Homozygous PKP2 deletion associated with neonatal left ventricle noncompaction

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1 Clin Genet 2017: 91: Printed in Singapore. All rights reserved Short Report 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Homozygous PKP2 deletion associated with neonatal left ventricle noncompaction Ramond F., Janin A., Di Filippo S., Chanavat V., Chalabreysse L., Roux-Buisson N., Sanlaville D., Touraine R., Millat G. Homozygous PKP2 deletion associated with neonatal left ventricle noncompaction. Clin Genet 2017: 91: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2016 Left ventricular noncompaction cardiomyopathy (NC) is a clinically heterogeneous disorder characterized by a trabecular meshwork and deep intertrabecular myocardial recesses that communicate with the left ventricular cavity. Several genetic causes of NC have been reported, with variable modes of inheritance, including autosomal dominant and X-linked inheritance, but relatively few responsible genes have been identified. A NGS workflow, based on a panel of 95 genes developed for sequencing most prevalent sudden cardiac death-causing genes, was used to make a rapid and costless molecular diagnosis in two siblings with a severe noncompaction cardiomyopathy starting prenatally and leading to rapid cardiac failure. For the first time, a total homozygous PKP2 deletion was identified. This molecular defect was further confirmed by MLPA and array-comparative genomic hybridization (CGH). Heterozygous PKP2 mutations are usually reported in a significant proportion of Arrhythmogenic Right Ventricular Cardiomyopathy cases. Our results show, for the first time, the involvement of PKP2 in severe cardiomyopathy with ventricular non compaction. Conflict of interest The full disclosure presents no conflict of interest. The authors alone are responsible for the content and writing of the paper. F. Ramond a,a.janin b,c,d,e,s.di Filippo f, V. Chanavat b,c, L. Chalabreysse g,n.roux- Buisson h,i, D. Sanlaville j, R. Touraine a and G. Millat b,c,d,e a Genetics Department, CHU-Hôpital Nord, Saint-Etienne, France, b Laboratoire de Cardiogénétique Moléculaire, Hospices Civils de Lyon, Lyon, France, c NGS sequencing platform for molecular diagnosis, Hospices Civils de Lyon, Lyon, France, d Université de Lyon, Lyon, F-69003, France, e Université Lyon 1, Lyon, France, f Paediatric Cardiology and Congenital Heart Disease Department, Cardiovascular Louis-Pradel Hospital, Hospices Civils de Lyon, Lyon, France, g Department of Pathology, Louis Pradel Hospital, Hospices Civils de Lyon, Lyon, France, h Grenoble Institut des Neurosciences, Equipe Muscle et Pathologies, INSERM U836, UJF, Grenoble, France, i CHU de Grenoble, Laboratoire de Biochimie Génétique et Moléculaire, Grenoble, France, and j Cytogenetics, Hospices Civils de Lyon, & Centre de Recherche en Neurosciences de Lyon, Equipe GENDEV, INSERM U1028; CNRS UMR5292; UCBL1, Lyon, France Key words: arrhythmogenic right ventricular dysplasia/cardiomyopathy cardiomyopathy left ventricular non compaction NGS sequencing PKP2 deletion Corresponding author: Gilles Millat, PhD, Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie Est, F Bron Cedex, France. Tel: +33(0) ; fax: +33(0) ; gilles.millat@chu-lyon.fr Received 21 December 2015, revised and accepted for publication 17 March

