Chromosomal microarray and whole‐exome sequence analysis in Taiwanese patients with autism spectrum disorder

Abstract Background Autism spectrum disorder (ASD) is defined as a group of genetically and clinically heterogeneous neurodevelopmental disorders. Interplay between de novo and inherited rare variants has been suspected in the development of ASD. Methods Here, we applied 750K oligonucleotide microarray analysis and whole‐exome sequencing (WES) to five trios from Taiwanese families with ASD. Results The chromosomal microarray analysis revealed three representative known diagnostic copy number variants that contributed to the clinical presentation: the chromosome locations 2q13, 1q21.1q21.2, and 9q33.1. WES detected 22 rare variants in all trios, including four that were newly discovered, one of which is a de novo variant. Sequencing variants of JMJD1C, TCF12, BIRC6, and NHS have not been previously reported. A novel de novo variant was identified in NHS (p.I7T). Additionally, seven pathogenic variants, including SMPD1, FUT2, BCHE, MYBPC3, DUOX2, EYS, and FLG, were detected in four probands. One of the involved genes, SMPD1, had previously been reported to be mutated in patients with Parkinson's disease. Conclusions These findings suggest that de novo or inherited rare variants and copy number variants may be double or multiple hits of the probands that lead to ASD. WES could be useful in identifying possible causative ASD variants.


| INTRODUCTION
Autism spectrum disorder (ASD), which belongs to a group of neurobehavioral syndromes, is characterized by significantly impaired social interaction and communication as well as by restricted, repetitive, and stereotyped patterns of behaviors, interests, and activities (Johnson, Myers, & American Academy of Pediatrics Council on Children With, 2007). The prevalence of ASD is estimated to be 1:59 children and 1:100 adults (Baio et al., 2018;Brugha et al., 2011). The rate of ASD is higher in males than in females (4:1), which is higher than those of Down syndrome and epilepsy. Developmental delays are observed in approximately 40% of individuals with ASD, and approximately 70% show some level of intellectual disability. ASD has strong genetic contributions, and singlegene disorders are recognized as causative in less than 20% of ASD cases (Herman et al., 2007). The most consistently reported single gene disorders associated with ASD are fragile X syndrome, Rett syndrome, and tuberous sclerosis. The prevalence of fragile X syndrome among subjects with ASD is 1.5%-3% (Clifford et al., 2007). The genetic etiology of ASD is complex.
Chromosomal microarray analysis (CMA) examines gross chromosomal structural abnormalities and can detect deletions and duplications as well as the size and presence of known genes within a chromosomal region. The most common microarray abnormalities in ASD involve the chromosome regions 15q11-q13, 16p11.2, and 22q11.2 (Carter & Scherer, 2013;Roberts, Hovanes, Dasouki, Manzardo, & Butler, 2014). In the clinical setting, CMA, which has a diagnostic yield ranging from 7.0% to 9.0%, is recommended as the first tier test for children and adults presenting with ASD (Battaglia et al., 2013;McGrew, Peters, Crittendon, & Veenstra-Vanderweele, 2012;Shen et al., 2010).
Technological improvements have led to tremendous advances in our understanding of the genetic basis of ASD over the past 10 years. Most genomic studies on ASD using next-generation sequencing (NGS) have focused on proteincoding regions and analyzed trio information to identify sequence-level de novo mutations (De Rubeis et al., 2014;Iossifov et al., 2014Iossifov et al., , 2012Neale et al., 2012;O'Roak et al., 2012;Sanders et al., 2012). Hundreds of genes have been implicated in the cause of ASD. The identification of new genes involved in ASD has made this condition a strong candidate for genome-based diagnostic testing, which consists of CMA and NGS, as well as whole-genome sequencing (WGS) and whole-exome sequencing (WES).
Recently, Guo et al. applied WGS, WES, and CMA to investigate genomic variants in ASD families and compared the performances of WGS and WES for use in diagnostic testing (Guo et al., 2019). The authors reported the diagnostic utility of WGS for detecting disorder-related variants (particularly multiple rare-risk variants that contribute to phenotypic severity in individuals with ASD), identifying genetic heterogeneity in multiplex ASD families and predicting novel ASD-associated genes for future study.
In this study, we aimed to define causative or susceptibility variants for ASD and their copy number variants by CMA. We studied five subjects who are typical of those seen in developmental pediatric clinics. The sample was stratified based on the clinical phenotype of the patients.

