We have described the molecular characterization of two 2p15–p16.1 deletions identified in unrelated individuals with AD.9
analysis using public databases revealed 108 repetitive elements, including SINEs and LINEs, in the regions of the breakpoints. A comparison of the repeats at the ends of each deletion revealed no regions of sequence homology equal to or greater than 60% for any stretch of sequence longer than 200
bp in Subject 1's deletion. For Subject 2's deletion, there was a 2960 nucleotide region, belonging to the LINE-1 repeat family with 87.9% homology at the proximal and distal breakpoints. LINE-1 retrotransposons have been implicated in genomic rearrangements, as they are significantly enriched at the breakpoints of CNVs compared with the genomic background.30
We suggest that the LINE-1 elements present at both breakpoints of the deletion in Subject 2 predispose to genomic instability through NAHR, and that this mode of deletion generation raises the possibility of recurrence of deletions in this region.
As our initial report,9
other smaller overlapping rearrangements involving the described 2p15–p16.1 region have been reported in controls, including a recurrent 2.9
Mb duplication in neurotypical individuals (58.2–61.1
). A subject with a de novo
duplication of band 2p16.1 (60.5–61.4
Mb) was found in one individual who did not physically resemble Subjects 1 and 2, and did not have an ASD (http://decipher.sanger.ac.uk/
; patient Id 1570). Finally, recurring de novo
microdeletions of various sizes (0.57~8
Mb) have been detected within the described 2p15–p16.1 region,10, 11, 12, 13
with one of four having a confirmed ASD. All subjects presented with several phenotypic traits overlapping those in our patients, including ID and facial dysmorphologies such as high palate, smooth upper vermillion border, everted lower lip, broad and high nasal root, and telecanthus.
The above findings strongly suggest that the 2p15–p16.1 region is unstable. To determine the frequency of 2p15–p16.1 deletions among individuals with ASDs, we tested 798 individuals with an ASD for the presence of microdeletions similar to those described in Subjects 1 and 2; none were found. Furthermore, no similar cases have been reported from five other published studies,31, 32, 33, 34, 35
with at least 3410 subjects with an ASD evaluated for CNVs. However, Liang et al13
reported a case with the 2p15–p16.1 deletion with autism. Thus, the 2p15–p16.1 microdeletion has a prevalence of less than 0.1% among individuals with an ASD.
We selected XPO1 and OTX1 as being of greatest interest for candidate gene testing. We found that SNP rs6735330 in XPO1 was significantly associated with autism in the family-based studies in all four cohorts even after FDR adjustment, with association being highly significant in ASD–CARC cohorts (PFDR=1.29 × 10−5), the AGRE cohort (PFDR=0.001), and the combined family sets (PFDR=2.34 × 10−9) (). The common allele was significantly associated with more severe deficits in social interaction, verbal communication, and repetitive behaviors (all PFDR values <0.01), suggesting that it is in high LD with a functional polymorphism or mutation in this gene, which could be a risk factor for autism in the families studied. Given the role of CMR1 in nuclear export of cargo RNAs with a nuclear export signal, including ribosomal RNAs, U snRNAs, SRPs, and specific mRNAs, it is possible that compromise in the transport of any of these molecules could affect brain development, and lead to behavioral features characteristic of persons with ASDs.
, although deleted in only 1/3 of subjects with autism (and 1/6 subjects with the deletion reported so far), is in the vicinity of the deletion breakpoints and has a function in forebrain and sensory organ development.36
We reasoned that there could be a position effect on the expression of this gene as has been shown for other genes near chromosome breakpoints.37, 38, 39, 40, 41, 42
The role of OTX1
in sensory organ development is interesting, considering that sensory impairments are common in individuals with autism. Leekam et al43
tested individuals with autism using the Diagnostic Interview for Social and Communication Disorders (DISCO),44
which uses 21 items grouped in seven domains (auditory, visual, touch, smell/taste, etc.), and found that they had a higher mean score than the control groups, with more than 90% presenting with symptoms in more than one sensory domain. In addition, the cortical migration defects detected by MR neuroimaging in both of our 2p15–p16.1 deleted subjects, despite only one having a deletion of this gene, made OTX1
a good target for candidate gene screening, assuming that its expression is affected by position effect variegation.
Although case-control studies did not provide evidence for an association between OTX1
with autism, the family-based studies found a very strong association between two of the three OTX1
SNPs (rs2018650 and rs13000344) and risk for ASD (). A highly significant association with ASD was also found for the rs2018650G–rs13000344C haplotype (PFDR
=2.63 × 10−11
). QTDT revealed that at least one copy of the common alleles of these SNPs was associated with more severe ASD symptoms, as determined by the ADI–R, providing evidence for an association between OTX1
and ASD. We also note that OTX1
is expressed in the cerebellum within the external germinal layer, a proliferative zone close to the pia, which is responsible for the production of granule cells. These cells differentiate into the granule cells of the cerebellar cortex, which are reduced in the brains of individuals with autism.45
The exception in our results for both OTX1 and XPO1 is with families from New York and the SIRFA families. It is of interest that many of the New York families are of Italian descent. The lack of association in the SIRFA sample may also be due to patient selection criteria, which excluded any patient displaying major dysmorphic features or CNS anomalies as determined by cranial MRI.
The lack of positive findings for both XPO1
in genome-wide association studies (GWAS) by others46, 47, 48, 49, 50, 51, 52, 53, 54
may reflect the greater genetic heterogeneity in these larger data sets, as we found a clear association with specific core ASD phenotypes. Re-analysis of GWAS separating those with more severe and milder ADI–R symptom scores should be done to test this possibility.
The absence of additional cases of 2p15–p16.1 deletion in our own ASD study cohort (n
=798), and at least 2586 cases from other published studies31, 32, 33, 34, 35
suggests that the involvement of 2p15–p16.1 with autism is a rare event. Given that the presence of dysmorphic features represents an exclusion criterion for several ASD studies, this may reduce the frequency of detection of 2p15–p16.1 deletions in other screening studies.
Our findings suggest that the newly described 2p15–p16.1 microdeletion syndrome represents a somatic and neurodevelopmental phenotype inclusive of autism, moderate to severe ID, CNS dysmorphology, and distinctive craniofacial dysmorphology. In addition, functional variants in the XPO1 and/or OTX1 genes in LD with the three SNPs showing strong association with autism, may contribute to autistic behaviors in not only the two 2p15–p16.1 deletion subjects described herein, but also in a larger proportion of individuals with ASD with more severe core ASD symptoms mapping to this region of chromosome 2.