Novel association of Dandy-Walker malformation with CAPN15 variants expands the phenotype of oculogastrointestinal neurodevelopmental syndrome

Abstract

Oculogastrointestinal neurodevelopmental syndrome has been described in seven previously published individuals who harbor biallelic pathogenic variants in the CAPN15 gene. Biallelic missense variants have been reported to demonstrate a phenotype of eye abnormalities and developmental delay, while biallelic loss of function variants exhibit phenotypes including microcephaly and craniofacial abnormalities, cardiac and genitourinary malformations, and abnormal neurologic activity. We report six individuals from three unrelated families harboring biallelic deleterious variants in CAPN15 with phenotypes overlapping those previously described for this disorder. Of the individuals affected, four demonstrate radiographic evidence of the classical triad of Dandy-Walker malformation including hypoplastic vermis, fourth ventricle enlargement, and torcular elevation. Cerebellar anomalies have not been previously reported in association with CAPN15-related disease. Here, we present three unrelated families with findings consistent with oculogastrointestinal neurodevelopmental syndrome and cerebellar pathology including Dandy-Walker malformation. To corroborate these novel clinical findings, we present supporting data from the mouse model suggesting an important role for this protein in normal cerebellar development. Our findings add six molecularly confirmed cases to the literature and additionally establish a new association of Dandy-Walker malformation with biallelic CAPN15 variants, thereby expanding the neurologic spectrum among patients affected by CAPN15-related disease.

Keywords: Dandy-Walker malformation; Purkinje cells; calpain; cerebellar dysgenesis; oculogastrointestinal neurodevelopmental syndrome; phenotype expansion.

Figure 1. Molecular consequence of CAPN15 variation and reported phenotype presentations.

Previously reported patients with mild neurodevelopmental phenotypes have predominantly missense variants in CAPN15, while novel frameshift variants and full gene deletions present with Dandy-Walker malformation. A previously reported frameshift variant c.2904+1_2905–45del (Mor-Shaked et al., 2021) leads to skipping of an exon and was reported with more severe brain involvement including cortical thinning. Cerebellar phenotypes were not specified in the siblings with this variant.

Figure 2.. Dandy-Walker malformation segregates with biallelic deleterious variants in CAPN15.

A) Pedigree for Family 1 demonstrating two affected siblings. Proband (III-3) and affected sister (III-2) both inherited biallelic CAPN15 variants and presented with Dandy-Walker malformation. Heterozygous carriers of the p.Leu865Pro variant do not present with typical syndromic features of oculogastrointestinal neurodevelopmental syndrome. Individuals II-2 and II-3, who are heterozygous for 16p13.3 deletion, present with intellectual disability, which is consistent with the ATR-16 phenotype (MIM #141750) associated with heterozygous deletion of this region. Unaffected sibling III-1 demonstrated absence of both the 16p13.3 deletion and the CAPN15 familial variant. B) Brain imaging for Family 1 demonstrating confirmed Dandy-Walker Malformation in both siblings. III-2 and III-3 show residual vermis (yellow arrows). Additional findings for both images: lambdoidal torcular elevation and large posterior fossa cysts splaying the cerebellar hemispheres. III-2 shows mild hydrocephalus; III-3 shows severe hydrocephalus.

Figure 3.. Frameshift variants found in Families 1 and 2 result in a loss of CAPN15 C-terminus.

RT-PCR (a) and Western Blot (b) of probands (A, A1, A2) and unaffected (U) heterozygous parents in Family 1 (p.Arg716Glyfs*99) and Family 2 (p.Gln254Profs*7). Reduced mRNA expression is found in affected homozygotes of Family 2, but not Family 1 compared to unaffected heterozygous parents. In both cases, there is a loss of full-length protein expression as indicated by a lack of staining by an antibody directed at the C-terminus of CAPN15.

Figure 4.. Several in silico analyses support the pathogenicity of the novel p.Leu865Pro missense variant.

(A) Leu865 is widely conserved across vertebrate species. Sequences obtained via 100 Vertebrates MultiZ alignment. (B) Alpha-fold predicted structure AF-O75080-F1 places Leu865 in a highly organized region central to the protein, interfacing between an alpha-helix and beta-sheet complex. (C) Several algorithms predict the variant to be deleterious, including measures of natural population variation, sequence constraint, and missense relative pathogenicity.

Figure 5.. Expression of CAPN15/SOLH in the maturing mouse cerebellum.

Parasagittal images of the cerebellar hemisphere lateral to the vermis from the IV/V lobule demonstrate CAPN15 co-expression (green) with calbindin (red), a Purkinje cell marker, during early postnatal development in the mouse at timepoints critical for cerebellar maturation (postnatal day 5, bottom left; postnatal day 11, bottom middle and bottom right). Additional faint non-nuclear CAPN15/SOLH staining can be seen in the molecular layer superior to the Purkinje cells, as well as the granule zone layer inferior to the Purkinje cells. CAPN15/SOLH expression persists into the adult mouse. Scale bars as follows: top left 150 μm, top middle 75 μm, top right 37 μm, bottom left 150 μm, bottom middle 150 μm, bottom right 20 μm.