European Review for Medical and Pharmacological Sciences 2019; 23: 1710-1721 Next-generation sequencing identifies a homozygous mutation in ACADVL associated with pediatric familial dilated cardiomyopathy S.J. CARLUS1, I.S. ALMUZAINI2, M. KARTHIKEYAN3, L. LOGANATHAN3, G.S. AL-HARBI1, A.M. ABDALLAH4, K.M. AL-HARBI1 1Pediatrics Department, Cardiogenetics Unit, College of Medicine, Taibah University, Al-Madinah, Kingdom of Saudi Arabia 2Department of Pediatric Cardiology, Al-Madinah Maternity and Children Hospital (MMCH), Al-Madinah, Kingdom of Saudi Arabia 3Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu, India 4West Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust, Birmingham, United Kingdom Abstract. – OBJECTIVE: Pediatric familial di- Key Words lated cardiomyopathy (DCM) is a rare and se- Pediatric familial dilated cardiomyopathy, Targeted vere heart disease. The genetics of familial DCM gene sequencing, ACADVL, Saudi Arabia, Consanguinity, are complex and include over 100 known dis- Molecular docking, Molecular dynamics. ease-causing genes, but many causative genes are unknown. We aimed to identify the causative gene for DCM in a consanguineous Saudi Ara- bian family with affected family members and a history of sudden death. Introduction PATIENTS AND METHODS: Affected (two chil- dren) and unaffected (one sibling and the mother) Dilated cardiomyopathy (DCM) is a heart family members were screened by next-gener- ation sequencing (NGS) for 181 candidate DCM disease characterized by ventricular dilation and genes and underwent metabolic screening. Fif- impaired myocardial contractility. DCM affects ty-seven clinically annotated controls and 46 DCM 1 in 2500 of the general population and is one of cases were then tested for the identified mutation. the most common cardiomyopathies causing pe- In silico structural and functional analyses includ- diatric heart failure1-4. The prognosis for children ing protein modeling, structure prediction and with DCM is often poor5, with approximately dynamic simulations were performed. RESULTS: 40% undergoing cardiac transplantation or death A homozygous missense mutation 3 in exon 15 of the acyl-CoA dehydrogenase very within five years of diagnosis . long chain gene (ACADVL; chr17:7127303; G>A) DCM is known to be caused by a variety of mu- was identified in affected subjects that substitut- tations in sarcomere, mitochondrial, RNA-bind- ed histidine for arginine at codon 450 (p.R450H). ing, Z-disk, cytoskeletal, sarcoplasmic reticulum The variant was heterozygous in the mother and and nuclear envelope proteins6. In children, DCM unaffected sister. The mutation was absent in 57 is usually autosomal dominant (AD)7; however, clinically annotated controls and 48 pediatric DCM cases. The mutation was predicted to cause a 14% of pediatric DCM cases are associated with significant and deleterious change in the ACADVL a wide spectrum of extracardiac features such protein structure that affected drug binding, sta- as neuromuscular, metabolic, or malformation bility, and conformation. Metabolic screening con- syndromes with X-linked, mitochondrial or auto- firmed VLCAD deficiency in affected individuals. somal recessive (AR) inheritance8. About 20-50% CONCLUSIONS: The ACADVL R450H mutation of DCM patients have familial DCM (FDCM)9, is an uncommon cause of the DCM phenotype which is often characterized by genetic heteroge- that appears to be autosomal recessive. Targeted NGS is useful for identifying the causative muta- neity and high variability even between members tion(s) in familial DCM of unknown genetic cause. of the same family; the age of the onset and dis- 1710 Corresponding Author: S. Justin Carlus, Ph.D; e-mail: [email protected] ACADVL mutation in pediatric familial dilated cardiomyopathy ease progression also differ considerably between Study Participants and Clinical individuals with FDCM. To date, mutations in Evaluation over 100 genes have been reported to cause Peripheral blood samples were collected from DCM, accounting for 50% of affected individuals four individuals: two affected DCM subjects and but leaving half without a known genetic basis10. an unaffected normal child and their mother Acyl-CoA dehydrogenase very long chain from a consanguineous Saudi Arabian family (ACADVL) mutations are known to cause AR attending the Department of Pediatric Cardiol- DCM and result in very long-chain acyl-coenzyme ogy, MMCH, Al-Madinah, Saudi Arabia. The A dehydrogenase (VLCAD) deficiency, pediatric mother provided written informed consent for cardiomyopathy and sudden death11. ACADVL is study participation. There was a family history about 5.4 kb long, located on chromosome 17p13.1, of sudden death of the father, reported to be from and