DOI: 10.1161/CIRCGENETICS.115.001263
Sudden Cardiac Arrest and Rare Genetic Variants in the Community
Running title: Milano et al.; SCA and rare genetic variants in the community
Annalisa Milano, PhD1*; Marieke T. Blom, PhD, MSE1*; Elisabeth M. Lodder, PhD1*; Daniel A. van Hoeijen, MSc1; Julien Barc, PhD1; Tamara T. Koopmann, PhD1; Abdennasser Bardai, MD, PhD1; Leander Beekman, BSc1; Peter Lichtner, PhD2; Maarten P. van den Berg, MD, PhD3; Arthur A.M. Wilde, MD, PhD1,4; Connie R. Bezzina, PhD1; Hanno L. Tan, MD, PhD1
1
Department Depa De part pa rtm rt me t of ment of Clinical & Experimental Cardiol Cardiology, olog ol o y, Heart Center, Am A Amsterdam stter erddam Medical Center, University Univ Un iversity off Amsterdam, iv Amst ster e dam, m Amsterdam, Ams m terd rdam am, the the Netherlands; Neth Ne ther erlandds;; 2Instit Institut i ut für H Humangenetik, uman um a ge g ne netik, H Helmholtz elmh el m oltz Z Zentrum, en ntr trum u , Technische U nive ni veersit ität it ä München, Mün ünch ch hen, en Mu Munich ch, Ge ch erm manyy; 3De Technische Universität Munich, Germany; Department epa partme ment me n off Cardiology, Card Card dio iolo ogy y, Un U University iver e si er sity ty Medical Med edic ical ic al Center Groningen, University Groningen, Groningen, the Netherlands; C enter Gronin ngeen, U niiversiity y of Groni ingen, G roning ngen, th ng he Ne Nether rla lannds; 4Pr Princess Princ cesss Al-J Al-Jawhara Jaw wharaa Alwhar AllBrah Br a im Cen ah en ntr t e off Exc celllencee in nR essea earcch of H erredittarry Disorders, Dissord derrs,, Jeddah, Jed edda ed dah, Kingdom Kin ngd dom m of Saudi Sauudii Arabia Sa Arabia Brahim Centre Excellence Research Hereditary *contributed *con *c ontr on trrib ibut uted ut ed eequally q al qu ally ly
Correspondence: C orre or resp re spon sp onde on denc de nce: nc e: Hanno L. Tan, MD, PhD
Connie R. Bezzina, PhD
Dept of Clinical & Experimental Cardiology
Dept of Clinical & Experimental Cardiology
Heart Center, Academic Medical Center
Heart Center, Academic Medical Center
University of Amsterdam
University of Amsterdam
Meibergdreef 9
Meibergdreef 9
1105 AZ Amsterdam
1105 AZ Amsterdam
the Netherlands
the Netherlands
Tel: +31 (0)20 5663264
Tel: +31 (0)20 5665403
E-mail: [email protected]
E-mail: [email protected]
Journal Subject Terms: Arrhythmias; Electrophysiology; Sudden Cardiac Death
1 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on January 25, 2016
DOI: 10.1161/CIRCGENETICS.115.001263
Abstract: Background - Sudden cardiac arrest (SCA) ranks among the most common causes of death worldwide. Because SCA is most often lethal, yet mostly occurs in individuals without previously known cardiac disease, the identification of patients at risk for SCA could save many lives. In unselected SCA victims from the community, common genetic variants (which are not disease-causing per se, but may increase susceptibility to VF) are associated with increased SCA risk. However, whether rare genetic variants contribute to SCA risk in the community is largely unexplored. Methods and Results - We here investigated the involvement of rare genetic variants in SCA risk at the population level, by studying the prevalence of six founder genetic variants present in the Dutch ppopulation (PLN-p.Arg14del, p ( p g , MYBPC3-p.Trp792fsX17, p p , MYBPC3-p.Arg943X, p g , MYBPC3-p.Pro955fsX95, PKP2-p.Arg79X, and the Chr7q36 idiopathic ventricu ventricular fibrillation ulaar fi fibr bril br illa il lati la tion ti risk-haplotype) isk-haplotype) in a cohort of 1440 unselected Dutch SCA victims included in the AmsteRdam AmsteRdam REsuscitation Study RE Esu susscit scitat attio ionn St tud udy (ARREST). The six studied d founder fouunder mutations ns were werre found found to be more prevalent the ARREST ethnically pr valent (1.1%) prev %) inn th he AR ARRE REST RE ST SCA SCA cohort co oho h rt compared com mpareed tto o aan n et thnic ical ic ally ly aand ndd ggeographically eogrrap eogr aphi hica hi c ll llyy matched controls matcched set of con matc ontrolls (0.4%, on %, n=1379, n=137 3779,, p95% of all individuals with out-of-hospital SCA in whom a resuscitation is attempted21. ECG recordings from the ambulance monitor/defibrillator or automated external defibrillator are used to determine whether VF had occurred. SCA cases are defined as individuals with a cardiac arrest in an out-of-hospital setting with VF. Individuals with an obvious non-cardiac cause of VF (e.g.,
4 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on January 25, 2016
DOI: 10.1161/CIRCGENETICS.115.001263
trauma, intoxication, drowning, suicide) are excluded. Individuals in whom only asystole (but no VT/VF) is recorded are also excluded, because we cannot ensure that cardiac arrest stemmed from cardiac causes, as asystole is the end stage of any cardiac arrest, and may be due to noncardiac causes (e.g., respiratory failure)22. Medical histories are obtained from the general practitioner and the immediate cause of SCA is obtained from hospital records. Genomic DNA is extracted from peripheral blood samples drawn during routine patient care, according to standard procedures. Written informed consent is obtained from all surviving SCA cases. Control cohorts Amsterdam area controls Amsterdam area controls, collected in a region overlapping the geographic captur uree ar ur aarea ea ooff capture ARRE EST ST,, were weere 11379 3 9 healthy volunteer blood don 37 nor ors of Dutch Europeann de ddescent scent at Sanquin ARREST, donors 2 Bloo B loood Supplyy (67% (67% male, mal ale, e, mean meaan age age 53 years) yeaars rs))23 . Blood
GoNL GoNL controls GoN unrelated the GoNL controls weree al all 500 un unre r laated individualss from th re he da ddatabase tabase ooff th ta the GoNL study which is comprised comp co mp pri rise sedd of w whole-genome-sequenced hole ho le-ge g no ge nome me-se sequ q en qu ence cedd in indi individuals divi vidu dual alss distributed dist di stri ribu bute tedd evenly even ev enly ly y ac acro across ross ss tthe he N Netherlands ethe et herl rlan ands ds (covering eleven of its twelve provinces) without selection on the basis of phenotype or disease16,24. PREVEND controls PREVEND controls were all 8261 individuals from the database of the PREVEND study, which is comprised of inhabitants of the city of Groningen in the north of the Netherlands17. Founder mutations: selection and genotyping The following six founder mutations were selected for study because of their recurrence (indicative of a founder effect) in DNA diagnostic testing of patients with inherited cardiac
5 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on January 25, 2016
DOI: 10.1161/CIRCGENETICS.115.001263
disorders at Cardiogenetics departments in the Netherlands11–15. These mutations were: three mutations associated with hypertrophic cardiomyopathy (HCM, mutations MYBPC3p.Trp792fsX17, MYBPC3-p.Arg943X, MYBPC3-p.Pro955fsX95); one mutation associated with arrhythmogenic right ventricular cardiomyopathy (ARVC, PKP2-p.Arg79X); one mutation associated with an overlap phenotype of dilated cardiomyopathy (DCM) and ARVC (PLNp.Arg14del), and a founder haplotype linked to idiopathic VF (IVF, the Chr7q36 IVF riskhaplotype), which was detected by either the chr7:154002240 c.