The proband was a male subject of 13 years of age. At physical examination he was found to be of small stature (height < 2.5 standard deviation (s.d.) below the norm average), of normal weight (0 s.d.) and head circumference (occipital frontal; 0 s.d.). He displayed mild dysmorphic features consisting of slightly low implanted protruding ears, long eyelashes, hypotelorism (outer canthal distance −2 s.d.), a high palate, large and irregular front teeth and a mildly triangular-shaped face. He was diagnosed with hypermetropic vision in both eyes (S + 4.0). In addition, a mild lumbar lordosis and a small difference in leg length were found, causing a slightly askew pelvis. Standard laboratory screening, including thyroid function, and additional clinical studies, including fundoscopy, an echo of the heart and kidneys, an electrocardiogram, an electroencephalogram and magnetic resonance imaging of the brain, revealed no structural abnormalities.
He fulfilled DSM-IV diagnostic criteria for autistic disorder according to clinical consensus and the results of the ADI-R and ADOS-G. He also showed problems with regard to his attention span and increased impulsivity. However, these symptoms did not meet sufficient criteria for attention deficit and hyperactivity disorder. His cognitive abilities were in the normal range (full IQ, 98; verbal IQ, 93; performance IQ, 105). Both parents and his two siblings did not display any dysmorphic features and had normal psychomotor development. None of the family members displayed autistic features.
The eldest brother was born with a congenital heart defect (single ventricle defect due to mitral valve atresia). Cardiac surgery was postoperatively complicated by renal insufficiency necessitating chronic treatment with diuretics and cardiotonic medications, and growth hormone later in life. At age 13, he died of acute heart failure, but an autopsy was not performed. The available pictures of him did not suggest facial dysmorphic features. Information from the parents indicated that he had reached psychomotor developmental milestones in time and had not displayed autistic behavior.
A karyotype performed on the proband indicated a deletion on chromosome 13q. Results of the additional serial fluorescent in situ hybridization experiments (RP11-209J19, 2n; RP11-77O2, n; RP11-67L17, n; RP11-520F9, 2n) narrowed the break points of the deletion to 52 524 189–52 955 445 (proximal) and 63 071 144–63 447 585 (distal). These findings were confirmed and further specified by the results of the 550 K Illumina SNP array, showing that the deletion spanned almost 10 Mb, starting at 52 784 996–52 805 428 (proximal), and ending at 63 062 087–63 082 454 (distal). The same deletion was found in the mother. Further, heterozygosity of the 17 SNPs was consistent with the deleted genomic region in the proband and his mother, and with the absence of the deletion in his father and his two siblings (data not shown). There were neither DNA for molecular analysis nor cells for karyotyping available from the deceased brother.
The deleted region contained only four genes: protocadherin 17 precursor, DIAPH3, tudor containing 3 and protocadherin 20 precursor. Sequencing of all the exons of these four genes revealed a non-synonymous single-nucleotide variant in exon 15 of DIAPH3 on the remaining allele. Subsequently, we found both the proband’s father and brother to be heterozygous for the same variant in DIAPH3 (see ). No other exonic variants were detected, except for one known untranslated SNP (rs339531), also in DIAPH3.
The identified point mutation (C > A) results in the substitution of proline by threonine at amino-acid position 614 (Pro614Thr) located in the formin homology 1 (FH1) domain of DIAPH3 (). This particular polymorphism was not reported in the genome databases. In addition, we did not identify this mutation in our screening of 128 control individuals of mixed ethnic descent, nor in the additional sample of 200 Dutch individuals without a lifetime psychiatric diagnosis. The DIAPH3 Pro614Thr was not observed in these 328 control subjects (656 chromosomes), suggesting it is a very low allele frequency.
Figure 2 Proband and consensus DNA and amino-acid sequence. Upper panel: consensus DNA and amino-acid sequence versus sequence results in the proband. Lower panel: formin homology 1 (FH1) domain in human DIAPH3 carrying a C > A point mutation that results (more ...)
Within DIAPH3 only one other nonsynonymous polymorphism with an allele frequency greater than 0.05 is reported by the genome databases (rs36084898, average heterozygosity: 0.148), leading to the substitution of asparagine by serine at position 363. This substitution occurs within the Rho GTPase-binding/ FH3 (GBD/FH3) domain of DIAPH3. However, this variant is unlikely to affect DIAPH3 function given that the GBD/FH3 domain of the DIAPH3 paralog DIAPH1 also contains serine at an analogous position (amino-acid position 336).
DIAPH3 is a regulator of actin dynamics, mediating assembly of unbranched actin filaments. The proline-rich FH1 domain of DIAPH3 is a binding site for the actin monomer-binding protein, profilin, thereby regulating actin nucleation.20
The probability of a functional effect of the point mutation was considered high given its location in the FH1 domain and our observation that the C > A mutation is situated in a short nucleotide sequence element that is highly conserved across vertebrates (chr13:59,443,088–59,443,116, LOD=56) and across a subset of mammalian species (Chr13:59,443,091–59,443,116, LOD = 17). Therefore, we examined whether the C > A mutation affects DIAPH3 function in the presence of an active form of the Rho family GTPase Rif (RifQL) in mouse 3T3 fibroblasts.21
In line with previous work, expression of the murine homologue of DIAPH3, mDia2 or Rif-QL alone did not lead to significant induction of filopodia formation as compared to controls (). In contrast, when mDia2 was co-transfected with RifQL, significantly more filopodia were observed (). Intriguingly, mutated mDia2 failed to induce filopodia formation in the presence of RifQL ). Overall, these data suggest that the C > A mutation has functional consequences for DIAPH3 function and that it interferes with the effect of DIAPH3 on actin dynamics and filopodia formation.
Figure 3 Mutant mDia2 does not induce filopodia in NIH 3T3 cells. (a and b) A small number of filopodia is found on NIH 3T3 cells transfected with empty vector (EV) or RifQL. (c) Coexpression of wild-type (WT) mDia2 and RifQL induces the formation of numerous (more ...)
To establish whether DIAPH3 may have a function in the developing and adult brain, we performed RNA in situ hybridization of Diaph3-specific probes on cryosections of various mouse embryonic stages and adult mouse brain. During embryonic development expression was mainly observed in the subventricular zone (SVZ) of the brain (). Initially the signal was present in the ventricular zone (VZ) of the entire brain up to the spinal cord (10.5 (not shown) and 12.5 d.p.c., ); at later stages expression became progressively restricted to the SVZ of the forebrain and roof of the midbrain (14.5 and 16.5 d.p.c., ). At 18.5 d.p.c. only a low level of expression was maintained in the SVZ of the forebrain (). In addition, from 14.5 d.p.c. onward, staining was observed in the granular cell layer of the cerebellum (). No signal was detected in the adult mouse brain. Outside the brain, Diaph3 expression was detected in the neural layer of the retina, the liver, the cortical region of the kidneys and in the developing teeth (not shown). Sense probed sections did not show significant staining at any of the stages analyzed.
Figure 4 Developmental expression of Diaph3 in the murine brain. All sections shown were hybridized with antisense probe; sense probed sections did not show significant staining. (a) At 12.5 and 10.5 days post coitum (d.p.c.) a signal was detected in the ventricular (more ...)