2 PKP2 gene deletion and non-compaction cardiomyopathy Left ventricular noncompaction cardiomyopathy (NC) has recently been classified as a distinct cardiomyopathy (1). It consists in a spongiform aspect of myocardium limited to a part of the left ventricle or less frequently of the right ventricle or diffuse to both ventricles. It can be isolated or associated with cardiac malformation or arrhythmia and has a variable prognosis. It has been ascribed to various heterogeneous genetic alterations, but less than 50% have a mutation identified (2). The majority of cases are sporadic, but familial recurrence has been described most often with autosomal dominant or X-linked inheritance, and sometimes transmitted as an autosomal recessive or mitochondrial trait (2). Here, we report a novel PKP2 homozygous gene deletion observed in two siblings with severe NC diagnosed prenatally and leading to rapid and lethal cardiac failure. Material and methods This study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained for all cases. A molecular analysis was performed by next generation sequencing (NGS) on a NextSeq500 sequencer (Illumina, San Diego, CA, USA), of a large panel of 95 genes associated with sudden cardiac death [ABCC9, ACTC1, ACTN2, AKAP9, ANK2, ANKRD1, BAG3, CACNA1C, CACNA2D1, CACNB2, CALM1, CALM2, CALM3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTNNA3, DES, DMD, DPP6, DSC2, DSG2, DSP, DTNA, EMD, FHL1, FKTN, FLNC, GJA1, GJA5, GLA, GPD1L, HCN4, JPH2, JUP, KCNA5, KCND3, KCNE1, KCNE1L, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, LAMP2, LDB3 (ZASP), LMNA, RANGRF, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEXN, NKX2-5, NOS1AP, NPPA, PDLIM3, PKP2, PLN, PRKAG2, PTPN11, RAF1, RBM20, RYR2, SCN10A, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SGCD, SLMAP, SNTA1, TAZ, TCAP, TGFB3, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TRDN, TRPM4, TTN, TTR, and VCL]. Libraries were constructed with the Kapa Library Preparation Kit for Illumina platforms (Kapa Biosystems, London, UK) according to the manufacturer s instructions. In silico analysis was further performed using a personalized Sophia Genetics pipeline optimized for both single nucleotide polymorphism (SNP)/indels and copy number variation (CNV) detection. Results The first case of this family was the second child of second degree-related parents without previous familial medical history (Fig. 1). After an unremarkable beginning of pregnancy, fetal hydrops was seen at 32 weeks, caused by fetal cardiac failure. Fetal anemia was excluded. Karyotype analysis and metabolic screening in amniotic fluid were normal. A diagnosis of left ventricular non compaction (NC) was made by Fig. 1. Pedigree of the family. The two affected sibs are marked with black symbols, the obligate carriers of the heterozygous deletion are marked with a central dot. The father s sibs are aged between 39 and 54 and the two grandmothers are 68 and 73 years old. Except for the two parents, the other family members were not further investigated. ultrasound examination. After birth, the female newborn was admitted to intensive care unit in a cardiopediatric department. Her echocardiography (ECG) was normal and she had no other symptoms than cardiac insufficiency related to diffuse myocardial noncompaction (Fig 2a). Cardiac failure progressed rapidly and was refractory to medication, leading to death at 12 days of age. Autopsy confirmed the diagnosis of noncompaction of both ventricles without other heart malformation (Fig. 2b). No other malformations or problems were seen. Histologically, some degree of disorganization in the arrangement of the cardiomyocytes, sometimes separated by interstitial edema, was seen together with endomyocardial fibrosis. Metabolic analyses were unremarkable. Cardiac ultrasound examination was normal in both parents. Two years later, a healthy son was born. Finally, during the fourth pregnancy, ultrasound examination at 29th week identified a recurrence of cardiac failure with spongious aspect of the myocardium in a male fetus, making the parents opt for termination of pregnancy. Autopsy was denied by the parents. Extensive molecular analysis was performed by NGS sequencing of a large panel of 95 genes associated with sudden cardiac death. The molecular analysis led to detect a total homozygous deletion of PKP2, further confirmed by an MLPA approach (MRC-Holland SALSA MLPA P168 AC-PKP2 probemix kit). The two affected siblings were carriers of the homozygous deletion, whereas each parent is carrier of the deletion in a heterozygous status. Array-comparative genomic hybridization (CGH) analysis in the index case also showed that the deletion is limited to the PKP2 gene solely (hg19, chr12:32,936,266_33,056,189) (Fig. 3). The two healthy brothers have not been tested yet because of their young age (3 and 8 years old) and the absence of clinical sign of cardiac disease. A further cardiologic examination (ECG and magnetic resonance imaging (MRI)) showed that both parents (35 and 41 years old) are free of any anomaly, and specifically free of arrhythmogenic right ventricular 127

