Biochemistry and Cell Biology Copy number variation in fetal alcohol spectrum disorder Journal: Biochemistry and Cell Biology Manuscript ID bcb-2017-0241.R1 Manuscript Type: Article Date Submitted by the Author: 09-Nov-2017 Complete List of Authors: Zarrei, Mehdi; The Centre for Applied Genomics Hicks, Geoffrey G.; University of Manitoba College of Medicine, Regenerative Medicine Reynolds, James N.; Queen's University School of Medicine, Biomedical and Molecular SciencesDraft Thiruvahindrapuram, Bhooma; The Centre for Applied Genomics Engchuan, Worrawat; Hospital for Sick Children SickKids Learning Institute Pind, Molly; University of Manitoba College of Medicine, Regenerative Medicine Lamoureux, Sylvia; The Centre for Applied Genomics Wei, John; The Centre for Applied Genomics Wang, Zhouzhi; The Centre for Applied Genomics Marshall, Christian R.; The Centre for Applied Genomics Wintle, Richard; The Centre for Applied Genomics Chudley, Albert; University of Manitoba Scherer, Stephen W.; The Centre for Applied Genomics Is the invited manuscript for consideration in a Special Fetal Alcohol Spectrum Disorder Issue? : Keyword: Fetal alcohol spectrum disorder, FASD, copy number variations, CNV https://mc06.manuscriptcentral.com/bcb-pubs Page 1 of 354 Biochemistry and Cell Biology 1 Copy number variation in fetal alcohol spectrum disorder 2 Mehdi Zarrei,a Geoffrey G. Hicks,b James N. Reynolds,c,d Bhooma Thiruvahindrapuram,a 3 Worrawat Engchuan,a Molly Pind,b Sylvia Lamoureux,a John Wei,a Zhouzhi Wang,a Christian R. 4 Marshall,a Richard F. Wintle,a Albert E. Chudleye,f and Stephen W. Scherer,a,g 5 aThe Centre for Applied Genomics and Program in Genetics and Genome Biology, The Hospital 6 for Sick Children, Toronto, Ontario, Canada 7 bRegenerative Medicine Program, University of Manitoba, Winnipeg, Canada 8 cCentre for Neuroscience Studies, Queen's University, Kingston, Ontario, Canada. 9 dDepartment of Biomedical and MolecularDraft Sciences, Queen's University, Kingston, Ontario, 10 Canada 11 eDepartment of Pediatrics and Child Health, Max Rady College of Medicine, Rady Faculty of 12 Health Sciences, University of Manitoba, Winnipeg, Canada. 13 fDepartment of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady 14 Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada 15 gDepartment of Molecular Genetics and McLaughlin Centre, University of Toronto, Toronto, 16 Ontario, Canada 17 Corresponding authors: Stephen W. Scherer ([email protected]) and Albert E. 18 Chudley ([email protected]) 19 1 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 2 of 354 20 Abstract: Fetal alcohol spectrum disorder (FASD) is characterized by a combination of 21 neurological, developmental, and congenital defects that may occur as a consequence of prenatal 22 alcohol exposure. Earlier reports showed that large chromosomal anomalies may link to FASD. 23 Here, we examined the prevalence and types of copy number variations (CNVs) in FASD cases 24 previously diagnosed by a multidisciplinary FASD team in sites across Canada. We genotyped 25 95 children with FASD and 87 age-matched, typically developing controls on the Illumina 26 Human Omni2.5 SNP array platform. We compared their CNVs to those of 10,851 population 27 controls, in order to identify rare CNVs (<0.1% frequency), which may include large unbalanced 28 chromosomal abnormalities, that might be relevant to FASD. In 12/95 (13%) of the FASD cases, 29 rare CNVs were found that impact potentially clinically-relevant developmental genes, including 30 the CACNA1H involved in epilepsy andDraft autism, the 3q29 deletion disorder, and others. Our 31 results show that a subset of children diagnosed with FASD have chromosomal deletions and 32 duplications that may co-occur or explain the neurodevelopmental impairments in a diagnosed 33 cohort of FASD individuals. Children suspected to have FASD with or without sentinel facial 34 features of fetal alcohol syndrome and neurodevelopmental delays should potentially be 35 evaluated by a clinical geneticist and possibly have genetic investigations as appropriate to 36 exclude other etiologies. 37 Key words: Fetal alcohol spectrum disorder, FASD, copy number variations, CNV. 38 Résumé: [will be translated into French by the journal translator] 39 Mots-clés: Fetal alcohol spectrum disorder, FASD, copy number variations, CNV. 2 https://mc06.manuscriptcentral.com/bcb-pubs Page 3 of 354 Biochemistry and Cell Biology 40 Introduction 41 Fetal alcohol spectrum disorder (FASD) is an umbrella term for a group of alcohol-related 42 disorders, characterizing the full range of damage from prenatal alcohol exposure (Chudley et al. 43 2005). It comprises fetal alcohol syndrome (FAS), partial FAS, alcohol-related 44 neurodevelopmental disorders, and alcohol-related birth defects, which are congenital anomalies 45 that can include growth deficits, facial dysmorphism, and/or malformations in multiple 46 physiological systems (Abdelmalik et al. 2013; Chudley et al. 2005; Douzgou et al. 2012; 47 Popova et