Our study reports the systematic resequencing of the coding regions of 111 X-linked candidate genes in a cohort of 285 ASD and SCZ patients, aimed at the identification of genes potentially involved in these disorders. Using various methods, we prioritized these genes from a larger group of 1125 X-linked genes and ~5000 potentially synaptic genes. For the X chromosome, only 28 genes were shared between the gene list from SynDB and that derived from proteomic studies. This non-concordance may be explained by three major reasons: (1) some synaptic proteins, such as membrane receptors, are difficult to purify and could be missed by proteomic studies, (2) the presence of a higher number of false positives in SynDB because of inclusion of genes based on structural predictions, and (3) a fraction of the proteins found through proteomic studies may also represent false positives, as they could be due to contamination of the synaptic fraction during the preparation. These results convinced us to use the complementarity of these two types of sources. Altogether, the gene lists extracted from SynDB and from proteomic studies comprised 65% of the genes identified through our manual search of PubMed. The combination of these three resources resulted in a comprehensive list of X chromosome potentially synaptic genes. This list also provides good candidate genes for the screening of other psychiatric and neurological disorders, such as bipolar disorder or Tourette’s syndrome.
Here, we identified several hundred variants, the majority of which had not been described in any public database. Tarpey et al.
who recently published a list of X-chromosome variants found in patients with MR, identified 134 of the variants reported here, which corresponded mainly to the polymorphism category. However, their cohort is not a pertinent control for our study, as MR and ASD are genetically related and are often clinically comorbid. We screened three times less nucleotides (75 954 657 bp vs ~200 000 000 bp) than Tarpey et al.
and found around three times less variants in coding regions (533 vs 1858). However, we identified significantly less truncating variants (6 vs 40). This result is not surprising, as their cohort included only families with X-linked MR. Consistent with their results, we observed that truncating variants in two genes (P2RY4
) seemed to be well tolerated in males and to not lead to any obvious phenotype. Contrary to the variant in P2RY4
, which removes only the C-terminal end of the protein, the variant in HS6ST2
removes almost all the protein. Possible functional redundancy with other heparan sulfate 6-O-sulfotransferase subtypes, such as HS6ST1 and HS6ST3, may explain the fact that HS6ST2 truncation is tolerated in male individuals.
Despite the probable function of rare variants with a recent origin in ASD and SCZ, we identified and confirmed only two de novo
damaging variants, which were found in IL1RAPL1
in two ASD girls. However, because of the particularity of X-linked transmission, we are aware that X-linked genes are not ideal to test the de novo
hypothesis. One nonsense and several missense variants were identified in the MAOB
gene in SCZ patients. MAOB catalyzes the oxidative deamination of xenobiotic and biogenic amines, such as the neurotransmitter dopamine or the neuromodulator phenylethylamine.75
Phenylethylamine is involved in the modulation of mood and, as it is structurally close to amphetamine, it may cause, when expressed at high levels, a similar type of psychosis.76
Moreover, low MAOB activity and elevated phenylethylamine in urine were described earlier in SCZ patients.77
Several studies failed to identify any association between polymorphisms in MAOB and SCZ; however, one positive association was recently found in the Spanish population.78
Nonetheless, one would not expect positive associations if disease-predisposing mutations are different rare mutations. Although no evident neurological phenotype was found in two boys who carry a deletion of a fragment of MAOB
and of the Norrie disease gene,79
knockout mice present elevated phenylethylamine in urine and an increased reactivity to stress.80
Regarding the possible implication of MAOB
in SCZ, the identification of one nonsense mutation in this gene in one SCZaff patient is potentially relevant, even if the cosegregation was not perfect. Further studies are needed to determine the effect of the three missense variants found in the four additional families on MAOB function.