has 20 exons12. VLCAD deficiency is a rare AR heart disease. The patients reside in Al-Madinah metabolic disorder that affects mitochondrial fatty in western Saudi Arabia. acid β-oxidation, which in turn impacts heart, liver, DCM was diagnosed according to published and muscle function12. VLCAD deficiency can be guidelines17 using the family history, physical and classified into three clinical phenotypes, of which clinical examination, electrocardiograms (ECG), the early-onset form is severe with a high incidence chest x-rays, and 2D-echocardiography. Doppler of cardiomyopathy and high mortality13. echocardiography was performed using a Hewl- Our previous surveys of the registry of a ett-Packard 1500 or 5500 echocardiographic sys- pediatric cardiology clinic at the Maternity and tem. The diagnosis of DCM was characterized by Children Hospital (MMCH), Al-Madinah, Saudi the presence of left ventricular dilation <45% and Arabia, revealed a very high prevalence of DCM left ventricular end-diastolic dimension >117%. in the pediatric heart patient population, and the Other cardiac or systemic causes of DCM includ- DCM mortality rate is high in infants in central ing coronary and valvular diseases were absent. and eastern Saudi Arabia14. Affected children were more likely to die if they were the offspring Dilated Cardiomyopathy Gene Panel of a consanguineous marriage14. Saudi Arabia has We previously developed an in-house cus- one of the highest rates of consanguinity in the tom gene panel covering 181 genes confirmed world, as marriages between first cousins and or presumed to be involved in cardiomyopathy close relatives are widely accepted and frequently pathology10; the complete gene list is available occur15, and consanguinity may be a risk factor in Table I. Briefly, this gene panel covers cod- for some congenital abnormalities16. ing exons, exon-intron boundaries, and known In this paper, we aimed to identify novel mu- variants in the intronic regions of target genes. tation(s) and gene(s) responsible for familial DCM The total target region length is 1108523 base using targeted NGS technology in a consanguin- pairs (bp) involving 3852 target regions. Target eous Saudi Arabian family with affected family regions include exons, 250 bp of adjacent introns, members. In doing so, we identified a homozygous and 5′- and 3′-UTR regions. All annotated coding missense mutation in ACADVL in affected individ- regions were extracted from hg19 from Ensembl uals with concomitant VLCAD deficiency on met- (http://www.ensembl.org) and the UCSC genome abolic screening. To provide functional insights, in browser (http://genome.ucsc.edu). silico analyses were used to predict the deleterious effect of the mutation and to understand its effects Targeted Gene Sequencing on protein structure and drug binding. For targeted gene sequencing, DNA was ex- tracted from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). DNA Materials and Methods quantity and quality were measured by reading the whole absorption spectrum (220-750 nm) with Ethics Statement a NanoDrop ND-2000 and calculating the DNA This study was fully in accordance with the eth- concentration and absorbance ratio at 260/280 ical standards of the Taibah University Ethics Re- nm. Each sample was quantified with a Qubit 3.0 search Committee, the Ethics Review Board of Ma- Fluorometer (Thermo Fisher Scientific, Waltham, dinah Maternity and Children Hospital (MMCH), MA, USA). Extracted DNA samples were evalu- and with the 1964 Helsinki Declaration and its later ated by loading 100 ng of DNA on a 0.8% agarose amendments or comparable ethical standards. gel for electrophoresis, which showed intact bands 1711 S.J. Carlus, I.S. Almuzaini, M. Karthikeyan, L. Loganathan, G.S. Al-Harbi, et al. of DNA without any degradation. Then, 2-3 µg of using Varminer (an in-house variant interpreta- genomic DNA per participant were processed for tion tool), and variant pathogenicity was assessed library preparation, with the library enriched using based on the 2015 American College of Medical a custom Nimblegen panel and sequenced as 2 x Genetics (ACMG) guidelines. 250 bp paired-end reads on an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) according Validation by Sanger Sequencing to the manufacturer’s protocol. Sanger sequencing was performed to confirm the presence of variants. Primer sequences were Bioinformatics Analysis designed using Primer3. The primer pairs used for Bioinformatics analysis was performed as pre- the Polymerase Chain Reaction (PCR) were: for- viously described18. After obtaining the data, we ward:5’-CCTGTGCTAGGAACCTGGAG-3’ and used the Genome Analysis Toolkit (GATK) best reverse: 5’CCCAGAGAGCTCCTTTCCTT-3’. The practice variant-calling