European Review for Medical and Pharmacological Sciences 2019; 23: 7582-7598 Putative role of Brugada syndrome in familial

P.E. M A LTESE1, E. ALDANOVA2, N. KRIUCHKOVA2, A. AVERIANOV2, E. MANARA3, S. PAOLACCI3, A. BRUSON1, R. MIOTTO3, M. SARTORI1, G. GUERRI3, M. ZUNTINI3, G. MARCEDDU3, S. TEZZELE3, K. TADTAEVA2, A. CHERNOVA2, N. AKSYUTINA2, S. NIKULINA2, S. NODARI4, M. BERTELLI1,3

1MAGI’s Lab, Rovereto (TN), Italy 2Department Internal Diseases I, Krasnoyarsk State Medical University, Krasnoyarsk, Russia 3MAGI Euregio, Bolzano, Italy 4Department of Clinical and Surgical Specialities, Radiological Sciences and Public Health, University of Brescia Medical School, Brescia, Italy

Abstract. – OBJECTIVE: Familial atrial fibril- because it is typically associated with various dis- lation (FAF), a not uncommon of the orders and other genetic cardiomyopathies1. By atrium, is characterized by heritability, early on- contrast, familial AF (FAF), also known as lone set and absence of other defects. The mo- AF, is not associated with other cardiovascular lecular and genetic basis is still not completely risk factors, affects more than one family mem- clear and genetic diagnosis cannot be achieved 2 in about 90% of patients. In this study, we pres- ber with early onset and is therefore considered ent the results of genetic screening by next gen- a heritable nosological entity in its own right. eration sequencing in affected Russian families. Familial AF is not uncommon and is also geneti- PATIENTS AND METHODS: Sixty subjects (18 cally heterogeneous: 16 genes and 4 loci are cur- probands and 42 relatives) with a clinical diag- rently known to be associated with the condition, nosis of FAF were enrolled in the study. Since as listed in the Online Mendelian Inheritance in AF frequently associates with other cardiomy- opathies, we included all genes that were known Man database (OMIM, http://omim.org/). In a to be associated with these disorders at the time cohort of 192 individuals (384 alleles) and con- 3 of our study. All probands were therefore sys- sidering 14 AF-associated genes, Olesen et al tematically screened for 47 genes selected from demonstrated that only 7.6% of alleles harboured the literature. a very rare variant that could justify a Mendelian RESULTS: Our study revealed that seven vari- hereditary pattern of the condition. ants co-segregated with the clinical phenotype Since an increasing number of genes are asso- in seven families. Interestingly, four out of six genes and three out of seven variants have al- ciated with AF, the most time- and cost-effective ready been associated with Brugada syndrome strategy is next generation sequencing (NGS), in the literature. which however is not currently performed for CONCLUSIONS: To our knowledge, this is routine . the first report of association of the CACNA1C, In the present study, we report the genetic CTNNA3, PKP2, ANK2 and SCN10A genes with characterization of 18 Russian probands with FAF; it is also the first study in Russian families. FAF and their relatives by NGS. Given the low Key Words: positivity rate to genetic testing and considering FAF, , , Next generation that AF can be associated with other conditions, sequencing. we also extended the analysis to all genes known to be involved in other cardiomyopathies, such as Brugada syndrome (BS), long-QT syndrome (LQTS) and arrhythmogenic right ventricular Introduction dysplasia (ARRVD). We therefore considered a total of 47 genes. Atrial fibrillation (AF) is the most common ar- Since AF is associated with increased mor- rhythmia encountered in clinical practice mostly tality, the genetic characterization of all family

7582 Corresponding Author: Elena Manara, MD; e-mail: [email protected] Putative role of Brugada syndrome genes in familial atrial fibrillation members is useful to identify individuals at risk el” manufacturer’s instructions. For the quantifi- and to decide appropriate clinical follow-up to cation and validation of the genomic library, we prevent serious complications. used the Qubit® 2.0 Fluorometer system (Life Technologies, Carlsbad, CA, USA) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Patients and Methods CA, USA). The analysis of raw read data in fastq format was performed using an in-house bioin- A total of 60 subjects, 18 probands and their formatics pipeline as described in our previous families, were examined in the clinics of Kras- paper on long-QT syndrome (LQTS) in Russian noyarsk, Russia. All patients and healthy sub- families5. The analysis was performed on the jects underwent electrocardiogram (ECG), Holter coding exons and 15 bp flanking sequence of a monitoring and blood sampling at the Federal list of 47 genes that were reported to be linked to Cardiological and Vascular Center (Krasnoyarsk) hereditary cardiomyopathies at the time of anal- and at the Department of Internal Medicine I ysis (Table I). and Pediatrics IPO, Krasnoyarsk State Medical Variants were selected for further study on the University “V.F. Voino-Yasenetsky”. Enrolment basis of previously described criteria5. criteria included: DNA samples from probands were analyzed by this method and all genetic variants were validated by Sanger sequencing using a Beck- 1 ECG characteristics: absence of P waves; man Coulter CEQ 8000 sequencer (Beckmann irregular R-R intervals; Coulter, Milan, Italy). The genotype-phenotype 2. Clinical presentation: AF as major clinical correlation of each variant was evaluated by fam- manifestation (phenotype) with early onset ily segregation study using the same method. (before age 60)4; The electropherograms of amplified fragments 3. Family history: at least one affected first or were analyzed using ChromasPro 1.5 (Technely- second-degree family member. sium Pty. Ltd., Brisbane, Queensland, Australia) and Sequencher 5.0 ( Codes®; Ann Arbor, All subjects underwent genetic counselling in MI, USA) software and compared to GenBank which the risks and benefits of genetic testing reference sequences with the Basic Local Align- were explained. They signed specific consent to ment Search Tool (BLAST; http://blast.ncbi.nlm. use of their clinical and genetic data for research nih.gov). Nonsense, frameshift and splice site and publication. variants were considered disease-causing. The work described in this paper was approved We consulted the Human Gene by the Krasnoyarsk State Medical University Professional Database (HGMD) (http://www. Ethics Committee (protocol no. 54/2014) and car- biobase-international.com/product/hgmd), the ried out in accordance with the Declaration of Exome Aggregation Consortium (ExAC) data- Helsinki. base (exac.broadinstitute.org/), the Exome Vari- Genetic testing protocols were developed at ant Server (EVS) database (http://evs.gs.washing- MAGI’s Laboratories (MAGI’s Lab, Rovereto ton.edu/EVS/) and the public database of single and MAGI Euregio, Bolzano, Italy). Genetic stud- nucleotide variants (dbSNP, www.ncbi.nlm.nih. ies were performed at the Russian-Italian Labora- gov/SNP/) in order to identify genetic variants tory of Medical Genetics (Krasnoyarsk, Russia) previously reported as pathogenic and to check where a total of 60 blood samples were sent for for allele frequencies. DNA extraction. Nucleotide variants were assessed for pathoge- Targeted resequencing was performed using nicity using the PolyPhen 2 and SIFT algorithms6,7 the Illumina commercial kit “TruSight One se- via the Variant Effect Predictor tool (http://www. quencing panel” on the MiSeq platform (Illu- ensembl.org/info/docs/tools/vep/index.html) and mina, San Diego, CA, USA). This kit makes it MutationTaster (http://www.mutationtaster.org)8. possible to perform enrichment and final analysis Intronic variants were checked for their potential of a panel of approximately 4800 genes (http:// to affect splicing using Human Splicing Finder www.illumina.com/ products/trusight-one-se- software Version 3 (http://www.umd.be/HSF3/ quencing-panel.ilmn). HSF.html). In-solution target enrichment was performed Genetic variants were classified according to according to the “TruSight One sequencing pan- the criteria of ACMG Standards and Guidelines9.