al. 2016). FASD is also associated with numerous co-morbid neurodevelopmental 48 disorders, including intellectual disabilities, microcephaly, autism spectrum disorder (ASD), and 49 attention deficit hyperactivity disorder (Abdelmalik et al. 2013; Douzgou et al. 2012; Weyrauch 50 et al. 2017). Draft 51 The reported incidence of FASD is 0.2 to 3.0 per 1,000 live births for most countries (Douzgou 52 et al. 2012). However, an incidence of up to 89.2 per 1,000 births was reported in regions of 53 South Africa where wine production is abundant (May et al. 2007). A recent review of the 54 global prevalence of FASD in children and youth reported a prevalence of 7.7 per 1000 55 population (95% CI, 4.9-11.7 per 1000 population). The highest rate was reported by the World 56 Health Organization European region with 19.8 per 1000 population, (95% CI, 14.1-28 per 1000 57 population)(Lange et al. 2017). 58 It is well established in the medical community that not all children exposed to alcohol 59 prenatally develop FASD (Abel 1995; Stoler 1999). This indicates that factors other than 60 prenatal alcohol exposure also contribute to FASD aetiology. The impact of the genetic 61 variations to the aetiology of FASD has been studied in animal models (reviewed in (Eberhart 62 and Parnell 2016; McCarthy and Eberhart 2014)). However, genetic studies on human are 3 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 4 of 354 63 restricted to case reports with large chromosomal anomalies (Stoler 1999) and twin studies 64 (Chasnoff 1985). For example, association of trisomy 21 or ring chromosome 6 with FASD has 65 been reported (Bingol et al. 1987; Muller et al. 1993; Romke et al. 1987). Large chromosomal 66 anomalies such as 22q11.2 microdeletion and microduplication, and 1q21 microduplications, 67 among others, were reported in 9% (7/90) of cases with FASD in a UK cohort (Douzgou et al. 68 2012). A retrospective study of younger and older siblings of patients with FAS clearly indicated 69 a greatly increased risk of FAS in younger siblings if an older sibling had been diagnosed with 70 FAS (Abel 1988). The contribution of CNVs to the aetiology of neurodevelopmental disorders 71 (Gazzellone et al. 2016; Lionel et al. 2011; Oskoui et al. 2015; Pinto et al. 2014; Pinto et al. 72 2010; Zarrei et al. 2017a), including those that may be comorbid with FASD (Popova et al. 73 2016), has been well documented. However,Draft until the current study, a genome-wide CNV 74 analysis in FASD cases had not been performed. 75 Our objective was to assess the role of CNVs and chromosomal abnormalities, at a genome- 76 wide level, in a clinically well-defined collection of individuals with FASD, to further define 77 genomic risk contribution in this common disorder. 78 Materials and methods 79 Participants 80 Ninety-five children diagnosed with FASD and eighty-seven aged-matched, typically 81 developing controls as internal control were recruited through the NeuroDevNet Canadian FASD 82 study cohort (Portales-Casamar et al. 2016; Reynolds et al. 2011) and the majority of clinical 83 centers used the Canadian FASD Guidelines for diagnosis (Chudley et al. 2005). Siblings of 84 some probands, if consented, were also recruited for this study (Table S1). Our FASD cases have 4 https://mc06.manuscriptcentral.com/bcb-pubs Page 5 of 354 Biochemistry and Cell Biology 85 a sex ratio of 0.73 (male: 40; female: 55; Table S1; 0.85 in internal controls). Research Ethics 86 Board approval was obtained from each participating centre, and all caregivers and participants 87 provided written informed consent. Saliva samples were collected from the children. Details of 88 saliva collection and storage is provided elsewhere (Portales-Casamar et al. 2016). 89 Genotyping, CNV calling, and detection of rare variants 90 DNA extracted from saliva using standard protocols was genotyped on two Illumina Human 91 Omni2.5 SNP array platforms (Omni2.5m-Quad; n=132 and Omni2.5m-8; n=50) at The Centre 92 for Applied Genomics in Toronto according to the manufacturer’s protocol. Relevant microarray 93 data are deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/; 94 GSE102650). Quality control and ancestryDraft assessment procedures were as described previously 95 (Gazzellone et al. 2016; Oskoui et al. 2015). Ancestry was determined using PLINK v1.07 96 (Purcell et al. 2007). 97 CNV calling was performed as previously described with slight modifications (Oskoui et al. 98 2015). In brief, three CNV calling algorithms were used for both cases and internal controls: i.e. 99 iPattern (Pinto et al. 2011), PennCNV (Wang et al. 2007), and QuantiSNP (Colella et al. 2007). 100 We then analyzed “stringent” CNVs, those detected by at least two methods. Detecting large 101 CNVs particularly sex chromosomal aneuploidies is challenging (Oskoui et al. 2015). Hence, 102 these large CNVs are sometimes fragmented. If found, they were merged and their identity 103 confirmed by examining the probe density and B allele frequencies in the region.