This study set out to identify the relationship of the genetics of syndromic and non-syndromic autism. The fact that only 6/339 probands (1.8%) carry a de novo
novel, coding non-synonymous variant or coding indel in the 21 genes examined is consistent with their clinical presentation, as the patients selected represented cases of idiopathic autism rather than syndromic autism (which would be the expected phenotype caused by loss-of-function mutations in most of the genes tested). While this suggests that the individual mutations causing syndromic versus non-syndromic autism may be separate from each other, the actual number of de novo
missense mutations in these genes is surprisingly high. It has been estimated that on average, a newborn carries 0.86 de novo
amino acid altering mutations (25
). Given this rate, our study of 21 genes in 339 probands should have revealed <1 (0.27) de novo
missense mutations among these genes. The fact that the actual number of de novo
mutations is much higher (22-fold increase for all tested genes) suggests that while severe loss-of-function mutations of given genes may cause syndromic autism, milder mutations of the same genes may be associated with non-syndromic autism. However, the comparison of de novo
mutation rates between our own cohort and the per generation estimate cited above is limited by the fact that they rely on different detection methods and statistical analyses.
The two de novo
mutations identified in TSC2
are clearly in genes known to cause syndromic autism. The other four de novo
variants were identified in HOXA1
, genes that are yet to be confirmed to be involved in autism or that show phenotypes on the autism spectrum. While a missense variant of HOXA1
was reported in association with autism (15
), most subsequent studies had failed to replicate an association of the gene to autistic phenotypes (26
). As part of this study, we identified a de novo
missense mutation of HOXA1
in one patient, and a small de novo
3 bp deletion in a polyhistidine tract of the protein in another. The latter was seen at relatively high frequency (36 of 339 probands and 10 of 376 controls) and likely represents a common variant.
Mutations in the forkhead-domain gene FOXP2
provided evidence that the gene is critical for human speech and language (14
), but the number of autistic patients identified with FOXP2
mutations has been very limited (32
). In this study, we identified two patients with de novo
mutations in FOXP2
. While one of the two adds an additional glutamine to a polyglutamine tract of the protein, which may represent a benign variant, the other represents a missense mutation (p.H603P) in a protein domain that is highly conserved throughout species. The two patients identified to carry de novo
mutations of FOXP2
were both diagnosed with AD. Testing of their communication skills by the communication domain of the Vineland Adaptive Behavioral Scale II (VABS-II) revealed low scores in both individuals (74 in individual 11 598, and 77 in individual 11 446), suggesting moderate to significant impairment of communicative skills in both probands. These findings strengthen the role of FOXP2
and its contributions to the ASDs.
As part of this study, 18 of 339 probands were found to be carriers of novel oligogenic heterozygous coding variants, even among the small number of genes analyzed. The occurrence of oligogenic heterozygous events is of particular interest, as it has been suggested before that autism could represent a complex genetic disorder that results from simultaneous genetic variations in multiple genes (4
). Following the same concept, a two-hit model for CNVs has been proposed for severe developmental delay (36
) and subsequently been discussed for epilepsy as well (37
). For autism, Pinto et al.
) reported the occasional combination of de novo
and inherited CNVs within a given family. While this study of 21 genes provides limited insight in the actual complexity of autism genetics, the data show significant increase in oliogogenic heterozygous combinations of novel coding variants in genes such as CACNA1C
among autistic probands compared with control individuals. Given the uncertain significance of the aforementioned 3 bp deletion in the polyhistidine tract of HOXA1, we re-analyzed our data set excluding this common variant. This would leave 14 oligogenic heterozygous events among 339 probands and 4 oligogenic heterozygous events among 376 controls, which is still highly significant by Fisher's exact test (P
Studying the parents and unaffected siblings for the presence of oligogenic events revealed that the vast majority of these combinations are unique to the proband. However, the fact that four parents and two siblings carried the same combinations of oligogenic heterozygosity reveals that at least some of these events on their own are not sufficient to cause autism. One might speculate that the accumulation of several, if not many of such hypomorphic mutations causes a genetic load, which will ultimately cross a given threshold and lead to clinical manifestation of the ASD in the respective individuals (Fig. ). Our study is limited by the small number of genes tested, and the full range of oligogenic heterozygous events contributing to the etiology of autism will only become evident once large scale, whole exome or whole genome data sets of sequences from autistic individuals are analyzed to evaluate for such combinatorial events. Also, while our study detected a significant difference in the incidence of oligogenic heterozygous variants between probands and controls for the aforementioned genes, it might be the case that controls have different heterozygous combinations with other genes that were not tested.
Figure 2. Proposed models of inheritance for ASDs. Left panel: syndromic autism is mostly caused by severe loss-of-function mutations of specific genes, with each gene causing a specific syndrome. Right panel: non-syndromic autism may be caused by milder mutations (more ...)
‘Synergistic heterozygosity’ has been described as a potential disease mechanism in some metabolic disorders, with the idea that concurrent partial defects in more than one pathway, or at multiple steps in one pathway may lead to disease, even though no complete deficiency in any one enzyme is present (39
). In the field of autism genetics, several hypomorphic variants may accumulate either in a specific signaling pathway, or a subcellular compartment (such as the synapse) to exceed a threshold and result in phenotypic manifestation. This would be consistent with the data from clinical studies whereby children from families in which both parents manifest sub-threshold autistic traits are more likely to show more severe impairment in reciprocal and social behavior (40
It is noteworthy that the average full-scale IQ of individuals with de novo
mutations in some of the 21 autism susceptibility genes was 71.6 (SD = 19.2), whereas the average full-scale IQ of those with oligogenic heterozygous events without de novo
mutations was 94.1 (SD = 22.2). While evidence is emerging that intellectual disabilities might be widely attributable to de novo
), cases of the high-functioning ASD may rather be attributable to co-inheritance of subtle, yet functionally significant variants in respective genes.
In summary, our data uncovered de novo mutations in 1.8% of the ASD patients we studied and suggest that oligogenic heterozygosity of coding non-synonymous variants and coding indels may constitute a novel pathogenic mechanism or risk for ASDs. The data from this study provide a framework upon which to expand investigations into oligogenic events in larger data sets. A model of oligogenic heterozygosity may offer at least a partial explanation for why traditional linkage analysis and mapping approaches have been rather unsuccessful in identifying genetic variants predisposing to ASDs. Whole exome sequencing analyzed in the context of genes involved in pathways critical for neuronal development and function is likely to be a productive approach to unravel oligogenic and combinatorial events that might increase an individual's risk for ASDs.