7583 P.E. Maltese, E. Aldanova, N. Kriuchkova, A. Averianov, E. Manara, et al.

Table I. Genes involved in hereditary cardiomyopathies.

Gene (OMIM ID:) name Phenotype (OMIM ID:) Inheritance

ABCC9 ATP-binding cassette sub-family Atrial fibrillation, familial, 12 (614050) AD (601439) C member 9 Cardiomyopathy, dilated, 1O (608569) NA Hypertrichotic osteochondrodysplasia (239850) AD AKAP9 A-kinase anchor protein 9 Long QT syndrome-11 (611820) AD (604001) ANK2 B Long QT syndrome 4 (600919) AD (106410) Cardiac arrhythmia, ankyrin-B-related (600919) AD CACNA1C channel, L type, alpha 1 Brugada syndrome 3 (611875) AD (114205) polypeptide isoform (601005)a AD CACNA2D1 Voltage-dependent calcium Brugada syndrome (NA) AD (114204) channel subunit alpha-2/delta-1 CACNB2 Voltage-dependent L-type Brugada syndrome 4 (611876) AD (600003) subunit beta-2 CALM1 Calmodulin 1 - Long QT syndrome 14 (616247) AD (114180) calcium-modulated protein , catecholaminergic AD polymorphic, 4 (614916) CASQ2 Calsequestrin-2 Ventricular tachycardia, catecholaminergic AR (114251) polymorphic, 2 (611938) CAV3 Caveolin 3 Cardiomyopathy, familial hypertrophic (192600) AD (601253) Creatine phosphokinase, elevated serum (123320) AD Long QT syndrome 9 (611818) AD Myopathy, distal, Tateyama type (614321) AD Rippling muscle disease 2 (606072) AD CTNNA3 alpha-3 Arrhythmogenic right ventricular dysplasia, AD (607667) familial, 13 (615616) DPP6 Dipeptidyl aminopeptidase-like , paroxysmal familial, 2 (612956) AD (126141) protein 6 DSC2 Desmocollin-2 Arrhythmogenic right ventricular dysplasia 11 (610476) AD, AR (125645) DSG2 Desmoglein-2 Arrhythmogenic right ventricular dysplasia 10 (610193) AD (125671) Cardiomyopathy, dilated, 1BB (612877) NA DSP Arrhythmogenic right ventricular dysplasia 8 (607450) AD (125647) Cardiomyopathy, dilated, with woolly hair and AR keratoderma (605676) Dilated cardiomyopathy with woolly hair, keratoderma, AD and tooth agenesis (615821) GJA5 Gap junction alpha-5 protein Atrial fibrillation, familial, 11 (614049) AD (121013) Atrial standstill, digenic (GJA5/SCN5A) (108770) AD GNAI2 Guanine nucleotide-binding Ventricular tachycardia, idiopathic (192605) AD (139360) protein G(i) subunit alpha-2 GPD1L Glycerol-3-phosphate Brugada syndrome 2 (611777) AD (611778) dehydrogenase 1-like protein HCN4 Potassium/ Brugada syndrome 8 (613123) AD (605206) hyperpolarization-activated Sick sinus syndrome 2 (163800) AD cyclic nucleotide-gated channel 4 JUP Junction Arrhythmogenic right ventricular dysplasia 12 (611528) AD (173325) Naxos disease (601214) AR KCNA5 Potassium voltage-gated Atrial fibrillation, familial, 7 (612240) AD (176267) channel subfamily A member 5 KCND3 Potassium voltage-gated Brugada syndrome 9 (616399) AD (605411) channel subfamily D member 3 KCNE1 Voltage-gated potassium Jervell and Lange-Nielsen syndrome 2 (612347) AR (176261) channel, Isk related subfamily, Long QT syndrome 5 (613695) AD member 1 Table continued

7584 Putative role of Brugada syndrome genes in familial atrial fibrillation

Table I (Continued). Genes involved in hereditary cardiomyopathies.