| Subjects with ASD
Five patients with a clinical diagnosis of ASD were enrolled in the study. Autism screening was performed using the Autism Behavior Checklist, Taiwanese version (ABC-T), which was modified from the third edition of the Autism Behavior Checklist of Autism Screening Instrument for Education Planning (Krug, Arick, & Almond, 1980). Family members were also enrolled for inheritance pattern analysis. Blood samples were obtained, and genomic DNA was extracted using the Nucleospin® Blood Kit (Macherey-Nagel, GmbH & Co. KG, Duren, Germany). This study was approved by the China Medical University Hospital (CMUH105-REC1-039).

| Single-nucleotide polymorphism (SNP) array analysis
DNA samples (250 ng) were hybridized to the Affymetrix CytoScan 750K array according to the manufacturer's instructions. The 750K array contained greater than 750,000 markers for copy number analysis and 200,000 SNP probes for genotyping. The following standard experimental procedures were performed: digestion, ligation, polymerase chain reaction (PCR), PCR purification, fragmentation, labeling, hybridization, washing, staining, and scanning. After hybridization, GeneChip Scanner 3000 7G, Affymetrix GeneChip Command Console software, and Affymetrix ChAS 2.0 software were used for scanning the arrays, extracting the images, and performing the analysis, respectively. All data had to pass quality control (QC) metrics including the median of the absolute values of all pairwise differences ≤ 0.30, SNPQC ≥ 15, and a waviness standard deviation ≤ 0.12.

| WES
In total, 100 ng of genomic DNA based on Qubit quantification was mechanically fragmented on a M220 focused ultrasonicator Covaris (Covaris, Woburn, MA, USA), and QC was performed using an Agilent Bioanalyzer 4200 (Agilent Technologies, Santa Clara, CA, USA) to ensure an average fragment size of 150-200 bp. End repair, A-tailing, adaptor ligation, and enrichment of DNA fragments were then performed. A 200-400 bp band was gel-selected, and exome capture was performed using a TruSeq Exome Library Preparation Kit (Illumina, San Diego, CA, USA). The DNA library was quantified in the Qubit 3.0 Fluorometer (Invitrogen) and Agilent 4200 Bioanalyzer (Agilent Technologies). Samples were sequenced on an Illumina NextSeq500 platform and 150-bp paired-end reads were generated.

| Variant validations and segregation analysis
We used PCR and Sanger sequencing to validate candidate variants from WES. Segregation analysis was carried out on family members. PCR primers were designed using Primer3 (http://bioin fo.ut.ee/prime r3-0.4.0/). Table S1 lists the designed primers. The products were directly sequenced with an ABI PRISM BigDye kit using an ABI 3130 DNA sequencer (Applied Biosystems). Sequencing results were analyzed using the software Chromas, version 2.23.
Patient 3, a 15-year-old male, had an ABC-T score of 20 (Table 3)  T A B L E 2 Summary of the rare variants in autism-related genes detected in this study  (Table 2). TSC2, LZTS2 and BIRC6 are involved in the negative regulation of Wnt and canonical Wnt signaling pathways (GO:0030178 and GO:0090090) and negative regulation of signaling transduction (GO:0009968). No pathogenic mutations were detected.
The mutations in our five patients were further confirmed by Sanger sequencing of DNA from both parents to determine the origins of mutation or to reveal de novo mutations.

