrhythmic (lethal) events.18Goldenberg I. Moss A.J. Long QT syndrome.J. Am. Coll. Cardiol. 2008; 51: 2291-2300Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar However, penetrance of lethal events linked to the chromosome 7q36 risk haplotype is very high; only 50% survival at 58 years. Therefore, any method that allows identification of the subset of the presymptomatic family members at such a high risk of arrhythmic events is potentially lifesaving. Currently, we use haplotype analysis to identify relatives at risk and treat accordingly (i.e., ICD implant). Furthermore, noncarriers of the risk haplotype in these families can be reassured of the absence of an increased risk of SCD for themselves and their offspring. This strategy is, at this time, applicable only to the families carrying this specific haplotype, and a reliable classification is made only in cases in which the entire shared haplotype (between D7S1491 and D7S2546) is present or absent. Because the causal mutation might be present anywhere on the shared segment, the risk status of asymptomatic individuals carrying part of the risk haplotype (as a consequence of a recombination event) is considered uncertain. Currently, we can speculate only on how overexpression of DPP6 causes IVF. DPP6 is predominantly expressed in brain, where it is a component of the neuronal K+ current ISA, but it is also considered an essential component of the native transient K+ current (ITO) channel complex in human heart.11Radicke S. Cotella D. Graf E.M. Ravens U. Wettwer E. Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative beta-subunit of human cardiac transient outward current encoded by Kv4.3.J. Physiol. 2005; 565: 751-756Crossref PubMed Scopus (106) Google Scholar Recent studies have shown that the characteristic kinetics of the native human cardiac ITO are recapitulated only when Kv4.3 is coexpressed with DPP6 in addition to the β-subunit KChIP2.11Radicke S. Cotella D. Graf E.M. Ravens U. Wettwer E. Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative beta-subunit of human cardiac transient outward current encoded by Kv4.3.J. Physiol. 2005; 565: 751-756Crossref PubMed Scopus (106) Google Scholar In particular, DPP6 significantly alters the inactivation kinetics of both Kv4.2 and Kv4.3 and promotes expression of these alpha subunits in the cell membrane, as observed in different heterologous expression systems.10Nadal M.S. Ozaita A. Amarillo Y. Vega-Saenz de Miera E. Ma Y. Mo W. Goldberg E.M. Misumi Y. Ikehara Y. Neubert T.A. et al.The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels.Neuron. 2003; 37: 449-461Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 11Radicke S. Cotella D. Graf E.M. Ravens U. Wettwer E. Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative beta-subunit of human cardiac transient outward current encoded by Kv4.3.J. Physiol. 2005; 565: 751-756Crossref PubMed Scopus (106) Google Scholar, 19Soh H. Goldstein S.A. I SA channel complexes include four subunits each of DPP6 and Kv4.2.J. Biol. Chem. 2008; 283: 15072-15077Crossref PubMed Scopus (22) Google Scholar Kv4 subunits appear to assemble in a 1:1 stoichiometry, meaning that such channels carry four subunits each of Kv4 and DPP6 subunits.19Soh H. Goldstein S.A. I SA channel complexes include four subunits each of DPP6 and Kv4.2.J. Biol. Chem. 2008; 283: 15072-15077Crossref PubMed Scopus (22) Google Scholar Studies using Kv4-DPP6 fusion proteins enforcing a 1:1 Kv4:DPP6 subunit ratio generated channel complexes with biophysical properties similar to those of naturally assembled channels. Of relevance to our finding of increased DPP6 expression in the patients, coexpression of additional free DPP6 to these Kv4-DPP6 fusion channels did not affect channel kinetics, although it is well possible that translocation of Kv alpha subunits at the cell membrane is promoted, given that some increase in current amplitude was observed.19Soh H. Goldstein S.A. I SA channel complexes include four subunits each of DPP6 and Kv4.2.J. Biol. Chem. 2008; 283: 15072-15077Crossref PubMed Scopus (22) Google Scholar The ITO current, which mediates early (phase 1) cardiomyocyte repolarization, is distributed heterogeneously across the ventricular wall, with ITO being more prominent in epicardium compared to endocardium. DPP6 mRNA expression, however, appears similar between epicardium and endocardium,11Radicke S. Cotella D. Graf E.M. Ravens U. Wettwer E. Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative beta-subunit of human cardiac transient outward current encoded by Kv4.3.J. Physiol. 