Gene (OMIM ID:) Protein name Phenotype (OMIM ID:) Inheritance

KCNE2 Voltage-gated , Atrial fibrillation, familial, 4 (611493) AD (603796) Isk related subfamily, member 2 Long QT syndrome 6 (613693) AD KCNE3 Potassium voltage-gated channel Brugada syndrome 6 (613119) AD (604433) subfamily E member 3 KCNH2 Potassium channel, Long QT syndrome 2 (613688) AD (152427) voltage-gated, H2 Short QT syndrome 1 (609620) AD Long QT syndrome 2, acquired, susceptibility AD to (613688) KCNJ2 Inwardly rectifying potassium Andersen syndrome (170390) AD (600681) channel Atrial fibrillation, familial, 9 (613980) AD Short QT syndrome 3 (609622) AD KCNJ5 Potassium inwardly-rectifying Hyperaldosteronism, familial, type III (613677) AD (600734) channel, subfamily J, member 5 Long QT syndrome 13 (613485) AD KCNJ8 ATP-sensitive inward rectifier Sudden infant death syndrome (NA) AD (600935) potassium channel 8 KCNQ1 KQT-like voltage-gated Atrial fibrillation, familial, 3 (607554) AD (607542) potassium channel 1 Jervell and Lange-Nielsen syndrome (220400) AR Long QT syndrome 1 (192500) AD Short QT syndrome 2 (609621) AD Long QT syndrome 1, acquired, susceptibility AD to (192500) LMNA Prelamin-A/C Cardiomyopathy, dilated, 1A (115200) AD (150330) Emery-Dreifuss muscular dystrophy 2, AD (181350) AD Emery-Dreifuss muscular dystrophy 3, AR (616516) AR Heart-hand syndrome, Slovenian type (610140) AD Lipodystrophy, familial partial, type 2 (151660) AD Malouf syndrome (212112) AD Muscular dystrophy, congenital (613205) AD Restrictive dermopathy, lethal (275210) AR NPPA Natriuretic peptides A Atrial fibrillation, familial, 6 (612201) AD (108780) Atrial standstill 2 (615745) AR NUP155 Nuclear pore complex protein Atrial fibrillation 15 (615770) AR (606694) Nup155 PKP2 -2 Arrhythmogenic right ventricular dysplasia 9 (609040) AD (602861) PRKAG2 5’-AMP-activated protein Cardiomyopathy, hypertrophic 6 (600858) AD (602743) kinase subunit gamma-2 Glycogen storage disease of heart, lethal congenital AD (261740) Wolff-Parkinson-White syndrome (194200) AD RYR2 2 Arrhythmogenic right ventricular dysplasia 2 (600996) AD (180902) Ventricular tachycardia, catecholaminergic AD polymorphic, 1 (604772) SCN10A protein Brugada syndrome (NA) AD (108980) type 10 subunit alpha Atrial fibrillation (NA) AD Long QT syndrome (NA) AD SCN1B Sodium channel subunit beta-1 Atrial fibrillation, familial, 13 (615377) AD (600235) Brugada syndrome 5 (612838) AD Cardiac conduction defect, nonspecific (612838) AD SCN2B Sodium channel subunit beta-2 Atrial fibrillation, familial, 14 (615378) AD (601327) SCN3B Sodium channel subunit beta-3 Sodium channel subunit beta-3 (613120) AD (608214) Brugada syndrome 7 (613120) AD SCN4B Sodium channel, voltage-gated, Atrial fibrillation, familial, 17 (611819) AD (608256) type IV beta subunit Long QT syndrome-10 (611819) AD

Table continued

7585 P.E. Maltese, E. Aldanova, N. Kriuchkova, A. Averianov, E. Manara, et al.

Table I (Continued). Genes involved in hereditary cardiomyopathies.

Gene (OMIM ID:) Protein name Phenotype (OMIM ID:) Inheritance

SCN5A Alfa polypeptide of voltage-gated Atrial fibrillation, familial, 10 (614022) AD (600163) sodium channel type V Brugada syndrome 1 (601144) AD Cardiomyopathy, dilated, 1E (601154) AD Heart block, nonprogressive (113900) AD Heart block, progressive, type IA (113900) AD Long QT syndrome-3 (603830) AD Sick sinus syndrome 1 (608567) AR Ventricular fibrillation, familial, 1 (603829) AD Sudden infant death syndrome, susceptibility to (272120) AR SNTA1 Syntrophin, alpha 1 Long QT syndrome 12 (612955) AD (601017) TGFB3 Transforming growth factor Arrhythmogenic right ventricular dysplasia 1 (107970) AD (606237) beta-3 proprotein Loeys-Dietz syndrome 5 (615582) AD TMEM43 Transmembrane protein 43 Arrhythmogenic right ventricular dysplasia 5 (604400) AD (612048) Emery-Dreifuss muscular dystrophy 7, AD (614302) AD TRDN Triadin Ventricular tachycardia, catecholaminergic polymorphic, AR (603283) 5, with or without muscle weakness (615441) TRPM4 Transient receptor potential Progressive familial heart block, type IB (604559) AD (606936) cation channel subfamily M member 4 TTN Cardiomyopathy, dilated, 1G (604145) AD (188840) Cardiomyopathy, familial hypertrophic, 9 (613765) AD Myopathy, myofibrillar, 9, with early respiratory AD failure (603689) Salih myopathy (611705) AR

AD: autosomal dominant; AR: autosomal recessive; NA: not available; OMIM: Online Mendelian Inheritance in Man.

