Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders

Autism spectrum disorders are a group of heterogeneous disorders with a strong genetic component but a complex genetic architecture. This complexity makes genetic diagnosis challenging, with a current diagnostic yield ranging from 15% to 30% [60,61]. Unbiased genome-wide molecular tools such as NGS, with a steadily lowering cost, have a proven efficacy, although they produce genetic and genomic information that cannot be properly interpreted yet. Here, we used WES and blood transcriptome by RNAseq in a selected group of males with idiopathic ASD to detect putative causal genetic variants of this complex disease. Segregation and recurrence analyses along with expression studies were used to better discriminate putatively pathogenic variants from innocuous rare variation.

WES identified several cases with likely monogenic forms of ASD, including four patients with de novo variants in strong candidate autosomal genes (11%) and two patients with inherited X-linked mutations (5.6%). Since our study did not include parental exome sequencing, our detection rate of monogenic cases may be underestimated. Autosomal LoF mutations were identified in SCN2A, MED13L, and KCNV1. SCN2A is one of the few genes found recurrently mutated in unrelated patients with ASD and intellectual disability, which is unlikely to occur by chance [10,36,37,62]. MED13L was previously associated with intellectual disability and heart defects, and a de novo splicing mutation was described in an autistic patient [13,63]. Recently, de novo deletions affecting coding regions of MED13L were found in two girls presenting a phenotype very similar to the patient we report here, including facial dysmorphism, hypotonia, and development delay, along with ASD [64]. Our findings are consistent with a role of MED13L in neurodevelopmental disorders. The third de novo and likely pathogenic variant affected KCNV1, coding for a potassium channel subunit mainly expressed in the brain and involved in the regulation of two other potassium channels (KCNB1 and KCNB2). Defects in voltage-gated potassium channels have been associated with a variety of neuropsychiatric disorders, including bipolar disorder, schizophrenia, and ASD [12,65-67]. Therefore, our data suggest a role for potassium voltage-gated channels in the etiology of ASD. Finally, we detected a de novo missense mutation in the CUL3, which is other of the few recurrently de novo mutated genes in ASD patients [11,39].

Regarding X-linked mutations, we identified alterations in two genes (MAOA and CDKL5) previously associated with ASD and intellectual disability. We found a splicing mutation in MAOA in a multiplex family with and X-linked pattern of inheritance, with two affected male siblings and a maternal history of psychiatric disease. MAOA encodes the protein monoamine oxidase A, which degrades amine neurotransmitters such as dopamine, norepinephrine, and serotonin [68]. Both affected siblings had consistent biochemical alterations of the catecholamine pathway, very mild in their carrier mother, then supporting the pathogenicity of the mutation. A MAOA truncating mutation was first described in a Dutch family in 1993, and recently, a second loss of function mutation was found in a family segregating ASD and behavioral problems [69,70]. Our work further strengthens the relation between MAOA and ASD. The other X-linked mutation affected a highly conserved amino acid in CDKL5 and is predicted to be deleterious by different algorithms [71,72]. It was inherited from the healthy mother and also detected in the unaffected sister, who preferentially inactivated the mutated allele. Mutations in CKDL5 were reported in X-linked infantile spasms syndrome (ISSX) [MIM 308350], atypical Rett syndrome (RTT) [MIM 312750], and Angelman syndrome-like [MIM 105830] [40-42,73,74]. Since the phenotype of our patient is less severe than the one described in males with CDKL5 mutations, it is possible that the missense variant found in this patient is a hypomorphic allele with a milder effect. The biased X inactivation documented in the sister’s proband could act as a protective factor in females explaining their unaffected status.