2005; 565: 751-756Crossref PubMed Scopus (106) Google Scholar and unless the risk haplotype disrupts this uniform expression, any increase in ITO resulting from increased DPP6 mRNA in the patients might not necessarily disturb the transmural ITO gradient. In such a scenario, any increase in ITO amplitude on the cardiac action-potential morphology would be expected to follow the background ITO levels in the different myocardial layers. In epicardium, a deeper phase 1 may ensue with the appearance of a J-wave on the electrocardiogram,20Yan G.-X. Antzelevitch C. Cellular Basis for the Electrocardiographic J Wave.Circulation. 2006; 93: 372-379Crossref Scopus (638) Google Scholar a phenomenon that has recently been shown to precede ventricular arrhythmias in patients with idiopathic VF (the “early repolarization syndrome”).17Haïssaguerre M. Derval N. Sacher F. Jesel L. Deisenhofer I. de Roy L. Pasquié J.L. Nogami A. Babuty D. Yli-Mayry S. et al.Sudden cardiac arrest associated with early repolarization.N. Engl. J. Med. 2008; 358: 2016-2023Crossref PubMed Scopus (1040) Google Scholar An additional increase in ITO potentially leads to ST-segment elevation. However, no electrophysiological abnormalities were observed on the baseline ECG in carriers of the risk haplotype and no differences in prevalence of early repolarization were observed between haplotype carriers and noncarriers. Importantly, arrhythmic episodes appeared not to be preceded by any discernable electrocardiographic abnormality. Any morphological action-potential changes that might be present are apparently not sufficient to inscribe on the ECG and, therefore, are as yet obscuring the nature of the arrhythmogenic substrate, which in fact might be very local, in the lower part of the right ventricular free wall. The increased DPP6 mRNA levels observed are expected to be the consequence of mutations in regulatory sequences of the gene. The c.1-340C→T variant on the risk haplotype could underlie this effect. It is in a stretch of bases conserved among dog, mouse, and rat, and it potentially creates a GATA-1-like binding site (TGATAC) on the reverse strand.21Hasegawa M. Nishiyama C. Nishiyama M. Akizawa Y. Mitsuishi K. Ito T. Kawada H. Furukawa S. Ra C. Okumura K. Ogawa H. A novel −66T/C polymorphism in Fc epsilon RI alpha-chain promoter affecting the transcription activity: possible relationship to allergic diseases.J. Immunol. 2003; 171: 1927-1933PubMed Google Scholar However, it also remains possible that on the risk haplotype, an as yet unidentified sequence change, which could even be located tens or hundreds of kilobases away from the initiation codon, could still underlie the increased DPP6 expression from the risk haplotype.22Kleinjan D.A. van Heyningen V. Long-Range Control of Gene Expression: Emerging Mechanisms and Disruption in Disease.Am. J. Hum. Genet. 2005; 76: 8-32Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar In total, 10 of the 45 families studied carry the risk haplotype, suggesting that approximately 20% of cases in our clinic (which serves as a referral center for a large area in The Netherlands) are due to a founder mutation. Additional studies in cohorts of other ethnicities will have to establish to which extent DPP6 underlies IVF in other populations. In conclusion, we provide evidence for a familial component in IVF and identified a risk locus at chromosome 7q36. Our data support a role for DPP6 as the causal gene, and we propose overexpression of this gene as the pathogenic mechanism. Identification of the genetic basis of IVF enables presymptomatic genetic diagnosis for a disorder of which the first and only symptom is potentially lethal arrhythmia. This study was supported by The Netherlands Heart Foundation, grants 2005T024 (C.R.B.) and 2003T302 (A.A.M.W.); the Interuniversity Cardiology Institute of The Netherlands, project 27; ZonMw, project 62000010; and Fondation Leducq Transatlantic Network of Excellence, grant 05CVD01 (Preventing Sudden Cardiac Death). None of the authors have a financial interest related to this work. We thank J.M. Ruijter for assistance with statistical analysis of the quantitative PCR experiments, G.J. te Meerman for advice on the haplotype-sharing analysis, D.M. Roden for helpful discussions, and A.V. Postma for help with the LOD-score calculations. We thank J.P. van Tintelen, I.M. van Langen, N. Hofman, B.W. van Paassen, and M. Bronk for patient counseling; T.A. Simmers, B.A.J.M. de Mol, and A.J. Six for patient referral, examination, and tissue sampling; and L.M.E. Stoets and F.A.M. Hennekam for genealogical research. We are very grateful to the patients and their families who cooperated in this study. The URLs for data presented herein are as follows:GenBank, http://www.ncbi.nlm.nih.gov/Genbank/NCBI genome browser, http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeOnline Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/UniSTS database, http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists
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