Results ing of these six variants are shown in Figure 2A. Results from the genotype-phenotype segrega- Next generation sequencing analysis was per- tion study and a summary of clinical features formed for the 47 known hereditary cardiomy- of probands and relatives are shown in Figure 1 opathy-related genes using a TruSight One se- and Table III, respectively. All the remaining 24 quencing panel (Illumina). The average number variants listed in Table II did not segregate with of mappable reads per sample was 10 M. On the disease (data not shown). average, 97% of the target bases of gene-disease subpanels were covered at least 10×, with a mean Family 2: The proband (R585) is a 30-year-old coverage of 120× per sample. male who suffered episodes of AF once ev- The NGS analysis revealed that 17 out of 18 ery two months and also complained of con- probands had a genetic variant in one of the genes tinuous weakness and dizziness. Symptoms included in the panel (Table II); the segregation began at age 18 years. In his family, only the study in these families (40 relatives) led us to maternal grandmother (R619) shows similar identify seven families with variants (39%, 7/18 cardiological features; she suffered AF attacks probands) probably related to the phenotype (Fig- once a month for more than 20 years. She also ure 1). Five variants found in four families had complains of irregular heartbeat, weakness, already been described in the literature, while dizziness and shortness of breath. Her moder- two, found in three unrelated probands and their ately abnormal10 left atrial diameter (Table III) affected relatives, were new. In three families, can be attributed to age and her long history of rare genetic variants were found in the CACNA1C arrhythmia11. gene. The other four families showed variants in Family 3: The male proband (R586), 33 years of the CTNNA3, PKP2, SCN10A, ANK2 and SCN5A age with onset at 21 years, complained of AF genes. Electropherograms from Sanger sequenc- episodes once every six months, stably for the

7586 Putative role of Brugada syndrome genes in familial atrial fibrillation 31 52 19 19 55 37 53 37 57 29 27 56 54 24 Reference ;

; ; ; ; ;

; Fam-1; Fam-1; Fam-3; Fam-3; Fam-5; Fam-9; Fam-9; Fam-9; Fam-9; Fam-6; Fam-4; Fam-9 Fam-11; Fam-11; Fam-11; Fam-18; Fam-18; Fam-14; Fam-16; Fam-18; Fam-13; Fam-15; Fam-12; Fam-17 Fam-14 Fam-18 Fam-18 Fam-12 Phenotype Fam-7; Fam-2; segregation Fam-8; Fam-2; Fam-2; Fam-3

NA NA NA MAF A:0.004 T:0.0011 T:0.0001 T:0.0001 T:0.0001 T:0.0027 T:0.0002 T:0.0009 T:0.0004 T:0.0004 A:0.0019 C:0.0009 A:0.0041 C:0.0004 G:0.0001 A:0.0062 A:0.0003 G:0.0005 A:0.0005 G:0.0008 A:0.0006 A:0.0004 C:0.00003 C:0.00006 A:0.00002 G:0.00008 G:0.000008 G:0.000008

In bold, variants classified in this study according to † †

VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS VUS Variant Pathogenic Likely benign Likely benign Likely benign Likely benign Likely benign Likely benign Likely benign Likely benign interpretation , variants segregating in affected family members NA NA refSNP and italics rs72650011 rs41261344 rs36210422 rs78086631 rs72648272 rs201035511 rs140714512 rs151327713 rs121912705 rs121912706 rs773891725 rs782475413 rs755579963 rs143682596 rs150016098 rs141207048 rs200318013 rs375428912 rs370516890 rs202187398 rs202160275 rs199473660 rs886043355 rs199889888 rs143900944 rs375209098 rs201306690 rs200586695 rs202098308 – – hgvs_p p.(Gln78*) p.Arg14Leu p.Ser755Thr p.(Ala71Val) p.Ser709Asn p.Arg176Trp p.Met365Val p.(Ala372Pro) p.Arg1193Gln p.Thr3744Asn p.(Val2014Ile) p.(Arg378His) p.(Ile1749Thr) p.(Val8259Ile) p.(Leu25351*) p.(Leu574Pro) p.(Tyr693Cys) p.(Pro7132Ser) p.(Gly1305Trp) p.(His8848Tyr) p.(Asn3848Ser) p.(Met1975Val) p.(Arg3906Trp) p.(Pro15705Leu) p.(Leu21508Val) p.(Arg21199Gln) p.(Leu16074Gln) p.(Arg30180Cys) p.(Arg28977Cys) c.-1C>A hgvs_c c.41G>T c.212C>T c.232C>T c.526C>T c.1721T>C c.1114G>C c.3913G>T c.1133G>A c.5246T>C c.3578G>A c.1093A>G c.2126G>A c.121-1G>T c.5923A>G c.2078A>G c.6040G>A c.2264G>C c.11716C>T c.47114C>T c.11231C>A c.48221T>A c.21394C>T c.76050delT c.90538C>T c.11543A>G c.26542C>T c.86929C>T c.24775G>A c.63596G>A c.64522T>G Refseq NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_133378 NM_001148 NM_001148 NM_170707 NM_021991 NM_005751 NM_005751 NM_001035 NM_006514 NM_000335 NM_004572 NM_004572 NM_000238 NM_000722 NM_000722 NM_ 201593 NM_001129827 NM_001129827 NM_001127384 NM_001127384 NM_001127384 AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AR AD AD AR AD AR AD AR AD AR AD AR AR AD AD AR AD AR AD AR AD AR AD AR AD AR Inheritance