The NS rare variants identified in this study were predicted to be more damaging than the NS polymorphisms, which suggests that they may include disease-causing mutations responsible for ASD or SCZ. The genetic follow-up on these variants is challenging, as most of our patients are sporadic cases. Moreover, because of the genetic complexity of ASD and SCZ, we do not expect to find a perfect segregation of variants with the disease, which hampers the interpretation of the results. Several genes in which we identified potentially damaging variants have a function in structural modulation of the synapse. The involvement of the IL1RAPL1, OPHN1, and MCF2 proteins in neurite outgrowth is well documented66,70,81
and evidence suggests that SLITRK2 and TM4SF2/TSPAN7 may also be implicated in this process.56,67
We also highlighted variants in genes involved in serotonin function (HTR2C
) and degradation (MAOB
) in SCZ patients.
Our gene scoring system aimed at selecting the best candidate genes seems to have performed well. MAOB was selected with a score equal to 6, which was the maximum score. Several of the genes in which we found potentially relevant variants in ASD (for example IL1RAPL1
, or MECP2
) were selected because of their involvement in NS-MR. Interestingly, most of our ASD individuals who carried mutations in these genes did not have MR. These results support a genetic link between MR and ASD, suggesting that the same genes and mutations may predispose to these two diseases, as was shown earlier for NLGN4.12
Therefore, the screening of genes involved in MR in ASD patients is warranted, even if the patients do not have MR. The study of factors (environmental or genetic) that modulate these phenotypes will be critical to understand the molecular pathways that underlie MR, ASD, or both.
The identification of two damaging variants in OPHN1
in one male with ASD and in another with COS suggests a genetic link between these two diseases. The COS patient was also diagnosed with pervasive developmental disorder as one-third of the individuals with COS. The hypothesis of the existence of common genes between ASD and SCZ is emerging82
and has been shown by the discovery that copy number variants of the NRXN1
synaptic gene are associated with these two diseases.83
Similarly, the SCZ-associated DISC-1
gene was also found to be involved in ASD.84
Interestingly, in concordance with our results, a recent study showed that several copy number variants are common between MR, ASD, and SCZ, supporting the existence of shared biologic pathways in these neurodevelopmental disorders.85,86
In conclusion, our results indicate that large-scale direct resequencing of synaptic candidate genes constitutes a promising approach to dissect the genetic heterogeneity of SCZ and ASD and to explore the hypothesis that a number of distinct individually rare penetrant variants are involved in the pathogenesis of these two syndromes. Indeed, the identification of an excess of potentially damaging rare variants in ASD and SCZ patients validated the usefulness of this approach. However, with the exception of mutations in IL1RAPL1
and maybe TM4SF2/TSPAN7
in ASD, it is difficult to make a definitive claim that the damaging variants identified here are disease causing. The truncating mutation in MAO
B, as well as other missense variants in different genes are promising, but further work involving functional testing will be needed to confirm the implication of these rare variants in SCZ and ASD. If the hypothesis of rare variants with classical Mendelian inheritance does not seem to entirely explain the complexity of the genetic factors involved in ASD, we succeeded, at least, in the identification of causative mutations in some ASD patients. However, the story seems to be more complicated for SCZ. The excess of patients accumulating several NS rare variants in the SCZ cohort in comparison with the ASD cohort and the lack of identification of a clear segregating damaging mutation in SCZ in our study suggest that SCZ may involve more an interaction between rare variants with moderate effects in different synaptic genes rather than addition of Mendelian inheritances. Interestingly, a recent genome- wide association study on SCZ reported that common polygenic variants could also contribute to the risk of SCZ.7
These common variants may work in concert with rare variants to manifest SCZ. However, we are limited in this conclusion because we analyzed only variants on the X chromosome. A similar approach targeting autosomal genes is needed to examine whether autosomal rare variants provide a similar picture to that of our X-chromosome study. More generally, for these diseases, the ‘rare variant with penetrant effect’ vs the ‘common variant with low effect’ hypotheses should not be viewed as exclusive hypotheses, but more as a continuum including also variants with rare frequency and having moderate effect. That is why direct resequencing of candidate genes, as well as copy number variants or genome-wide association study analyses, could be viewed as complementary approaches to dissect the genetic susceptibilities to SCZ and ASD.