| DISCUSSION
In this study, WES was performed to identify possible ASD causal variants in five Taiwanese families; one novel de novo variant in one trio and rare variants in each trio were successfully identified. These genes are involved mainly in the negative regulation of Wnt and canonical Wnt signaling pathways, negative regulation of signaling transduction and endoplasmic reticulum calcium ion homeostasis. We detected no association of the ABC-T score with a particular pathway. However, possible causal variants may be missed if located within a noncoding region; thus, WGS will be necessary in future studies.
Three ASD patients (ASD25, ASD26, and ASD27) were found to carry a novel missense variant of four genes (JMJD1C, TCF12, BIRC6, and NHS) ( Table 2). JMJD1C encodes a putative histone demethylase and is involved in the epigenetic control of gene transcription. This study identified a variant of JMJD1C, c.6344T > G, which results in the substitution of phenylalanine by cysteine (p.F2115C). The p.F2115C mutation is in the JmjC domain, a domain family that is part of the cupin metalloenzyme superfamily. Mutations in this gene are associated with Rett syndrome and intellectual disability (Saez et al., 2016). TCF12 encodes a member of the basic helix-loop-helix E-protein family that recognizes the consensus-binding site (E-box) CANNTG. This study identified a variant, c.770G > A, which results in substitution of an arginine by histidine (p.R257H) in TCF12. BIRC6 encodes an inhibitor of apoptosis protein with baculoviral inhibition of apoptosis protein repeat (BIR) and ubiquitin-conjugating enzyme E2, catalytic (UBCc) domains. This study found a variant of BIRC6, c.6600G > T, which results in substitution of a glutamine by histidine (p.Q2200H). NHS encodes a protein with four conserved nuclear localization signals that function in brain development. This study identified a variant, c.20T > C, which results in substitution of isoleucine by threonine (p.I7T) in NHS. Mutations in this gene are associated with Nance-Horan syndrome (Shoshany et al., 2017). These variants were not found among the 277,264 alleles in the GnomAD database and were predicted to be damaging in silico by SIFT and to be likely damaging by Polyphen2.
Most of the variants identified in this study were found in autosomal genes, whereas one was identified in the X-Y pseudoautosomal gene, ASMT, which has been reported to be associated with the autism phenotype and sleep disturbance (Cai et al., 2008;Wang et al., 2013). In the present study, we identified one reported missense variant, pG151S, in the Taiwanese population with ASD. Additionally, we detected no obvious dominant or recessive compound heterozygous mutations in ASD-related genes.
By considering pathogenic mutations with ClinVar, we found variants in four of five probands (80%). The pathogenic mutations were detected in SMPD1, FUT2,BCHE,MYBPC3,DUOX2,EYS,and FLG2 in four different patients (Table 4). SMPD1 encodes a lysosomal acid sphingomyelinase that converts sphingomyelin to ceramide. Defects in this gene are a cause of Parkinson's disease (Mao et al., 2017). FUT2 encodes a Golgi stack membrane protein and is highly associated with the development of inflammatory bowel disease (Wu et al., 2017). BCHE encodes a cholinesterase enzyme and is a member of the type-B carboxylesterase/lipase family of proteins. Some of the genetic variants are prone to the development of prolonged apnea following administration of the muscle relaxant succinylcholine (Panhuizen, Snoeck, Levano, & Girard, 2010). BCHE p.T343fs has been reported in colon adenocarcinomas and esophageal carcinomas. MYBPC3 encodes the cardiac isoform of myosin-binding protein C. Mutations in MYBPC3 are one cause of familial hypertrophic cardiomyopathy (Aurensanz Clemente et al., 2017). MYBPC3 is one of the American College of Medical Genetics and Genomics genes. DUOX2 encodes a glycoprotein and a member of the NADPH oxidase family. DUOX2 mutations are the most powerful genetic predisposing factors for thyroid dyshormonogenesis (Chen et al., Rare variants in autism-related genes (as shown in Table 2) are involved in several pathways. 2018). EYS is mutated in autosomal recessive retinitis pigmentosa (Mucciolo et al., 2018). FLG2 encodes an intermediate filament-associated protein that functions in aggregation and the collapse of keratin intermediate filaments in mammalian epidermis. Mutations in this gene are associated with ichthyosis vulgaris and atopic dermatitis (Hassani et al., 2018).
In conclusion, we report on five ASD patients with rare variants and one patient with a de novo variant. However, this association study was performed with only a small number of cases; therefore, further studies with larger sample sizes are needed.