List and characteristics the of heterozygous variants identifiedFAF probands. in

AKAP9 Gene AKAP9 ANK2 ANK2 CACNA1C CACNA1C CACNA2D1 CTNNA3 CTNNA3 CACNB2 CACNA2D1 JUP TTN CTNNA3 KCNH2 TTN TTN TTN TTN LMNA SCN10A TTN TTN TTN RYR2 PKP2 PKP2 TTN SCN5A TTN TTN 7587 theStandardscriteria ACMG of andGuidelines thatevaluation;differ infromnormal thelastClinVar type, variants classified in this study not differ that do from last ClinVar the evaluation. Protein substitutions not in parenthesis were functionally characterized; In bold AD; autosomal dominant; AR, autosomal recessive; MAF, minor allele frequency; VUS, variant of unknown significance. unknown significance. of variant VUS, frequency; allele minor MAF, recessive; AR, autosomal dominant; autosomal AD; Table II. Table P.E. Maltese, E. Aldanova, N. Kriuchkova, A. Averianov, E. Manara, et al.

Figure 1. Family pedigree illustrating cosegregation of gene and atrial fibrillation phenotypes. P, proband; *Documented clinical evaluation; E+ and E-, positive and negative to genetic test, respectively; ● Obligate carrier.

last 10 years. He feels tired and debilitated. The age 58 and 54 years, respectively. Signs and mother (R598), 67 years of age, has had similar evolution were quite similar, with one AF cardiac features since she was 50 (Table III). episode per year accompanied by debilitation, Since onset, she has suffered AF attacks about dizziness and shortness of breath. once a month. She also complains of headache, Family 14: Two members of this family were as- irregular heartbeat, shortness of breath and sessed clinically and genetically: a 78-year-old persistent fatigue. woman (R655) and her 38-year-old daughter Family 9: The 34-year-old male proband (R65) (R656), both diagnosed with lone paroxysmal was diagnosed with idiopathic paroxysmal AF AF. The proband’s daughter had her first AF with onset at 25 years of age and recurrent pal- episode lasting more than one minute at age pitations and dizziness as main symptoms. In 26, provoked by transesophageal echocardi- the family, only the 66-year old mother (R629) ography. The mother suffered her first AF has been diagnosed with paroxysmal AF (at episode at age 59 years (Table III). Since then, age 50 years) (Table III). In the last 5 years, clinical course and associated symptoms have she has suffered AF attacks twice a year. She been similar in the two patients. complains of headache, dizziness and fatigue. Family 17: This 30-year-old male proband (R596) Family 12: Only two members were evaluated in is the patient with the earliest onset of all affected this family, the proband (R652), an 80-year-old subjects in the seven families. His first attack of woman, and her younger sister (R653), age 78 AF occurred at age 16 years. Attacks have con- years (Table III). The proband and her sister tinued with a frequency of one or two per year. were diagnosed with lone paroxysmal AF at The proband’s 34-year-old sister (R597) man-

7588 Putative role of Brugada syndrome genes in familial atrial fibrillation

Figure 2. A, Sequence chromatograms of the four variants identified in AF patients.B, Amino acid (aa) conservation across species of the novel CACNA1C variant showing that Cav1.2 Ala71 is highly conserved.

ifested similar disease features with late onset Probands from family 2 and family 3 (R619 at age 28. Since the first attack, the frequency and R586, respectively) showed the p.(Ala71Val) has been similar to that of her brother and both variant in exon 2 of the CACNA1C gene, which complain of associated weakness and dizziness encodes an alpha-1 subunit of a voltage-depen- (Table III). Their father died of . dent calcium channel or Cav1.2. Genetic variants Family 18: The 51-year-old female proband in specific sites (i.e., exon 8 or 8a) of this gene are (R615) suffered her first episode of AF when known to be associated with Timothy Syndrome, she was 35. Since then, she has had episodes a very rare condition in which LQTS is accompa- once every 6 months, accompanied by debil- nied by physical and neurological involvement12, itation and dizziness. Her 71-year-old mother BS13 and LQTS type 8 without extracardiac ab- (R617) showed a similar course and symptoms normalities14,15. The amino acid alanine in posi- but with later onset at age 59 years (Table III). tion 71 is highly conserved through evolution, suggesting that it may be important for protein function (Figure 2B). Discussion The p.(Ala71Val) variant is located near the N-terminus domain of the protein in a highly Genetic screening of Russian families having conserved region of the Cav1.2 protein. Only members with AF produced interesting results. four variants in exon 2, namely p.Ala28Thr,

7589 P.E. Maltese, E. Aldanova, N. Kriuchkova, A. Averianov, E. Manara, et al. 7590 335 333 375 333 333 383 367 320 394 396 383 342 399 397 390 360 405 QTc (msec) Table continued Table QT 320 320 320 320 320 380 380 360 360 360 360 360 340 300 340 400 400 (msec) 80 80 80 80 80 80 80 80 80 80 80 80 80 80 100 100 100 QRS (msec) 180 160 160 140 140 160 180 160 160 180 140 160 160 160 160 160 140 PQ (msec) Electrocardiogram

P 80 80 80 80 80 80 60 80 80 80 60 60 60 80 80 60 100 (msec) 76 55 57 78 65 65 70 72 65 65 65 68 82 66 60 66 60 HR (beats/min)

– – – 75 71 76 70 70 72 69 67 65 70 68 68 66 66 EF %

– – – 3.7 3.7 3.7 3.6 3.0 3.6 3.6 3.3 3.3 3.3 3.3 3.3 2.6 4.5 cm LAD* Echocardiogram – – – – – – – – 1/month 1/month 1 per year 1 per year 1/6 months1/6 1/6 months1/6 1/6 months1/6 1/2 months1/2 AF attack frequency 1/6-8 months

PA PA PA AF IPA LPA LPA LPA LPA LPA LPA Healthy Healthy Healthy Healthy Healthy Healthy Healthy Type of Type

– – – – – – – 18 21 52 58 59 50 25 50 26 54 onset Age of . Clinical characteristics AF of families.

