Genomic analysis of 116 autism families strengthens known risk genes and highlights promising candidates

Abstract

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a strong genetic component in which rare variants contribute significantly to risk. We performed whole genome and/or exome sequencing (WGS and WES) and SNP-array analysis to identify both rare sequence and copy number variants (SNVs and CNVs) in 435 individuals from 116 ASD families. We identified 37 rare potentially damaging de novo SNVs (pdSNVs) in the cases (n = 144). Interestingly, two of them (one stop-gain and one missense variant) occurred in the same gene, BRSK2. Moreover, the identification of 8 severe de novo pdSNVs in genes not previously implicated in ASD (AGPAT3, IRX5, MGAT5B, RAB8B, RAP1A, RASAL2, SLC9A1, YME1L1) highlighted promising candidates. Potentially damaging CNVs (pdCNVs) provided support to the involvement of inherited variants in PHF3, NEGR1, TIAM1 and HOMER1 in neurodevelopmental disorders (NDD), although mostly acting as susceptibility factors with incomplete penetrance. Interpretation of identified pdSNVs/pdCNVs according to the ACMG guidelines led to a molecular diagnosis in 19/144 cases, although this figure represents a lower limit and is expected to increase thanks to further clarification of the role of likely pathogenic variants in ASD/NDD candidate genes not yet established. In conclusion, our study highlights promising ASD candidate genes and contributes to characterize the allelic diversity, mode of inheritance and phenotypic impact of de novo and inherited risk variants in ASD/NDD genes.

Fig. 1. Rare de novo coding variants in cases and unaffected siblings.

a Rare coding de novo variants per individual in our cohort (ASD cases=144, unaffected siblings=55). b Distribution of rare de novo coding variants in cases and unaffected siblings: the pie charts represent rare de novo coding variants split by predicted functional consequences, represented by different colours. PTVs and missense variants are divided into two and three tiers of predicted functional severity, represented by different shade, based on the LOEUF (<0.6, ≥0.6) and MPC metrics (MPC ≥ 2 (DmisB), 1 ≤ MPC < 2 (DmisA), 0 ≤ MPC < 1), respectively.

Fig. 2. Enrichment for de novo and inherited pdSNVs in SynGO Genes.

Visualisation of gene set enrichment analyses (GSEA) of genes harbouring pdSNVs (left) and synonymous variants (right) in affected individuals, each compared to a background set of brain-expressed genes. All Cell Components (CC) or Biological Process (BP) related terms with gene annotations in SynGO are plotted in a circular fashion, with the highest hierarchical term (“synapse” for CC or “process in synapse” for BP) in the centre and each layer of subclasses in outward concentric rings. Over-represented synaptic terms are indicated with different colours, according to the Q-value, and are reported in detail in Supplementary Table 7. The CC and BP plots of genes affected by rare pdSNVs (left) show an enrichment of synaptic terms, while no enrichment emerged from the genes hosting rare synonymous SNVs (right).

Fig. 3. Contribution of de novo and inherited pdSNVs to high confidence ASD/NDD genes.

De novo and inherited pdSNVs include PTVs in genes with LOEUF score <0.6 (PTV_LOEUF), missense variants with MPC ≥ 2 (DmisB) and missense variants with 1 ≤ MPC < 2 (DmisA). Contribution of each variant type identified in ASD individuals and unaffected siblings for a list of genes previously associated to ASD (a) and NDD (b). The list of ASD genes comprised 185 genes associated at FDR ≤ 0.05 and 135 genes with FDR < 0.1 (88 of which were common between the two lists). In our cohort, pdSNVs were identified in 97 ASD genes (a). The list of NDD genes included 452 genes from a list of 664 genes associated at FDR ≤ 0.05, after the exclusion of the genes already included among the 232 ASD genes. In our cohort, pdSNVs were identified in 139 NDD genes (b). **, genes with FDR ≤ 0.001; *, genes with FDR ≤ 0.05; §, genes with FDR < 0.1; dotted line indicates a putative de novo PTV_LOEUF.

Fig. 4. BRSK2 exons, domains and reported variants.

Schematic representation of the BRSK2 gene structure (a) and protein domains (b), illustrating potentially damaging variants reported in this and previous studies. Protein domains include protein kinase domain (containing the active site, AS), ubiquitin-associated domain (UBA), proline-rich domain (Pro-Rich), and kinase-associated 1 (KA1) domain. Splice variants are shown above the schematic representation of the MANE transcript (upper panel), and protein-altering variants are shown below the schematic representation of BRSK2 (PTVs in red, missense variants in black). Confirmed de novo variants are highlighted in bold. The two variants identified in this study (p.(Ala158Thr), p.(Asp540GlufsTer9)) are underlined.