Contribution of Multiple Inherited and Shared Rare Variants to Autism Spectrum Disorder (ASD) in a Family with 3 Affected Siblings

Background: Familial rare mutations can be found in 3-5% of ASDs. However, how they contribute to ASD remains largely unknown. As part of the iTARGET Autism project (http://www.itargetautism.ca/) and the MSSNG project (https://www.mss.ng/), we have recruited multiple families with ≥3 affected children to address this question.

Objectives: To analyze the genotype-phenotype correlation of inherited rare mutations shared by affected children in a multiplex ASD family.

Methods: We used whole genome sequencing (WGS) and high resolution DNA microarray to detect inherited rare mutations (SNVs, Indels, and CNVs) in a multiplex family with 3 affected children.

Results:

(1). Clinical investigations: This family has 3 boys all affected with ASD. Boy-1 (14-54A, 15 y/o) has ADHD, anemia, decreased pain sense, and no outwardly syndromic features. Boy-2 (14-53A, 14 y/o) has slightly coarse facies, showing the most obvious cognitive deficits and greater severity of ASD, and no anemia (nor a thalassemia). Boy-3 (‘14-55A, 8 y/o) has anemia since birth, astigmatism and no outwardly syndromic features. Family history: Dad is an α-thalassemia carrier; Dad’s nephew has PDD-NOS. Mom is a β-thalassemia carrier. Mom’s father is anemic with Asperger’s syndrome. Mom's paternal aunt and uncle both exhibit symptoms of OCD and anxiety disorder (unconfirmed).

(2). CNVs findings: An 18Kb deletion involving HBA1, HBA2, HBQ1 and HBM (alpha hemoglobin locus) was identified in Boy-1 by DNA microarray. WGS confirmed this deletion was from the father. Array results for Boy-2 and Boy-3 are normal.

(3). SNVs/Indels findings: Two inherited, likely pathogenic rare variants were found in all 3 affected boys: compound heterozygous variants in RELN (p.Val1153Ile and p.Ser630Arg) and a maternal missense variant in SHANK2 (p.Pro1184Ser). They are each strong ASD risk genes with both de novo and inherited mutations frequently found in affected or unaffected parents, suggesting other genetic or epistatic factors may co-act to cause ASD. Further to this hypothesis, additional gene variants are shared by all 3 boys, including compound heterozygous variants in SCN10A, maternal variants in DLG1, and paternal variants in KMT2C. Although these individual variants are of uncertain clinical significance, their co-occurrence in all 3 boys with ASD add to the mutation burden potentially predisposing to the ASD in children of parents with positive family histories. We also found a maternal variant in an ASD candidate gene, ASH1L (p.Gln433Pro), unique to Boy-2. Whether this missense mutation may contribute to the more severe ASD phenotype in this boy needs further investigation. A maternal pathogenic LOF mutation in HBB (c.126_129delCTTT, p.Phe42Leufs) was identified in Boy-3 as his mother is a β -thalassemia carrier. A HBA1/HBA2-involved deletion was found in Boy-1 and his father (an α -thalassemia carrier). Both Boy-1 and Boy-3 have anemia, while Boy-2, without anemia, does not have either of these two mutations.

Conclusions: Multiple inherited rare mutations, each with subthreshold or subtle clinical impact, may collectively contribute an additive effect or provide a 2^nd-hit, or multiple-hits to render pathogenicity in ASDs.