Case reports of two siblings with autism spectrum disorder and 15q13.3 deletions

Abstract Background Copy number variations (CNVs) have been implicated in psychiatric and neurodevelopmental disorders. Especially, 15q13.3 deletions are strongly associated with autism spectrum disorder (ASD), intellectual disability (ID), schizophrenia (SCZ), attention deficithyperactivity disorder (ADHD), and mood disorder. Case Presentation We present two siblings with ASD. They had a father with bipolar disorder (BD). Patient 1 is a 21‐year‐old female with ASD and mild ID, who had language delay and repetitive behavior in childhood, social difficulties, and refused to go to school because of bullying. She was hospitalized in a psychiatric hospital several times. Patient 2 is a 19‐year‐old male with ASD and ADHD. He did not have developmental delay, but had social difficulties and impulsiveness, then refused to go to school because of bullying. He was treated by a psychiatrist for anxiety and disrupted sleep rhythms. Array comparative genomic hybridization was performed for the siblings and parents. 15q13.3 deletions were detected in the siblings and their healthy mothers. No other pathogenic CNVs were detected. We performed whole‐genome sequencing of the family and identified 13 rare missense variants in brain‐expressed genes, which may be responsible for the phenotypic differences between the siblings and their mother. Conclusions This study shows incomplete penetrance and variable expressivity in 15q13.3 deletions. We detected second‐hit variants that may explain the phenotypic differences within this family. In addition, detecting 15q13.3 deletions may lead to early diagnosis and a better prognosis with careful follow‐up.


| Participants
All the members of this family were of Japanese ancestry. Patients were diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition criteria for ASD, ID, and ADHD.

| Genetic analysis
Genomic DNA was extracted from blood (Patient 1, Patient 2, and the mother) or saliva (the father) samples. aCGH was performed using NimbleGen 720 k Whole-Genome Tiling Arrays (Roche NimbleGen).
We generated CNV calls with Nexus Copy Number software, v9.0 (BioDiscovery). 1 Previously, we confirmed that CNV calls from this array are highly accurate, with a validation rate of >99%. We also confirmed the CNV calls by whole-genome sequencing (WGS) which explained next (Table S1).
To identify the genetic variants responsible for the phenotypic differences between the siblings and their mother, we performed WGS on this family using Complete Genomics technology. This sequencing was based on a nanoarray-based short-read sequencing-by-ligation technology. 13 Ingenuity Variant Analysis bioinformatic software was used to detect the functional variants. Variants were kept only if they met the call confidence criteria of call quality ≥180, with read depth ≥ 10, and occurred outside the top 1% most exonic variable 100 base windows in healthy public genomes. Of these variants, we excluded those with a minor allele frequency ≥1% of the genomes in the Tohoku Medical Megabank Organization (ToMMo). We searched for paternal variants that the two siblings had in common. Finally, variants were only kept for the following analysis if they were nonsynonymous variants (i.e., missense variants, nonsense variants, frameshift variants, in-frame indels). The pathogenicity of missense variants was assessed by PolyPhen-2. 14 All genomic locations are given in hg19 coordinates. We used the Human Brain Transcriptome database (https://hbatl as.org/pages/ hbtd) to evaluate brain-expressed genes.

| Phenotypic analysis
We retrospectively collected clinical data of the two siblings with 15q13.3 deletions from their medical records. The data included developmental history, family history, medical history, psychiatric symptoms, history of hospitalizations, and medications. Based on the data, the severity of symptoms was graded as one of four levels by a board-certified research psychiatrist: -(none), + (mildly present), ++ (moderately present), and +++ (strongly present). We performed aCGH for the siblings and their parents; 15q13.3 deletions were detected in both siblings and their healthy mother. As neither of the two patients had other pathogenic CNVs, we hypothesized that rare (<1%) second-hit variants (single nucleotide variants and insertions/deletions) may explain the phenotypic differences.

| C A S E PR E S E NTATI O N
We performed WGS of the siblings and their parents to identify potential second-hit variants from rare variants inherited from their father with BD. Seventeen missense variants were detected in both affected siblings, all of which were predicted to be pathogenic by PolyPhen-2 and inherited from their father. Among these variants, 13 were located in brain-expressed genes (e.g., KCND3, SEC24B, and TSPYL4; Table S2). Maternally inherited variants and variants that the siblings did not share are shown in Tables S3 and S4.

| DISCUSS ION
15q13.3 deletions have incomplete penetrance in neuropsychiatric diagnosis (80.5%), 12 including ASD (~35%). 15 Consistent with this, 15q13.3 deletion in Patients 1 and 2 was inherited from their healthy mother ( Figure 1A). In addition, the variable expressivity of 15q13.3 deletions is reported. 3 This family showed variable expressivity; Patient 1 had ASD, ID and Patient 2 had ASD, ADHD ( Figure 1A). To Although their CNV was the same, the phenotype of these patients was different at some points; only patient 1 had ID, and only patient 2 had ADHD. The severity of each psychiatric symptom was graded with 3 levels by a board-certified research psychiatrist, based on the ADI-R results: + (mildly present), ++ (moderately present), and +++ (strongly present). ADHD, attention deficit hyperactivity disorder; ADHD; DSM-5, Diagnostic and Statistical Manual of Mental Disorders 5th Edition; ASD, autism spectrum disorder; BD, bipolar disorder; FSIQ, Full Scale IQ; ID, intellectual disability; WAIS-III, Wechsler Adult Intelligence Scale-III.