While the definition of the potential pathogenicity for ASD mutations could be relatively straightforward in de novo and Mendelian cases assuming full penetrance, a greater challenge is the classification of putatively pathogenic heterozygous mutations and rearrangements inherited from unaffected parents. These variants, presumably with incomplete penetrance and variable expression, are thought to contribute to disease risk in an oligogenic model with probable environmental contribution. One of the most common criteria to define potential pathogenicity is the recurrence of mutations in the same gene in unrelated patients. In our small cohort, we detected two additional patients with rare inherited missense mutations in genes with de novo LoF mutations in other cases (SCN2A and MED13L), suggesting that the inherited mutations could also be contributing to the disease. While amorphic alleles might be highly penetrant, hypomorphic missense mutations might have a milder effect just increasing the global burden of risk for ASD. Moreover, the study of common functional consequences of rare variation pointed towards a set of relevant pathways only overrepresented in ASD patients. Among these, there were the PI3K/Akt signaling and the axon guidance which were previously associated to ASD by linkage studies, rare CNVs, genomic mutations, and comorbid ASD conditions [45-48].

Using CNV detection tools on WES data, we identified small rearrangements that were missed by previous molecular karyotype in eight cases, all of them inherited from unaffected progenitors. Some of these rare CNVs could also contribute to the disease in a multiple-hit model, such as the duplications found in ASMT as previously proposed [50-52]. Although we did not observe a significant effect on expression in the individual analysis of these rare CNVs, a global analysis showed a tendency for higher expression of genes in duplication type CNVs compared to those in deletions, as expected and described for other ASD-related rearrangements [20,75].

The incorporation of the peripheral blood transcriptomic data provided an important additive value to the identification of molecular biomarkers of ASD, leading to identification of additional mutations and transcriptional consequences. Despite that blood is not the ideal target tissue to study ASD, it is commonly used in neurodevelopmental disorders since it can be easily available for diagnostic testing [76]. In our study, approximately 30% of the rare variants were sufficiently expressed in blood, and 88% of these had a concordant calling in both techniques. By isoform analysis, we found an aberrant transcript in PTEN, due to a de novo intronic mutation that activates a cryptic splice site. The patient (ASD_36) presents macrocephaly, a feature that is consistently found in ASD patients with mutations in this gene (MIM 605309). Thus, transcriptome sequencing of blood cells was essential to achieve a diagnosis in an additional patient, reaching a final diagnosis yield of 19%. Moreover, the integrative approach also enabled the identification of rare inherited variants with functional consequences that could contribute to the phenotype. As previously suggested, the joint study of genomic and transcriptomic data can be crucial to unravel the mechanism of complex diseases [77,78]. We detected alteration in expression levels in 1.7% of expressed rare variants, inherited in all cases. Overexpression of MECP2 was found in a patient who had a rare SNP variant in the same gene. Duplication of MECP2 causes a known duplication syndrome almost exclusively in males with moderate to severe intellectual disability. Overexpression of the gene in peripheral leucocytes was previously described in ASD patients [79] and was also related to aggressive social behavior in schizophrenia [80]. We also identified three rare mutations with concurring overexpression of candidate genes ANK3 [81,82], CREBBP [83,84], and SEMA6B [85] in a single patient. They were inherited from both progenitors and they could contribute to ASD in an additive manner. Additionally, we also detected monoallelic expression of the wild-type allele associated with ten rare inherited truncating mutations suggesting non-sense mediated decay (NMD) in which the functional haploinsufficiency could contribute to the phenotype such as the alteration in ALG9 [MIM 606941] and RIT1 [MIM 609591] previously involved in neuropshychiatric conditions [86-88]. Finally, allele-specific expression analysis revealed the alteration of 68 genes (or specific transcript variants) with monoallelic expression but no cis-element responsible for it. This phenomenon was found to be more common for autosomal and X-linked genes in ASD patients than in controls in the brain and other tissues [89,90]. Allele-specific expression can be caused by unidentified cis-acting elements, including genetic or genomic mutations in the promoter or regulatory regions and epigenetic marks. Thus, some of identified genes with allele-specific expression might contribute to ASD. In fact, two of them have been previously associated (MTOR) [91] and/or are great candidates (FUS and TAF1C) [92,93]. Ongoing efforts to define the extent of expression variation in large numbers of healthy controls such as Geuvadis and GTEx will help to better clarify the deregulated genes found in individual patients that are related to disease.