Construction of copy number variation landscape and characterization of associated genes in a Bangladeshi cohort of neurodevelopmental disorders

Abstract

Introduction: Copy number variations (CNVs) play a critical role in the pathogenesis of neurodevelopmental disorders (NDD) among children. In this study, we aim to identify clinically relevant CNVs, genes and their phenotypic characteristics in an ethnically underrepresented homogenous population of Bangladesh. Methods: We have conducted chromosomal microarray analysis (CMA) for 212 NDD patients with male to female ratio of 2.2:1.0 to identify rare CNVs. To identify candidate genes within the rare CNVs, gene constraint metrics [i.e., "Critical-Exon Genes (CEGs)"] were applied to the population data. Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) was followed in a subset of 95 NDD patients to assess the severity of autism and all statistical tests were performed using the R package. Results: Of all the samples assayed, 12.26% (26/212) and 57.08% (121/212) patients carried pathogenic and variant of uncertain significance (VOUS) CNVs, respectively. While 2.83% (6/212) patients' pathogenic CNVs were found to be located in the subtelomeric regions. Further burden test identified females are significant carriers of pathogenic CNVs compared to males (OR = 4.2; p = 0.0007). We have observed an increased number of Loss of heterozygosity (LOH) within cases with 23.85% (26/109) consanguineous parents. Our analyses on imprinting genes show, 36 LOH variants disrupting 69 unique imprinted genes and classified these variants as VOUS. ADOS-2 subset shows severe social communication deficit (p = 0.014) and overall ASD symptoms severity (p = 0.026) among the patients carrying duplication CNV compared to the CNV negative group. Candidate gene analysis identified 153 unique CEGs in pathogenic CNVs and 31 in VOUS. Of the unique genes, 18 genes were found to be in smaller (<1 MB) focal CNVs in our NDD cohort and we identified PSMC3 gene as a strong candidate gene for Autism Spectrum Disorder (ASD). Moreover, we hypothesized that KMT2B gene duplication might be associated with intellectual disability. Conclusion: Our results show the utility of CMA for precise genetic diagnosis and its integration into the diagnosis, therapy and management of NDD patients.

Keywords: Autism Diagnostic Observation Schedule-Second Edition (ADOS-2); autism spectrum disorder (ASD); chromosomal microarray analysis (CMA); copy number variation (CNV); critical exon gene (CEG); neurodevelopmental disorders (NDDs); variant of uncertain significance (VOUS).

FIGURE 1

Summary of CNVs identified in 212 NDD patients. (A) Percentage of patients carrying pathogenic, VOUS, and benign CNVs. Of all samples assayed, 12.26% patients carried pathogenic CNVs, where 7.07% carried a pathogenic deletion and 5.19% a pathogenic duplication. (B) Percentage of pathogenic, VOUS, and benign CNVs. Of the total 1,581 CNVs, 1.7% are pathogenic, 13.54% are VOUS, and the remaining 84.76% are benign. (C) Bars indicate the total number of CNVs in deletion and duplication. The green line represents the number of unique genes impacted by the corresponding variants. (D) Percentage of male and female groups in the full cohort impacted by pathogenic CNVs. p-value was calculated by Fisher’s exact test. (E) and (F) size distribution of pathogenic and VOUS CNVs. The average length of pathogenic deletion and duplication is 5365.26 kb and 17,451.72 kb, respectively, whereas the average length of VOUS deletion and duplication is 129.17 kb and 476.76 kb, respectively.

FIGURE 2

Circos (Krzywinski et al., 2009) plot illustrating neurodevelopmental disorder map showing the frequency of rare and common CNVs. (A) Frequency distribution of all rare CNVs throughout chromosomes 1 to 22. Dots in the red, green, and blue circles indicate frequency of >30 kb deletion, >50 kb duplication, and LOH CNVs, respectively. Within a colored circle, the outermost sub-circle contains the maximum value of 0.01, and the inner sub-circles contain values less than 0.01 but more than 0.001. (B) Frequency distribution of all common CNVs throughout chromosomes 1 to 22. Dots in the green, purple, and blue circles indicate frequency of >30 kb deletion, >50 kb duplication, and LOH CNVs, respectively. Each colored circle contains frequency values more than 0.01.

FIGURE 3

KEGG and Gene ontology pathway enrichment analysis of pathogenic CNVs identified in the study cohort. (A) Impacted genes within the CNV breakpoints of all pathogenic deletions identified “GO:0019787,” “GO:0060548,” and “GO:0099003” pathways to be highly significant. (B) Impacted genes within the CNV breakpoints of all pathogenic duplications identified “GO:0044419,” “GO:0030163,” “GO:1901214,” and “GO:0099536” pathways to be highly significant. The false discovery rate (FDR) and p-value cut-off were 0.01 and 0.001, respectively. P-values are denoted using a color gradient (low p-values with darker colors).

FIGURE 4

Summary of variants in 18 CEGs from various databases. (A) and (B) List of variants present in 18 CEGs from DECIPHER (Bragin et al., 2014) and ClinVar (Landrum et al., 2016) databases, respectively. (C) Summary of 529 rare SNVs in the PSMC3 gene from the gnomAD (Karczewski and Francioli, 2017) database. (D) Schematic representation of the overlapping CNVs in our patients (#119 and #121) and previously reported cases. A close view of chromosome band 11p11.2 is displayed on the top. The comparison of the deleted regions in our patients with the previously reported CNVs identified a minimum overlapping critical region of 7.7 kb (chr11:47,418,769-47,426,473) disrupting PSMC3, in common. Red and blue rectangles symbolize PSMC3-involving gross deletion and duplication, respectively, and the orange single bar symbolizes PSMC3-involving SNP. CEG, critical exon gene; SNV, short-nucleotide variation; SNP, single-nucleotide polymorphism; gnomAD = The Genome Aggregation Database.

FIGURE 5

Schematic representation of the overlapping CNVs in our patient (#106) and previously reported cases. A close view of chromosome band 19q12-q13.2 is displayed on the top. The comparison of the duplicated region in our patients with the previously reported CNVs identified a minimum overlapping critical region of 38.4 kb (chr19:35,700,296-35,738,700), disrupting two genes, ZBTB32 and KMT2B, in common. Red and blue rectangles symbolize KMT2B-involving gross deletion and duplication, respectively.