Int J Clin Exp Pathol 2018;11(7):3732-3743 www.ijcep.com /ISSN:1936-2625/IJCEP0076955 Original Article Next-generation sequencing identified genetic variations in families with fetal non-syndromic atrioventricular septal defects Jinyu Xu1, Qingqing Wu1, Li Wang1, Jijing Han1, Yan Pei1, Wenxue Zhi2, Yan Liu3, Chenghong Yin4, Yuxin Jiang5 Departments of 1Ultrasound, 2Pathology, 3Obstetrics, 4Internal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China; 5Department of Ultrasound, Peking Union Medical College Hospital, Beijing, China Received March 29, 2018; Accepted May 9, 2018; Epub July 1, 2018; Published July 15, 2018 Abstract: Atrioventricular septal defects (AVSDs) account for approximately 5% of all congenital heart disease (CHD). About half of AVSDs are diagnosed in cases with trisomy 21 (Down’s syndrome, DS). However, many AVSDs occur sporadically and manifest as non-syndromic. The pathogenesis is complex and has not yet been fully eluci- dated. In the present study, we applied two advanced applications of next-generation sequencing (NGS) to explore the genetic variations in families with fetal non-syndromic AVSDs. Our study was mainly divided into two steps: (1) low-pass whole-genome sequencing (WGS) was used to detect the genome-wide copy number variations (CNVs) for included subjects; (2) whole-exome sequencing (WES) was used to detect the gene mutations for the subjects without AVSD-associated CNVs. A total of 17 heterozygous de novo CNVs and 19 heterozygous de novo gene muta- tions were selected, and 15 candidate genes were involved in these variations. Among these heterozygous de novo variations, most have potential pathogenicity for AVSDs, but the others require further investigation to confirm their pathogenicity. Our study not only shows the genetic diversity and the etiological complexity of AVSDs but also shows the rationality and practicability of this sequential genetic detection and analysis strategy. Keywords: AVSD, copy number variation, gene mutation, whole-genome sequencing, whole-exome sequencing Introduction AVSDs are often associated with other cardiac defects, such as Tetralogy of Fallot (TOF), dou- Congenital heart disease (CHD) is the most ble outlet right ventricle, and transposition of common congenital malformation seen at birth the great arteries [4, 5]. The clinical presenta- as well as the most common congenital defect tion and prognosis in AVSDs depend on the spe- contributing to death in the first year. The prev- cific morphology of the defects and the associ- alence of CHD is approximately 8 per 1000 ated anomalies. Untreated patients with AVSDs births worldwide [1]. Atrioventricular septal may present with cyanosis, breathlessness, defects (AVSDs) or atrioventricular (AV) canal recurrent respiratory infection, growth retarda- defects account for approximately 5% of all tion, variable heart murmur, or even congestive CHD [2, 3]. This group of defects is caused by cardiac failure, pulmonary hypertension, and abnormal development of endocardial cush- death in early life [3, 4]. Many parents who are ions. Meanwhile, endocardial cushions are diagnosed with fetal AVSDs may choose to ter- minate the pregnancy to reduce the economic involved in the formation of the atrial septum, and psychological burdens of their families. At the ventricular septum, and the mitral and tri- the same time, some parents may seek genetic cuspid valves during embryonic development. counseling to assess the genetic defects of the Therefore, AVSDs are manifested by varying malformed fetuses and the risk of recurrence in degrees of AV valvular and septal abnormali- their next pregnancies. ties, including atrial septal defect (ASD) and ventricular septal defect (VSD), and are classi- About half of AVSDs are diagnosed in cases fied as “partial, intermediate, or complete”. with trisomy 21 (Down’s syndrome, DS) [6, 7]. NGS in families with fetal non-syndromic AVSDs Deletions on chromosome 21 on a trisomic raphy and confirmed by post-mortem autopsy background may reduce the risk for AVSDs [8]. in Beijing Obstetrics and Gynecology Hospital, Some genes may act as susceptibility factors China. Fetuses with identified chromosomal for AVSDs in DS patients, such as CRELD1 gene karyotype abnormalities or extracardiac malfor- [9]. However, many AVSDs occur sporadically mations were excluded. Umbilical cord blood and manifest as non-syndromic. The pathogen- samples were collected from prenatal samples, esis is complex and has not yet been fully and fetal tissues were collected from abortus- elucidated. es. Meanwhile, peripheral blood samples were collected from the parents. All samples sent to In recent years, a relationship between sub- the MyGenostics medical laboratory (Beijing, chromosomal anomalies and CHD has been PRC) for analysis. The study was approved by strongly suggested [10]. These subchromo- the ethics committee of the hospital. Informed somal anomalies are known as copy number consent for storage and subsequent analysis variations (CNVs) and defined as copy number was obtained from all