Biallelic loss-of-function variants in EXOC6B are associated with impaired primary ciliogenesis and cause spondylo-epi-metaphyseal dysplasia with joint laxity type 3.

Spondylo-epi-metaphyseal dysplasias with joint laxity, type 3 (SEMDJL3) is a genetic skeletal disorder characterized by multiple joint dislocations, caused by biallelic pathogenic variants in the EXOC6B gene. Only four individuals from two families have been reported to have this condition to date. The molecular pathogenesis related to primary ciliogenesis has not been enumerated in subjects with SEMDJL3. In this study, we report two additional affected individuals from unrelated families with biallelic pathogenic variants, c.2122+15447_2197-59588del and c.401T>G in EXOC6B identified by exome sequencing. One of the affected individuals had an intellectual disability and central nervous system anomalies, including hydrocephalus, hypoplastic mesencephalon, and thin corpus callosum. Using the fibroblast cell lines, we demonstrate the primary evidence for the abrogation of exocytosis in an individual with SEMDLJ3 leading to impaired primary ciliogenesis. Osteogenesis differentiation and pathways related to the extracellular matrix were also found to be reduced. Additionally, we provide a review of the clinical and molecular profile of all the mutation-proven patients reported hitherto, thereby further characterizing SEMDJL3. SEMDJL3 with biallelic pathogenic variants in EXOC6B might represent yet another ciliopathy with central nervous system involvement and joint dislocations.

A homozygous hypomorphic BNIP1 variant causes an increase in autophagosomes and reduced autophagic flux and results in a spondylo-epiphyseal dysplasia.

BNIP1 (BCL2 interacting protein 1) is a soluble N-ethylmaleimide-sensitive factor-attachment protein receptor involved in ER membrane fusion. We identified the homozygous BNIP1 intronic variant c.84+3A>T in the apparently unrelated patients 1 and 2 with disproportionate short stature. Radiographs showed abnormalities affecting both the axial and appendicular skeleton and spondylo-epiphyseal dysplasia. We detected ~80% aberrantly spliced BNIP1 pre-mRNAs, reduced BNIP1 mRNA level to ~80%, and BNIP1 protein level reduction by ~50% in patient 1 compared to control fibroblasts. The BNIP1 ortholog in Drosophila, Sec20, regulates autophagy and lysosomal degradation. We assessed lysosome positioning and identified a decrease in lysosomes in the perinuclear region and an increase in the cell periphery in patient 1 cells. Immunofluorescence microscopy and immunoblotting demonstrated an increase in LC3B-positive structures and LC3B-II levels, respectively, in patient 1 fibroblasts under steady-state condition. Treatment of serum-starved fibroblasts with or without bafilomycin A1 identified significantly decreased autophagic flux in patient 1 cells. Our data suggest a block at the terminal stage of autolysosome formation and/or clearance in patient fibroblasts. BNIP1 together with RAB33B and VPS16, disease genes for Smith-McCort dysplasia 2 and a multisystem disorder with short stature, respectively, highlight the importance of autophagy in skeletal development.

A data set of variants derived from 1455 clinical and research exomes is efficient in variant prioritization for early-onset monogenic disorders in Indians.

Given the genomic uniqueness, a local data set is most desired for Indians, who are underrepresented in existing public databases. We hypothesize patients with rare monogenic disorders and their family members can provide a reliable source of common variants in the population. Exome sequencing (ES) data from families with rare Mendelian disorders was aggregated from five centers in India. The dataset was refined by excluding related individuals and removing the disease-causing variants (refined cohort). The efficiency of these data sets was assessed in a new set of 50 exomes against gnomAD and GenomeAsia. Our original cohort comprised 1455 individuals from 1203 families. The refined cohort had 836 unrelated individuals that retained 1,251,064 variants with 181,125 population-specific and 489,618 common variants. The allele frequencies from our cohort helped to define 97,609 rare variants in gnomAD and 44,520 rare variants in GenomeAsia as common variants in our population. Our variant dataset provided an additional 1.7% and 0.1% efficiency for prioritizing heterozygous and homozygous variants respectively for rare monogenic disorders. We observed additional 19 genes/human knockouts. We list carrier frequency for 142 recessive disorders. This is a large and useful resource of exonic variants for Indians. Despite limitations, datasets from patients are efficient tools for variant prioritization in a resource-limited setting.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

Biallelic variants p.Arg1133Cys and p.Arg1379Cys in COL2A1: Further delineation of phenotypic spectrum of recessive Type 2 collagenopathies.

The phenotypic spectrum of Type 2 collagenopathies ranges from lethal achondrogenesis Type 2 to milder osteoarthritis with mild chondrodysplasia. All of them are monoallelic except for the two recent reports showing that biallelic variants in COL2A1 can cause spondyloepiphyseal dysplasia congenita in two children. Here we report two additional families with homozygous variants, c.4135C>T (p.Arg1379Cys) and c.3190C>T (p.Arg1133Cys) in COL2A1 resulting in two distinct skeletal dysplasia phenotypes of intermediate severity. Though all six patients from four families exhibit a spondylo-epimetaphyseal dysplasia, they demonstrate a wide variation in severity of short stature and involvement of epiphyses, metaphyses, and vertebrae. We hypothesize that the variants are likely to be hypomorphic, given the underlying mechanisms of disease causation for known heterozygous variants in COL2A1. With this report, we provide further evidence to the existence of autosomal recessive Type 2 collagenopathy.

An emerging ribosomopathy affecting the skeleton due to biallelic variations in NEPRO.

Cartilage hair hypoplasia (CHH), anauxetic dysplasia 1, and anauxetic dysplasia 2 are rare metaphyseal dysplasias caused by biallelic pathogenic variants in RMRP and POP1, which encode the components of RNAse-MRP endoribonuclease complex (RMRP) in ribosomal biogenesis pathway. Nucleolus and neural progenitor protein (NEPRO), encoded by NEPRO (C3orf17), is known to interact with multiple protein subunits of RMRP. We ascertained a 6-year-old girl with skeletal dysplasia and some features of CHH. RMRP and POP1 did not harbor any causative variant in the proband. Parents-child trio exomes revealed a candidate biallelic variant, c.435G>C, p.(Leu145Phe) in NEPRO. Two families with four affected individuals with skeletal dysplasia and a homozygous missense variant, c.280C>T, p.(Arg94Cys) in NEPRO, were identified from literature and their published phenotype was compared in detail to the phenotype of the child we described. All the five affected individuals have severe short stature, brachydactyly, skin laxity, joint hypermobility, and joint dislocations. They also have short metacarpals, broad middle phalanges, and metaphyseal irregularities. Protein modeling and stability prediction showed that the mutant protein has decreased stability. Both the reported variants are in the same domain of the protein. Our report delineates the clinical and radiological characteristics of an emerging ribosomopathy caused by biallelic variants in NEPRO.

Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia.

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.