3 Ramond et al. (a) A B VSD VSD RA LA LA C Non-compacted D SF= 17.6% (b) Fig. 2. Imaging of the myocardial noncompaction of the first index case (a) Echocardiography: (A) four chamber view; (B) long axis view of the left ventricle; (C) short axis view of ventricles; and (D) TM-mode of left and right ventricles. (LA, left atrium,, left ventricle, SF, left ventricle shortening fraction, RA, right atrium,, right ventricle, VSD, ventricular septal defect). (b) Autopsy: cross-section of the heart showing spongiform aspect of both ventricles (, left ventricle;, right ventricle). cardiomyopathy/dysplasia (AC/D) according to the revised task force criteria (Appendix S1, Supporting Information). Signal-averaged electrocardiogram (SAECG) was not performed in regard to the normality of other investigations. Discussion A total homozygous PKP2 deletion was identified in two siblings with a severe noncompaction cardiomyopathy starting prenatally and leading to rapid cardiac failure. PKP2 mutations have been usually identified at heterozygous state in families with AC and more recently in patients with Brugada syndrome (3 14). AC is characterized by myocardial fibro-fatty infiltration of the myocardium and ventricular arrhythmias with elevated risk of syncope and sudden cardiac death in adolescents and young adults (3/4 before 40 years of age). Some patients develop heart failure at a later age. AC is associated with truncating or missense mutations that spread over all the coding sequence (6). 128

4 PKP2 gene deletion and non-compaction cardiomyopathy Fig. 3. Array comparative genomic hybridization (CGH) analysis (Agilent 180k microarray) showing the 120 kb homozygous deletion encompassing the whole PKP2 gene in proband s DNA compared with two control DNAs, in a mirror view. Left panel: blue line indicating the 12p11.21 position of the deletion on the chromosome whole view. Right panel: detailed view of the deleted region, showing the absence of hybridization of 12 contiguous probes, from position chr12:32,936,266 to position 33,056,189 (hg19). It has been recently shown that truncating PKP2 mutations are associated with a later age of onset of AC than missense ones, without difference in the severity of the pathology (7). Deletions of the PKP2 gene have previously been described at heterozygous state in patients with AC (9, 12, 13). It is noteworthy that knock-out mice homozygous for pkp2 null/null have early lethality related to impairment of myocardium development (15). A homozygous PKP2 mutation, c.2484c>t (p.gly828gly), modifying the splicing of exon 12 and leading to a frameshift (last coding exon is exon 14) has been previously identified in an adult woman with AC (16). However, normal splicing was conserved in a minor quantity. In this family, heterozygous carriers are asymptomatic, but we have to stress out that in families with AC inherited as dominant trait, penetrance is reduced. Our case is the first description of homozygous null mutation of PKP2 in human. We consider very likely that this homozygous loss of PKP2 gene is responsible for the severe phenotype of NC in this family, as learned from the homozygous null mouse model (15). Both parents were asymptomatic at 35 and 41 years of age (absence of AC according to the revised Task Force criteria, or Brugada syndrome) (17). They are healthy, of normal stature and free of any dysmorphic feature. Furthermore, parents confirmed the absence of familial history of cardiac dysrhythmia, sudden death or any other medical condition, whereas several elderly individuals are obligate heterozygotes for this PKP2 deletion (Fig. 1). Incomplete penetrance is a well-known phenomenon in families with PKP2 point mutations and many studies have suggested a digenic or compound heterozygosity mechanism underlying the penetrance of AC (18). Regarding our family, it is noteworthy that no mutation in other genes of our panel has been found in the proband s DNA, but of course, asymptomatic parents have not been studied with this panel. Heterozygous deletion of the entire PKP2 gene has previously been described in three families. In two families, only one adult patient is symptomatic and there is no information on the genotype of adult relatives (6, 8). In a large family, among nine adult heterozygote carriers, six were diagnosed with AC, one has minor signs of AC and two were without signs (7). The 122 kb deletion identified in this previously published family is very similar to our present one, and therefore unlikely to explain a difference in penetrance. The principal difference between this family and ours is the reason for molecular testing and along these lines, the presence in the former family, of a modifying or even principal factor in trans, is the most probable explanation that we cannot confirm or rule out at this stage because no other AC gene has been identified on chromosome 12. Therefore regarding the risk of AC, our data further illustrates the difficult interpretation of the incidental finding of a single mutation. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. Acknowledgements This work was supported by French Ministry of Research (Diagnosis Network on Neuromuscular Diseases). 129

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