-340C>T, or the chr7:g.154056404.TA/- variant, both of which are unique to the risk haplotype.12,25 Genotyping for the six founder mutations was conducted in the ARREST case cohort and he Amsterdam area control cohort, and performed on the MassARRAY system uusing sing si ng MALDIMAL ALDI DIDI the TOF ma mass s spe spectrometry p ctro pe rome m try with the iPLEX Gold che chemistry emi m stry (Sequenom Inc, Incc, San Diego CA, USA). Prim Pr mers weree ddesigned esig es igne ig need us usin ingg As in Assa sayy De sa Desi sign si gn ner 44.0.0.2 .0.00.22 with wit ithh iPLEX iPLE iP L X Gold Gold default def e ault ltt parameters. par aram amet am etter ers. s. The The Primers using Assay Designer PCR PC R primers used ed are li listed isted in Suppl Supplementary plem pl em mentary ry Tab Table blee 1.. Au A Automated to omateed geno genotype oty ypee ccalling a lingg w al was ass ddone onee with Typer Analyzer Analyzeer 4. 44.0.22.67. 0 22.667. We 0. We included incl c uded DNA NA A from a known know kn own carrier carr rrrie ierr for each mutation as a p si po siti tive ve control. con ontr trol ol.. Genotype Geno Ge noty ty ype p clustering clu lust ster erin ingg was was vi visu sual ally ly y cchecked heck he cked ed bby y an eexperienced xpper erie ienc nced ed evaluator. eva valu luat ator or.. Once Once positive visually carriership of a founder mutation was identified, this was subsequently validated by PCR-Sanger sequencing. Statistical analysis We used Mantel-Haenszel tests stratified according to age group (age 0.05). We additionally looked up the prevalence of the six founder mutations in the GoNL database of 500 unrelated Dutch subjects. No control from GoNL carried any of the founder mutations. We further focused on the most commonly encountered founder mutation in ARREST, the PLNp.Arg14del mutation. We looked up the prevalence of this mutation in the database of the PREVEND study, which consisted of 8267 individuals recruited from the region where PLNp.Arg14del is thought to have originated and where it is therefore expected to be most prevalent in the general population17,26. We found that PLN-R14del occurred significantly more often in ARREST than in PREVEND (0.56% versus 0.07%, Fisher’s exact p
Sudden Cardiac Arrest and Rare Genetic Variants in the Community
Running title: Milano et al.; SCA and rare genetic variants in the community
Annalisa Milano, PhD1*; Marieke T. Blom, PhD, MSE1*; Elisabeth M. Lodder, PhD1*; Daniel A. van Hoeijen, MSc1; Julien Barc, PhD1; Tamara T. Koopmann, PhD1; Abdennasser Bardai, MD, PhD1; Leander Beekman, BSc1; Peter Lichtner, PhD2; Maarten P. van den Berg, MD, PhD3; Arthur A.M. Wilde, MD, PhD1,4; Connie R. Bezzina, PhD1; Hanno L. Tan, MD, PhD1
1
Department Depa De part pa rtm rt me t of ment of Clinical & Experimental Cardiol Cardiology, olog ol o y, Heart Center, Am A Amsterdam stter erddam Medical Center, University Univ Un iversity off Amsterdam, iv Amst ster e dam, m Amsterdam, Ams m terd rdam am, the the Netherlands; Neth Ne ther erlandds;; 2Instit Institut i ut für H Humangenetik, uman um a ge g ne netik, H Helmholtz elmh el m oltz Z Zentrum, en ntr trum u , Technische U nive ni veersit ität it ä München, Mün ünch ch hen, en Mu Munich ch, Ge ch erm manyy; 3De Technische Universität Munich, Germany; Department epa partme ment me n off Cardiology, Card Card dio iolo ogy y, Un U University iver e si er sity ty Medical Med edic ical ic al Center Groningen, University Groningen, Groningen, the Netherlands; C enter Gronin ngeen, U niiversiity y of Groni ingen, G roning ngen, th ng he Ne Nether rla lannds; 4Pr Princess Princ cesss Al-J Al-Jawhara Jaw wharaa Alwhar AllBrah Br a im Cen ah en ntr t e off Exc celllencee in nR essea earcch of H erredittarry Disorders, Dissord derrs,, Jeddah, Jed edda ed dah, Kingdom Kin ngd dom m of Saudi Sauudii Arabia Sa Arabia Brahim Centre Excellence Research Hereditary *contributed *con *c ontr on trrib ibut uted ut ed eequally q al qu ally ly
Correspondence: C orre or resp re spon sp onde on denc de nce: nc e: Hanno L. Tan, MD, PhD
Connie R. Bezzina, PhD
Dept of Clinical & Experimental Cardiology
