We report a boy with postnatal microcephaly, minor dysmorphic features, mental retardation and autism associated with a 3.3 Mb deletion involving ~43 genes in chromosome 1p34.2p34.3. The deletion is large and de novo, leading us to hypothesise that it is responsible for his abnormal phenotype. However, the specific genes responsible for different features of the 1p34.2p34.3 deletion phenotype are unknown.
Only a few patients with interstitial deletions of 1p34.2p34.3 have been reported. The closest match is the recent report of a Dutch boy with a 4.1 Mb deletion that completely overlaps the deletion we report, with loss of an additional 14 genes including the glucose transporter gene SLC2A1
Overall, he has a more severe phenotype than our patient with severe mental retardation and hypotonia, ‘profound’ microcephaly and epilepsy, as well as heterotopia and ponto-cerebellar atrophy on brain imaging. The more severe phenotype is not surprising given the severe phenotype associated with loss of SLC2A1
alone (OMIM 606777). Three other patients have been reported with deletions involving this general region. One boy with a much larger ~17 Mb visible deletion had severe developmental delay, poor growth, and microcephaly.36
Two sibs who inherited a juxtaposed inversion (inv 1p22.3p34.1) and deletion (1p34.1p34.3) of this region presented with behaviour disorders, although their intelligence and appearance were normal.37
A detailed genotype–phenotype analysis involving 1p34.2p34.3 in these patients is provided in the supplementary materials.
Based on the reported altered regulation of RIMS3
(see below) and our pathway analysis, we hypothesise that mutations in the synaptic protein RIMS3 is the primary contributor to the autism phenotype in the 1p34.2p34.3 microdeletion patient. A large number of autism related genes are involved in synapse function, neuronal cell adhesion, or both.38
Several of these have been implicated in autism based on cytogenetic rearrangements, CNVs or rare mutations, including NLGN3
,10 14 15 SHANK3
RIMS3 belongs to the RIM protein family that function as important components of the presynaptic machinery for synaptic vesicle fusion and neurotransmitter release.39
Expression analysis of RIMS3
in the rat demonstrated that it is exclusively expressed in the brain40
and protein studies indicated that RIMS3 was detected only in rostral brain regions but not in spinal cord, hindbrain, or midbrain.40
Overexpression of RIMS3
has been demonstrated to greatly facilitate Ca2+
triggered exocytosis, supporting a role for RIMS3
as a regulator of exocytosis in the synaptic membrane.40
One recent study demonstrated that expression of RIMS3
is dysregulated in lymphoblastoid cells from autism patients with either maternal duplications of 15q11q13 or fragile X syndrome.24
Others have reported increased expression of RIMS3
in schizophrenia,41 42
a neuropsychiatric disorder hypothesised to have shared genetic aetiology with autism.43
Thus, several lines of evidence converge to support the hypothesis that abnormal RIMS3
function due to chromosomal imbalances or rare mutations may underlie autism. However, we cannot exclude the possibility that other genes within or near the ~ 3.3 Mb 1p34.2p34.3 deletion may contribute to autism.
To address whether RIMS3 point mutations underlie autism, we sequenced the complete RIMS3 coding region in 512 unrelated autism patients. In total, five coding variants were identified in autism patients that were absent in ~460 controls. None of these variants were de novo and all were inherited with no bias between maternal versus paternal transmission. We used several in silico programs to assess the functional impact of these variants on protein function, and identified one variant (p.E177A) that was predicted to be deleterious. This variant segregated with the autism phenotype in two sibs, was not identified in 1161 controls, and is highly conserved among eutherian mammals.
The p.E177A substitution resides in a C2
B domain that is known to bind to several proteins including α-liprins, SNAP-25, and voltage gated calcium channels.44 45
However, the critical residues for some of these interactions are downstream of p.E177 and this amino acid is not conserved among the RIM family of proteins.40
α-Liprins are known to interact directly with CASK, a calcium/calmodulin dependent serine protein kinase that belongs to the membrane associated guanylate kinase family. CASK regulates the trafficking, targeting and signalling of numerous presynaptic proteins including neurexins, APBA1 (Mint1) and ion channels,46
and is also implicated in brain development.47
Interestingly, a C2
B domain is also found in Doc2-α (Double C2-like domains, α), a protein that belongs to the same superfamily as RIMS3. DOC2A
is one of ~24 genes located within the 16p11.2 microdeletion-duplication region that has recently been detected in ~1% of patients with autism.8 11 12
Although the p.E177A variant was not found in a large number of controls and was predicted to be deleterious using SIFT and SNAP, further studies are needed to prove whether the p.E177A substitution is pathogenic or not, as protein simulation programs do not substitute for experimental confirmation.29 48
Only two of the three in silico methods we used predicted a deleterious effect from the p.E177A variant. This is not surprising, given that each program varies in methodology, including differences in classification approaches (eg, machine learning vs rule based systems) and input information (eg, sequence data, functional annotation, protein structure, solvent accessibility).29 31
We also identified one variant in a control subject that was predicted to be deleterious. Only limited phenotype data are available for this subject, so we cannot determine whether it is a benign or weakly pathogenic variant. Detailed phenotypic assessment of this subject would help distinguish between these possibilities.
In summary, our data demonstrate that heterozygous loss of one or more of the 43 genes located within a ~3.3 Mb interval on chromosome 1p34.2p34.3 are associated with abnormal development, including mental retardation, postnatal microcephaly and autism. Furthermore, our data suggest but do not prove conclusively that RIMS3 is an autism causative or contributory gene. To our knowledge, this is the first study to suggest that mutations in a RIM protein may underlie autism or other neurodevelopmental disorders. The identification of putative mutations in RIMS3 is consistent with the identification of CNVs and intragenic mutations of other pre- and post-synaptic proteins in patients with autism, and lends further support to the hypothesis that abnormalities in synaptic development and function underlie autism in some (and perhaps many) patients. Functional studies of RIMS3 variants such as p.E177A should provide additional insight into the role of pre-synaptic proteins in autism. Finally, the rare association of microcephaly and autism suggests that other gene(s) in the region are associated with brain growth.