De Novo Variants in WDR37 Are Associated with Epilepsy, Colobomas, Dysmorphism, Developmental Delay, Intellectual Disability, and Cerebellar Hypoplasia

Abstract

WD40 repeat-containing proteins form a large family of proteins present in all eukaryotes. Here, we identified five pediatric probands with de novo variants in WDR37, which encodes a member of the WD40 repeat protein family. Two probands shared one variant and the others have variants in nearby amino acids outside the WD40 repeats. The probands exhibited shared phenotypes of epilepsy, colobomas, facial dysmorphology reminiscent of CHARGE syndrome, developmental delay and intellectual disability, and cerebellar hypoplasia. The WDR37 protein is highly conserved in vertebrate and invertebrate model organisms and is currently not associated with a human disease. We generated a null allele of the single Drosophila ortholog to gain functional insights and replaced the coding region of the fly gene CG12333/wdr37 with GAL4. These flies are homozygous viable but display severe bang sensitivity, a phenotype associated with seizures in flies. Additionally, the mutant flies fall when climbing the walls of the vials, suggesting a defect in grip strength, and repeat the cycle of climbing and falling. Similar to wall clinging defect, mutant males often lose grip of the female abdomen during copulation. These phenotypes are rescued by using the GAL4 in the CG12333/wdr37 locus to drive the UAS-human reference WDR37 cDNA. The two variants found in three human subjects failed to rescue these phenotypes, suggesting that these alleles severely affect the function of this protein. Taken together, our data suggest that variants in WDR37 underlie a novel syndromic neurological disorder.

Keywords: CG12333; Drosophila; WD40 repeats; WDR37 domains; bang sensitivity; wdr37.

Figure 1

Clinical Features of Probands All probands exhibit facial dysmorphisms. (A and B) Proband 1 (P1) has a tall forehead, broad nasal bridge, flat philtrum with a thin vermillion border, low-set ears, and small jaw. (C and D) Proband 2 (P2) has hypertelorism, ptosis, long eyelashes, left iris coloboma, prominent nasal bridge, short smooth philtrum, thin upper lip, downturned corners of the mouth, and low-set ears with thick prominent helices. (E) Proband 3 (P3) has excess nuchal skin, visible epicanthal folds, broad nasal bridge, and smooth philtrum. (F and G) Proband 4 (P4) has epicanthal folds, ectropion of lower lids, wide mouth with downturned corners, smooth philtrum, and prominent nose.

Figure 2

Brain MRI Images in Probands 1, 2, 4, and 5 Shown are probands 1 (A–E), 2 (F–J), 4 (K–O), and 5 (P–T). Midline sagittal images show mild (K) or severe (A, F, P) cerebellar vermis hypoplasia; the horizontal white bars mark the expected lower edge of the vermis near the obex. Axial T2-weighted images through the posterior fossa (B, G, L, Q) show enlarged 4th ventricle and small cerebellar hemispheres with a striking foliar dysplasia (white arrowheads). Axial T2-weighted images through the cerebral hemispheres (third and fourth columns) show diffuse shallow sulci or simplified gyral pattern, reduced white matter volume, and mild ventriculomegaly. Coronal T2-weighted images show striking hippocampal hypoplasia and dysplasia in 3 of 5 subjects (black arrows in E, J, T). Note: for proband 3, only limited MRI images were available for review. For comparison, please refer to the studies of Sanchez et al. for MRI images in control individuals.

Figure 3

Conservation of WDR37 and Generation of wdr37-Null Allele (A) Alignment of WDR37 with wdr37. Protein domains of WDR37 were identified through UniProt and SMART databases. The variants identified in the probands are marked with a red box and variant residue is indicated above the variant amino acid. WD40 repeats are shaded yellow and the coiled coil region is shaded blue. (B) Generation of wdr37^GAL4Δ allele. Primer sites to verify the insertion are indicated on the construct. (C) RT-PCR confirmation of wdr37-null alleles. RNA is isolated and reverse transcribed from two independent lines of wdr37^GAL4Δ / Def(3R)6179 and yw flies as control. Correct amplicons are detected only in the yw animals.

Figure 4

Expression of wdr37 in Adults and Larvae (A) Expression of UAS-GFP under the control of wdr37^GAL4Δ is monitored in larval and adult body. (B) Expression domain of wdr37 is monitored in adult brain by using the indicated transgenes. Co-staining with neuronal marker (ElaV) and glial marker (repo) indicate that the gene is expressed in large subsets of neurons and glia.

Figure 5

Adult Phenotypes of wdr37-Null Mutants (A) wdr37 (wdr37^GAL4Δ /Df)-null flies exhibit bang sensitivity. Df corresponds to Df(3R)Exel6179. (B) Males of the indicated genotype in the presence of wild-type (CantonS) females are visualized for 30 min. Wild-type males (on the left) can maintain their grip of female abdomen during copulation whereas the wdr37^GAL4Δ/ Df(3R)6179 males often lose their grip and are unable to maintain normal copulatory posture. (C) Overexpression of the UAS-WDR37 human reference cDNA under the control of wdr37^GAL4Δ is toxic at 25°C and 22°C (with <5% that survive and are referred to as “escapers”); this toxicity is suppressed at 18°C. The UAS-WDR37 lines with the human variant cDNAs lose this toxicity. (D) wdr37-null flies (wdr37^GAL4Δ/Df) show negative geotaxis but fail to hang on the surface of the vial for 30 s; this phenotype is rescued by the full-length UAS WDR37 reference cDNA (Ref) but not rescued by neither of the variant cDNAs (p.T125I or p.S129C). (E) The loss of wdr37 leads to increased bang sensitivity, which is rescued by the human reference cDNA (Ref), but not the variants p.T125I or p.S129C. Note that the duration of bang sensitivity response is shorter at experimental temperature. (F) The reference human cDNA (Ref) can rescue the copulation defect whereas p.S129C and p.T125I variant cDNAs fail to rescue copulation defect. Ref indicates line with UAS-WDR37 full-length cDNA human reference, p.T125I indicates variant UAS-WDR37 with the p.T125I variant, and p.S129C indicates variant UAS-WDR37 with the p.S129C variant. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, and ^∗∗∗∗p ≤ 0.0001. ns indicates not significant.