Dept of Clinical & Experimental Cardiology
Heart Center, Academic Medical Center
Heart Center, Academic Medical Center
University of Amsterdam
University of Amsterdam
Meibergdreef 9
Meibergdreef 9
1105 AZ Amsterdam
1105 AZ Amsterdam
the Netherlands
the Netherlands
Tel: +31 (0)20 5663264
Tel: +31 (0)20 5665403
E-mail: [email protected]
E-mail: [email protected]
Journal Subject Terms: Arrhythmias; Electrophysiology; Sudden Cardiac Death
1 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on January 25, 2016
DOI: 10.1161/CIRCGENETICS.115.001263
Abstract: Background - Sudden cardiac arrest (SCA) ranks among the most common causes of death worldwide. Because SCA is most often lethal, yet mostly occurs in individuals without previously known cardiac disease, the identification of patients at risk for SCA could save many lives. In unselected SCA victims from the community, common genetic variants (which are not disease-causing per se, but may increase susceptibility to VF) are associated with increased SCA risk. However, whether rare genetic variants contribute to SCA risk in the community is largely unexplored. Methods and Results - We here investigated the involvement of rare genetic variants in SCA risk at the population level, by studying the prevalence of six founder genetic variants present in the Dutch ppopulation (PLN-p.Arg14del, p ( p g , MYBPC3-p.Trp792fsX17, p p , MYBPC3-p.Arg943X, p g , MYBPC3-p.Pro955fsX95, PKP2-p.Arg79X, and the Chr7q36 idiopathic ventricu ventricular fibrillation ulaar fi fibr bril br illa il lati la tion ti risk-haplotype) isk-haplotype) in a cohort of 1440 unselected Dutch SCA victims included in the AmsteRdam AmsteRdam REsuscitation Study RE Esu susscit scitat attio ionn St tud udy (ARREST). The six studied d founder fouunder mutations ns were werre found found to be more prevalent the ARREST ethnically pr valent (1.1%) prev %) inn th he AR ARRE REST RE ST SCA SCA cohort co oho h rt compared com mpareed tto o aan n et thnic ical ic ally ly aand ndd ggeographically eogrrap eogr aphi hica hi c ll llyy matched controls matcched set of con matc ontrolls (0.4%, on %, n=1379, n=137 3779,, p95% of all individuals with out-of-hospital SCA in whom a resuscitation is attempted21. ECG recordings from the ambulance monitor/defibrillator or automated external defibrillator are used to determine whether VF had occurred. SCA cases are defined as individuals with a cardiac arrest in an out-of-hospital setting with VF. Individuals with an obvious non-cardiac cause of VF (e.g.,
4 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on January 25, 2016
DOI: 10.1161/CIRCGENETICS.115.001263
trauma, intoxication, drowning, suicide) are excluded. Individuals in whom only asystole (but no VT/VF) is recorded are also excluded, because we cannot ensure that cardiac arrest stemmed from cardiac causes, as asystole is the end stage of any cardiac arrest, and may be due to noncardiac causes (e.g., respiratory failure)22. Medical histories are obtained from the general practitioner and the immediate cause of SCA is obtained from hospital records. Genomic DNA is extracted from peripheral blood samples drawn during routine patient care, according to standard procedures. Written informed consent is obtained from all surviving SCA cases. Control cohorts Amsterdam area controls Amsterdam area controls, collected in a region overlapping the geographic captur uree ar ur aarea ea ooff capture ARRE EST ST,, were weere 11379 3 9 healthy volunteer blood don 37 nor ors of Dutch Europeann de ddescent scent at Sanquin ARREST, donors 2 Bloo B loood Supplyy (67% (67% male, mal ale, e, mean meaan age age 53 years) yeaars rs))23 . Blood
