Using approaches that specifically analyze both the function and accumulation of rare variants in disease cohorts, we have found a number of candidate genes associated with ASD and schizophrenia in two cohorts with sample sizes substantially smaller than those required for GWA studies. Population genetic approaches are designed to deal with data where sample sizes are limited compared to the sizes required in GWA studies. In both the ASD and schizophrenia cohorts, GRIN2B
contain a statistically significant excess of rare missense variants, while the excess of rare missense variants in CACNAF1
was restricted to the ASD cohort. The involvement of a particular gene (e.g. GRIN2B
) in the etiology of both disorders may reflect a critical role for neurodevelopment processes and suggests a pleiotropic effect 
. Population genetic models incorporating demographic and selection processes, implemented in prfreq
, corroborated this result for MAP1A
, and identified three new candidate genes: GRIN3B
Nominal P Values of Identified Candidate Genes Exhibiting Excesses of Rare Variants.
Most of our mapped genes (, Table S5
) have been previously implicated in neurological disorders or in neurodevelopment. MAP1A
, a member of the microtubule-associated MAP1 proteins family, is predominantly expressed in adult neurons and is involved in axon and dendrite development. Among other interactions, MAP1A
participates in the linking of DISC1 to microtubules 
. DISC1 is a protein that was described as related to the pathogenesis of schizophrenia by linkage analysis of a Scottish family 
and confirmed among Finnish cohorts 
. The association between schizophrenia and eleven genes interacting with DISC1 (including MAP1A
) was explored in Finish families, with significant results in three of the candidates but not for MAP1A 
. Among calcium channel classes of genes, such as CACNA1F
, we found a significant excess of rare variants and two segregating inframe indels at CDS position 807 falling in a glutamic acid-rich coiled domain. CACNA1F
has been previously associated to schizophrenia 
and mutations have also been described for other neurological disorders 
. Finally, two independent meta-analyses corroborate our findings for a role of GRIN2B
in the etiology of schizophrenia 
codes a subunit of the glutamate and N-methyl-D-aspartate (NMDA) receptor. There exist several NMDA receptors that are constructed with one or more isoforms of the NR1 subunit (GRIN1
) in different combinations with NR2 (GRIN2A
genes) and NR3 subunits (GRIN3A
. Some studies have suggested a relationship of decreased expression and abnormalities in NMDA receptors with schizophrenia 
. Additionally, we observed key results for other NMDA related genes. GRIN3B
had an excess of detrimental variants, two different analyses revealed significantly higher mapp scores in GRIN3A
in the ASD cohort, while we observed a nonsense mutation in both GRIN2C
in our cohorts. In the case of GRIN2B
, we also observed a coding indel which results in an amino acid insertion at cds position 1,353. This collection of rare and functional changes in the GRIN
gene family points to an important role of NMDA receptors in these neurological disorders.
For some cases in our study, different methods identified different loci associated with ASD and Schizophrenia. Observing signficant disease associations for the same genes using all the methods employed would be ideal, however it is not expected as each statistical test evaluates a different component of variation within the data. Furthermore, the ability to use each test varies by chromosome (X vs. autosomal) and cohort. The Li and Leal collapsing method 
evaluates the accumulation of rare missense variants in genes in cases relative to controls, and is independent of silent variants. The ratio of missense to silent mutations is cohort specific and is used to identify genes that have an excess of missense mutations conditional on the number of silent mutations, relative to the cohort-wide average. As an example, a gene can have a minimum of two rare missense variants and generate a significant result with the collapsing method, but might not have sufficient power to test the ratio of rare missense to silent variants.
In contrast, we do expect genes with high missense to silent variant ratios, to have negative γmkprf estimates, and to contribute to more negative γprfreq estimates overall. We observed these patterns for the GRIN2B gene () in both diseases which was supported by a significant collapsing method test result. Like GRIN2B, CACNA1F also shows concordance among methods within the ASD cohort; including a nominally significant missense to silent variant ratio, a significant collapsing method result, and a negative shift in the γmkprf estimate relative to the control cohort. Finally, MAP1A has a significant ratio of missense to silent variants relative to the rest of the genes in both cohorts, and appears to have a signficant impact on the overall γprfreq estimates for both the ASD and SCZ cohorts. Due to limited resequencing data in controls, we were unable to apply the collapsing method to MAP1A. In conclusion, for GRIN2B, MAP1A, and CACNA1F, we see concordant results among multiple methods when data availability allows testing by the different methods.
Previous studies demonstrate that genes associated with Mendelian disease or cancer have more negative population selection parameters compared to genes implicated in complex diseases 
. This pattern may be explained by a late-onset effect, or by a potential enrichment of positively selected genes, among the loci involved in complex disease 
. Another explanation is that genes which accumulate either rare inherited or de novo
mutations are also more likely to accumulate rare mutations which individually have either a high impact, as in the case of Mendelian disorders, or have an intermediate impact in aggregate.
In this paper we hypothesized that rare variants in neurologically expressed genes are enriched in ASD and schizophrenia cohorts. Our findings support a rare allele-major effect model as we have uncovered significant excess of rare variants in our disease cohorts. It remains an open question if ASD and schizophrenia are caused by variants found in a reduced set of genes such as DISC1 or NMDA receptor related genes, or in a larger number of genes associated to the same functional class or pathway/network.