Family ID, sex R598, F Mother Fam-3 Fam-3 M R586 (P), R587, F R587, Brother F R622, Sister Fam-9 M (P), R65 R619, F R619, Grandmother R623, M R623, Maternal uncle F R621, Mother R642, M Father F R629, Mother F R641, Brother R626, F R626, First cousin R585 (P), M (P), R585 Fam-2

Fam–12 F (P), R652

R653, R653, F Sister Fam-14 F (P), R655 R656, F Daughter Table III. Table Putative role of Brugada syndrome genes in familial atrial fibrillation 336 362 354 367 320 360 QTc (msec) QT 320 320 360 340 340 340 (msec) 80 80 80 80 80 80 QRS (msec) 160 180 160 160 180 160 PQ (msec) Electrocardiogram

P 80 80 80 80 80 60 (msec) 70 65 68 66 60 60 HR (beats/min)

75 78 70 70 65 68 EF % 3.3 3.3 3.3 3.3 3.3 3.2 cm LAD* Echocardiogram – – 1/6 months1/6 1/6 months1/6 AF attack frequency 1/6-12 months 1/6-12 1/6–12 months1/6–12

AF LPA LPA LPA LPA Healthy Healthy Type of Type

– – 16 35 59 28 onset Age of . Clinical characteristics AF of families.

Family ID, sex R597, F R597, Sister Fam-17 M R596 (P), Fam-18 F (P), R615 R595, F R595, Mother R617, F R617, Mother R618, M R618, Son

7591 Table IIITable (Continued) proband; P: atrial AF: fibrillation;lone paroxysmal;idiopathic PA: paroxysmal; paroxysmal;LPA: IPA: LAD:left atrial diameter;3.0-4.0 cm *Normal and cm ranges are 2.7-3.8 womenfor and men, respectively (Bold indicates font abnormal ejection EF: fraction; values); HR: heart corrected rate. QTc: interval QT (Bazett’s Formula). P.E. Maltese, E. Aldanova, N. Kriuchkova, A. Averianov, E. Manara, et al. p.(Ala34Val), p.(Gly37Arg) and p.Ala39Val, are The variant SCN10A p.Arg14Leu found in two described in association with non-syndromic affected members of family 12 is a known variant long-QT (LQT)15, sudden cardiac death16, sud- already associated with BS in various indepen- den arrhythmic death syndrome17 and BS as- dent reports24-26. Although the frequency of this sociated with shorter-than-normal QT interval, variant did not differ between BS patients and respectively13. Two of them were also char- controls25, functional evaluation of the mutant acterized functionally: the p.Ala28Thr variant protein has shown that it causes loss-of-function showed an in vitro gain-of-function effect that of Nav1.5 current, which can be expected to re- prolonged duration in line with duce excitability and lead to development of the the associated LQT phenotype15; p.Ala39Val arrhythmogenic substrate responsible for inherit- has been described in the literature associated ed cardiac arrhythmia syndromes, including BS with BS and the authors demonstrated that the and AF24. observed loss of current was due to a defect in The two affected subjects in family 14 share trafficking of mature Cav1.2 channels from the the variant p.(Ala372Pro) in the PKP2 gene, endoplasmic reticulum/Golgi complex to the cell a likely disease-causing variant, associated in membrane13. the literature with ARRVD27,28. The variant in Our study shows that p.(Ala71Val) segregated proband R655 is in compound heterozygous with phenotype in family 3, whereas segregation state with p.(Met365Val), previously described in family 2 suggests that the variant may have in- in association with BS in at least two differ- complete . Although incomplete pen- ent reports26,29. However, R655 did not show etrance of CACNA1C variants has already been a more severe phenotype than her daughter described18, involvement of this variant in onset (R656). This finding, in addition to the result of of the disease in both families can only be postu- the segregation study, indicates that if there is lated from the above observations and therefore any association between PKP2 and AF in this the predicted pathogenicity needs to be demon- family, it can only be attributed to the variant strated by functional studies. p.(Ala372Pro). Genetic testing of family 9 revealed that the The p.Arg1193Gln variant in the SCN5A gene proband (R65) has the CACNA1C, p.(Val2014Ile) found in the proband of family 17 (R596) is a variant. This variant was shown by Burashnikov variant well-known in the literature due to its et al19 to cause loss of calcium channel current great ethnic heterogeneity. The earliest reports function. Interestingly, Burashnikov et al19 de- from two different research groups linked this scribed the variant in a patient who showed a variant with LQTS30 and sudden unexplained BS type I ECG after sodium block challenge and nocturnal death syndrome, a typical presentation had a family history of sudden death of unknown of BS in individuals from southeast Asia31. The cause at an early age. two groups performed functional characteriza- Although electrical remodelling led by chang- tion but obtained different results. The functional es in calcium current channel density has been evaluation performed by Wang et al30 showed associated with persistence of AF20, and although that the mutant channel generates a persistent there is increasing evidence suggesting that mi- and non-activating current leading to prolonged croRNAs are responsible for decreasing L-type duration. p.Arg1193Gln Ca2+ current (ICa) through downregulation of was therefore described as a “gain-of-function” CACNA1C mRNA expression21,22, no genetic variant consistent with previously defined vari- variants in CACNA1C has ever previously been ants causing LQT type 3. The functional charac- described in association with lone AF23. terization of p.Arg1193Gln performed by Vatta In our report, three out of seven families et al31 showed that the variant accelerates fast showed a genetic variant in the CACNA1C gene, inactivation of the sodium channel, resulting and one of these variants is already associat- in a reduced sodium current consistent with a ed with BS in the literature. A primary role of “loss-of-function” variant: this explained the BS changes in cell Ca2+ loading in the onset of AF, phenotype. Finally, independent research groups as distinct from the maintenance of AF, remains definitively associated this variant with both car- to be demonstrated. A reason for the original as- diac conditions, showing that distinct phenotypes sociation could be the fact that CACNA1C is not depend on the background splice variant used usually evaluated in genetic association studies of for expression32,33. These results suggest caution AF and the results may have been missed. in interpreting findings of arrhythmia variants