GoNL GoNL controls GoN unrelated the GoNL controls weree al all 500 un unre r laated individualss from th re he da ddatabase tabase ooff th ta the GoNL study which is comprised comp co mp pri rise sedd of w whole-genome-sequenced hole ho le-ge g no ge nome me-se sequ q en qu ence cedd in indi individuals divi vidu dual alss distributed dist di stri ribu bute tedd evenly even ev enly ly y ac acro across ross ss tthe he N Netherlands ethe et herl rlan ands ds (covering eleven of its twelve provinces) without selection on the basis of phenotype or disease16,24. PREVEND controls PREVEND controls were all 8261 individuals from the database of the PREVEND study, which is comprised of inhabitants of the city of Groningen in the north of the Netherlands17. Founder mutations: selection and genotyping The following six founder mutations were selected for study because of their recurrence (indicative of a founder effect) in DNA diagnostic testing of patients with inherited cardiac
5 Downloaded from http://circgenetics.ahajournals.org/ at Queen's University--Kingston (CANADA) on January 25, 2016
DOI: 10.1161/CIRCGENETICS.115.001263
disorders at Cardiogenetics departments in the Netherlands11–15. These mutations were: three mutations associated with hypertrophic cardiomyopathy (HCM, mutations MYBPC3p.Trp792fsX17, MYBPC3-p.Arg943X, MYBPC3-p.Pro955fsX95); one mutation associated with arrhythmogenic right ventricular cardiomyopathy (ARVC, PKP2-p.Arg79X); one mutation associated with an overlap phenotype of dilated cardiomyopathy (DCM) and ARVC (PLNp.Arg14del), and a founder haplotype linked to idiopathic VF (IVF, the Chr7q36 IVF riskhaplotype), which was detected by either the chr7:154002240 c.-340C>T, or the chr7:g.154056404.TA/- variant, both of which are unique to the risk haplotype.12,25 Genotyping for the six founder mutations was conducted in the ARREST case cohort and he Amsterdam area control cohort, and performed on the MassARRAY system uusing sing si ng MALDIMAL ALDI DIDI the TOF ma mass s spe spectrometry p ctro pe rome m try with the iPLEX Gold che chemistry emi m stry (Sequenom Inc, Incc, San Diego CA, USA). Prim Pr mers weree ddesigned esig es igne ig need us usin ingg As in Assa sayy De sa Desi sign si gn ner 44.0.0.2 .0.00.22 with wit ithh iPLEX iPLE iP L X Gold Gold default def e ault ltt parameters. par aram amet am etter ers. s. The The Primers using Assay Designer PCR PC R primers used ed are li listed isted in Suppl Supplementary plem pl em mentary ry Tab Table blee 1.. Au A Automated to omateed geno genotype oty ypee ccalling a lingg w al was ass ddone onee with Typer Analyzer Analyzeer 4. 44.0.22.67. 0 22.667. We 0. We included incl c uded DNA NA A from a known know kn own carrier carr rrrie ierr for each mutation as a p si po siti tive ve control. con ontr trol ol.. Genotype Geno Ge noty ty ype p clustering clu lust ster erin ingg was was vi visu sual ally ly y cchecked heck he cked ed bby y an eexperienced xpper erie ienc nced ed evaluator. eva valu luat ator or.. Once Once positive visually carriership of a founder mutation was identified, this was subsequently validated by PCR-Sanger sequencing. Statistical analysis We used Mantel-Haenszel tests stratified according to age group (age 0.05). We additionally looked up the prevalence of the six founder mutations in the GoNL database of 500 unrelated Dutch subjects. No control from GoNL carried any of the founder mutations. We further focused on the most commonly encountered founder mutation in ARREST, the PLNp.Arg14del mutation. We looked up the prevalence of this mutation in the database of the PREVEND study, which consisted of 8267 individuals recruited from the region where PLNp.Arg14del is thought to have originated and where it is therefore expected to be most prevalent in the general population17,26. We found that PLN-R14del occurred significantly more often in ARREST than in PREVEND (0.56% versus 0.07%, Fisher’s exact p