parents. changes, including deletions, duplications, or multiallelic variation events of genomic regions DNA library construction ranging from 1 kilobase (Kb) to several mega- bases (Mb). CNVs can be identified using chro- The RelaxGene Blood DNA System (Tiangen mosomal microarray analysis (CMA), which is Biotech, Beijing, PRC) and the Universal Geno- based on gene chip technology and limited by mic DNA Kit (CWBiotech, Beijing, PRC) were probe spacing and density. Recently, several used to extract genomic DNA from the blood studies have demonstrated the possibility of and tissue samples, respectively. The quality using low-pass whole-genome sequencing and concentration of the genomic DNA were (WGS) to detect CNVs [11, 12]. Low-pass WGS evaluated by Nanodrop 2000 (Thermo Fisher, is an application of next-generation sequencing MA, USA). The ratio of A260/280 was between (NGS) that can detect genome-wide CNVs, even 1.8 and 2.0, and the concentration was greater those beyond the probe’s range of CMA [13]. than 30 ng/μL. The genomic DNA was broken However, there is very little research on the into fragments of 100-500 base pairs (Bp) detection of CNVs in AVSD cases without DS using the Covaris S220 DNA sonication system [14]. As another approach of NGS, whole-exome (Covaris, MA, USA). The fragments were end- sequencing (WES) has been more and more polished, adenylated, and ligated with adaptors used to explore the gene mutations of some in turn. Proper reaction systems and cycles of diseases. However, this research is only just polymerase chain reaction (PCR) amplification beginning for AVSDs [15]. were carried out using the GeneAmp PCR System 2720 (Applied Biosystems, CA, USA) In our study, we applied NGS to explore the for enrichment of ligated DNA fragments. All genetic variations in fetuses with non-syndrom- enzymes and buffers were from MyGenostics ic AVSDs and normal chromosome karyotypes. (Baltimore, MD, USA). All operations were car- Our study was mainly divided into two steps: (1) ried out according to the manufacturers’ rec- Low-pass WGS was used to detect the genome- ommendations. The final DNA library products wide CNVs for included subjects; (2) WES was were quantitatively detected using NanoDrop used to detect the gene mutations for the 2000 and 1% agarose gel electrophoresis. The subjects without AVSD-associated CNVs. To concentration of normal DNA library products exclude benign family genetic factors and to was greater than 30 ng/μL, and the ratio of analyze the sources of the meaningful genetic A260/280 was between 1.8 and 2.0. The main variations, we applied family study, and the bands of the DNA library fragments were about same steps were completed on the healthy 280-400 Bp. parents. Low-pass WGS and data analysis Materials and methods NGS was carried out on the Nextseq 500 sys- Subject enrollment tem (Illumina, CA, USA) to generate 150 Bp paired-end reads (a target depth of 0.6×) for The study subjects were fetuses with non-syn- each prepared DNA library according to the dromic AVSDs diagnosed by fetal echocardiog- manufacturer’s recommendations. Reads were 3733 Int J Clin Exp Pathol 2018;11(7):3732-3743 NGS in families with fetal non-syndromic AVSDs Table 1. Phenotypic characteristics of the 50 Genome Analysis Toolkit (version 3.7) software AVSD fetuses tools [18]. Variants were first prioritized based Sex n (%) on their frequency in the 1000 Genomes Pro- ject (1000 g 2015aug_all), Exome Sequencing Male 28 (56.0) Project (ESP6500, ExAC_ALL, ExAC_EAS) and Female 22 (44.0) an inhouse database of 800 healthy Chinese AVSD type n (%) Han adults, with rare (minor allele frequency < Partial 21 (42.0) 0.05) variants receiving priority [19, 20]. Vari- Intermediate 11 (22.0) ants in AVSD-associated genes (described in Complete 18 (36.0) detail below) were selected for further analy- Associated cardiac defects n (%) sis and annotated by different bioinformatics Yes 27 (54.0) tools: The Sorting Intolerant Form Tolerant (SI- No 23 (46.0) FT), PolyPhen-2, Mutation Taster, GERP++ [21- Abbreviations: AVSD, atrioventricular septal defect. 24]. AVSD-associated gene list aligned to the National Center for Biotechnology In order to identify potential candidate CNVs Information human reference genome build 37 and gene mutations associated with AVSDs, we (HG19) using Burrows-Wheeler Aligner (version compiled a list of 375 human genes with a 0.7.10) [16]. Quality control and removal of putative role in the development of AVSDs duplicated reads were carried out using Picard using Phenolyzer. We used the disease or phe- (picard-tools-1.119). Finally, the mapped reads notype terms “heart septal defect”, “heart ven- were produced. The exact CNV breakpoint tricular septal defect”, “heart atrial septal sequences were calculated using the binary defect”, “atrioventricular canal/septal defect”, segmentation algorithm to determine candi- “endocardial cushion defect”, and selected the date CNV regions and the copy ratio. A CNV was “seed genes” sorted by Phenolyzer (Supple- defined as a deletion or a duplication when its mentary Table 1) [25].