7592 Putative role of Brugada syndrome genes in familial atrial fibrillation in genetic studies and highlight the contribution In conclusion, six of the AF families here of interaction of environment and genetic back- reported carry variants in genes commonly as- ground with genetic variants32. sociated with BS (CACNA1C, PKP2, SCN10A Given the relatively common occurrence, and SCN5A). BS is characterized by a right with a certain ethnic specificity (i.e., the vari- bundle branch block pattern with ST-segment ant is common in Asians with a MAF of 8%, elevation in leads V1 to V3. Interestingly, none rare in whites with a MAF of 0.3% and unseen of our six probands showed ECG patterns com- in Hispanics and blacks)34, this functional ge- patible with BS or LQTS (Figure 3), and none netic variant was described as a risk factor for had any organic heart disease. It is known from cardiac in the general population. the literature that some drugs can provoke elec- The variant segregated with disease phenotype trocardiographic changes consistent with BS in in AF family 17 here described. Moreover, the patients with AF38. Drug challenges to clarify proband and her sister inherited the variant from the phenotypes were not performed for ethical their father, obligate carrier of the genetic vari- reasons. ant, who died of cardiac arrest before the start AF and BS share some pathogenic genes, i.e., of the study (Figure 1). This evidence, together SCN1B, SCN3B and SCN5A (data from OMIM); with the above mentioned literature, seems to however, the association with the BS gene CAC- increase the potential role of this variant in NA1C described here is new. sudden deaths due to , while at the Variants in PKP2, ANK2 and SCN10A seem same time underlining a new association with somehow involved in AF. In particular, PKP2 lone AF. is known to influence atrial volume39 and to be In family 18, the two affected subjects, the associated with AF in the setting of ARRVD/ proband R615 and her daughter R617, share two cardiomyopathy40. SCN10A variants seem to be variants with possible influence on the patho- associated with late sodium current and alter- genic phenotype: a truncated variant p.(Gln78*) ations in heart conduction41 and to modulate risk in CTNNA3, a gene associated with ARRVD-13 of AF42. ANK2 variants are associated with LQTS (OMIM disease 615616), and a substitution and cardiac arrhythmia. However, the association p.(Thr3744Asn) in ANK2, a gene associated with with FAF in these two cases is new. Involvement cardiac arrhythmia or LQTS 4 (OMIM disease of the ARRVD-associated gene, CTNNA3, in AF 600919). ARRVD is characterized by progressive is new and requires further study. fibro-fatty replacement of the right with The present study has a number of limitations. structural and functional abnormalities of the First, segregation studies were only performed ventricles, electrocardiographic depolarization/ in families with two affected members. Second, repolarization changes, re-entrant arrhythmias, we only selected variants shared by affected and sudden death as main clinical features35. In members, i.e., we chose to disregard variants proband R615, 12-lead ECG showed no specific described as pathogenic in the literature if they signs of ARRVD but cardiac magnetic resonance were only found in probands and not in affect- was not performed. ed relatives. We decided to use the Mendelian CTNNA3 encodes for catenin alpha-3, an 895 inheritance model to simplify interpretation of amino-acid protein; the variant found in our fam- the results, but we cannot exclude the possibility ily introduces a stop codon in position 78 and can that phenotype differences between members be classified as pathogenic on the basis of autoso- of the same family could be driven by a com- mal dominant transmission of the gene described mon pathogenic variant (shown in this report) in ARRVD patients. that acts in a different genetic setting. There is ANK2 encodes for ankyrin-2, a 220 KDa pro- growing conviction that heritable arrhythmia tein involved in the localization and membrane syndromes are oligogenic or even polygenic stabilization of ion transporters and channels diseases43 and some of the genes evaluated in in cardiomyocytes. It has been shown that our study have been demonstrated to behave in mice heterozygous for a null variant in ANK2, this way18,44. Likewise in family 18, we cannot which disrupts cell organization of various ion exclude the possibility that both ANK2 and CT- pumps and channels, manifest arrhythmia36,37. NNA3 are involved in the resulting phenotype. In addition, the present variant has been de- Third, the study was conducted exclusively on scribed as pathogenic in a patient with cardiac Russian patients and it may not be possible to arrhythmia36. generalize the results to other populations.

7593 P.E. Maltese, E. Aldanova, N. Kriuchkova, A. Averianov, E. Manara, et al.

Figure 3. Absence of ST-segment elevation in the AF probands.

Conclusions lar pathologies and mortality, especially sudden death and heart failure45-48. The mechanisms by To the best of our knowledge, this is the first which AF increases risk are not completely clear, report of lone AF families with genetic variants but we are sure that genetic studies will shed light and genes known to be associated with other in- on them. herited cardiac diseases, although the differences Sudden cardiac death is caused by ventricular in their phenotypes remain unclear. In light of our arrhythmias and Brugada syndrome is a right results, we suggest that hereditary cardiomyopa- ventricular disease. In any case, genetically it is thies may share a number of genes and variants, not strange that genes associated with ventricu- and phenotype differences could be explained lar disease be associated with atrial phenotypes by the different genetic background in which and such evidence is not new. Indeed, Francis et they act. This is also the first report in Russian al49 remarked that the arrhythmogenic substrate families. of Brugada syndrome may not be restricted Clinically, the result of this study is inter- to the ventricles. Even more importantly, Al- esting for identification of subjects at risk of hassani et al50 observed a family with lone AF sudden death. This hypothesis needs to be characterized by a large deletion in PKP2, a verified in a larger population of subjects with gene normally associated with arrhythmogenic BS and FAF. right ventricular cardiomyopathy. The authors In support of the hypothesis, many recent stud- suggest that in certain patients and families, ies suggest that atrial fibrillation is effectively cardiomyopathy gene variants may manifest associated with increased risk of cardiovascu- preferentially with atrial rather than ventricular

7594 Putative role of Brugada syndrome genes in familial atrial fibrillation phenotypes, and that this could depend on the 3) Olesen MS, Andreasen L, Jabbari J, Refsgaard L, fact that genes causative for ventricular cardio- Haunsø S, Olesen SP, Nielsen JB, Schmitt N, Svend- sen JH myopathy may serve similar functions in the . Very early-onset lone atrial fibrillation pa- tients have a high prevalence of rare variants in atria, and pathogenic variants in these genes genes previously associated with atrial fibrillation. may manifest with isolated atrial phenotypes, Hear Rhythm 2014; 11: 246-251. potentially secondary to differential penetrance 4) Kopecky SL, Gersh BJ, McGoon MD, Whisnant JP, in the atria and ventricles. Holmes DR, Ilstrup DM, Frye RL. The natural history It seems clear that besides implementing the of lone atrial fibrillation. A population-based study normal measures for reducing the risk of throm- over three decades. N Engl J Med 1987; 317: 669- boembolic stroke, it is also necessary to prevent 674. 5) Maltese PE, Orlova N, Krasikova E, Emelyanchik E, all these other complications. This can be do- Cheremisina A, Kuscaeva A, Salmina A, Miotto R, ne through a multidimensional therapeutic pro- Bonizzato A, Guerri G, Zuntini M, Nicoulina S, Ber- gramme that considers all the comorbidities that telli M. Gene-targeted analysis of clinically diag- frequently accompany atrial fibrillation46. In the nosed long QT russian families. Int Heart J 2017; near future, genetic data may help in the man- 58: 1-7. agement of AF by improving the identification 6) Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, of individuals at risk for heart failure and sudden Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damag- cardiac death. ing missense mutations. Nat Methods 2010; 7: The success rate (39%) of genetic testing ob- 248-249. tained with our approach indicates that AF is 7) Kumar P, Henikoff S, Ng PC. Predicting the ef- genetically heterogeneous and that other genes, fects of coding non-synonymous variants on pro- not evaluated in this study, may be implied in the tein function using the SIFT algorithm. Nat Protoc pathogenesis. Although a comprehensive gene 2009; 4: 1073-1081. panel for FAF is still far from completion, since 8) Schwarz JM, Cooper DN, Schuelke M, Seelow D. Mu- the molecular and pathophysiological foundations tationtaster2: mutation prediction for the deep-se- quencing age. Nat Methods 2014; 11: 361-362. are still not fully understood51, the NGS approach 9) Richards S, Aziz N, Bale S, Bick D, Das S, Gasti- remains the best choice for this kind of study. er-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Familial forms of the disease are not uncommon Voelkerding K, Rehm HL. Standards and guidelines and it is therefore important to identify individ- for the interpretation of sequence variants: a joint uals at risk for the purpose of prevention or for consensus recommendation of the American Col- determining appropriate treatments and planning lege of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med clinical follow-up. 2015; 17: 405-424. 10) Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise J, Solomon S, Spencer KT, St. John Sut- Conflict of Interest ton M, Stewart W. Recommendations for cham- The Authors declare that they have no conflict of interests. ber quantification. Eur J Echocardiogr 2006; 7: 79-108. 11) Sanfilippo AJ, Abascal VM, Sheehan M, Oertel LB, Harrigan P, Hughes RA, Weyman AE Acknowledgements . Atrial enlarge- We thank Helen Ampt for reviewing the manuscript. This ment as a consequence of atrial fibrillation. A work was funded by a grant from the Autonomous Province prospective echocardiographic study. Circulation of Trento (Grant No. S503/2016/238507). 1990; 82: 792-797. 12) Splawski I, Timothy KW, Sharpe LM, Decher N, Ku- mar P, Bloise R, Napolitano C, Schwartz PJ, Joseph RM, Condouris K, Tager-Flusberg H, Priori SG, San- References guinetti MC, Keating MT. CaV1.2 calcium channel dysfunction causes a multisystem disorder in- cluding arrhythmia and autism. Cell 2004; 119: 1) Tsai CT, Lai LP, Hwang JJ, Lin JL, Chiang FT. Molec- 19-31. ular genetics of atrial fibrillation. J Am Coll Cardi- 13) Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, ol 2008; 52: 241-250. Sanguinetti MC, Aizawa Y, Guerchicoff A, Pfeiffer R, 2) Darbar D, Herron KJ, Ballew JD, Jahangir A, Gersh Oliva A, Wollnik B, Gelber P, Bonaros EP, Burash- BJ, Shen WK, Hammill SC, Packer DL, Olson TM. nikov E, Wu Y, Sargent JD, Schickel S, Oberheiden R, Familial atrial fibrillation is a genetically heteroge- Bhatia A, Hsu LF, Haïssaguerre M, Schimpf R, Borg- neous disorder. J Am Coll Cardiol 2003; 41: 2185- grefe M, Wolpert C. Loss-of-function mutations in 2192. the cardiac calcium channel underlie a new clini-

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