Two sisters with RSPRY1-related spondyloepimetaphyseal dysplasia.

Biallelic variants in RSPRY1 have been found to result in spondyloepimetaphyseal dysplasia. Two siblings presenting with short stature, facial dysmorphism, progressive vertebral defects, small epiphysis, cupping and fraying of metaphyses, brachydactyly, and short metatarsals harbored a homozygous missense variant c.1652G>A;p.(Cys551Tyr) in the RSPRY1 gene. The phenotype in our patients resembles spondyloepimetaphyseal dysplasia, Faden-Alkuraya type. Thus, our study provides further evidence to support the association of RSPRY1 variants with spondyloepimetaphyseal dysplasia. We observed joint dislocation as a novel clinical feature of this condition.

Multi-gene panel sequencing in highly consanguineous families and patients with congenital forms of skeletal dysplasias.

Skeletal dysplasias (SKDs) are a heterogeneous group of more than 750 genetic disorders characterized by abnormal development, growth, and maintenance of bones or cartilage in the human skeleton. SKDs are often caused by variants in early patterning genes and in many cases part of multiple malformation syndromes and occur in combination with non-skeletal phenotypes. The aim of this study was to investigate the underlying genetic cause of congenital SKDs in highly consanguineous Pakistani families, as well as in sporadic and familial SKD cases from India using multigene panel sequencing analysis. Therefore, we performed panel sequencing of 386 bone-related genes in 7 highly consanguineous families from Pakistan and 27 cases from India affected with SKDs. In the highly consanguineous families, we were able to identify the underlying genetic cause in five out of seven families, resulting in a diagnostic yield of 71%. Whereas, in the sporadic and familial SKD cases, we identified 12 causative variants, corresponding to a diagnostic yield of 44%. The genetic heterogeneity in our cohorts was very high and we were able to detect various types of variants, including missense, nonsense, and frameshift variants, across multiple genes known to cause different types of SKDs. In conclusion, panel sequencing proved to be a highly effective way to decipher the genetic basis of SKDs in highly consanguineous families as well as sporadic and or familial cases from South Asia. Furthermore, our findings expand the allelic spectrum of skeletal dysplasias.

Indian patients with CHST3-related chondrodysplasia with congenital joint dislocations.

CHST3-related chondrodysplasia with congenital joint dislocations (CDCJD, #MIM 143095), is a rare genetic skeletal disorder caused by biallelic loss of function variants in CHST3. CHST3 is critical for the sulfation of chondroitin sulfate. This study delineates the clinical presentation of nine individuals featuring the key symptoms of CDCJD; congenital joint (knee and elbow) dislocations, short trunk short stature progressive vertebral anomalies, and metacarpal shortening. Additional manifestations include irregular distal femoral epiphysis, supernumerary carpal ossification centers, bifid humerus, club foot, and cardiac abnormalities. Sanger sequencing was carried out to investigate molecular etiology in eight patients and exome sequencing in one. Genetic testing revealed five homozygous variants in CHST3 (four were novel and one was previously reported). All these variants are located on sulfotransferase domain of CHST3 protein and were classified as pathogenic/ likely pathogenic. We thus report on nine individuals with CHST3-related chondrodysplasia with congenital joint dislocations from India and suggest monitoring the health of cardiac valves in this condition.

Clinical, genetic and structural delineation of RPL13-related spondyloepimetaphyseal dysplasia suggest extra-ribosomal functions of eL13.

Spondyloepimetaphyseal dysplasia with severe short stature, RPL13-related (SEMD-RPL13), MIM#618728), is a rare autosomal dominant disorder characterized by short stature and skeletal changes such as mild spondylar and epimetaphyseal dysplasia affecting primarily the lower limbs. The genetic cause was first reported in 2019 by Le Caignec et al., and six disease-causing variants in the gene coding for a ribosomal protein, RPL13 (NM_000977.3) have been identified to date. This study presents clinical and radiographic data from 12 affected individuals aged 2-64 years from seven unrelated families, showing highly variable manifestations. The affected individuals showed a range from mild to severe short stature, retaining the same radiographic pattern of spondylar- and epi-metaphyseal dysplasia, but with varying severity of the hip and knee deformities. Two new missense variants, c.548 G>A, p.(Arg183His) and c.569 G>T, p.(Arg190Leu), and a previously known splice variant c.477+1G>A were identified, confirming mutational clustering in a highly specific RNA binding motif. Structural analysis and interpretation of the variants' impact on the protein suggests that disruption of extra-ribosomal functions of the protein through binding of mRNA may play a role in the skeletal phenotype of SEMD-RPL13. In addition, we present gonadal and somatic mosaicism for the condition.

Genome sequencing identifies a large non-coding region deletion of SNX10 causing autosomal recessive osteopetrosis.

Autosomal recessive osteopetrosis (ARO) is a rare genetic disorder caused by impaired osteoclast activity. In this study, we describe a 4-year-old boy with increased bone density due to osteopetrosis, autosomal recessive 8. Using genome sequencing, we identified a large deletion in the 5'-untranslated region (UTR) of SNX10 (sorting nexin 10), where the regulatory region of this gene is located. This large deletion resulted in the absence of the SNX10 transcript and led to abnormal osteoclast activity. SNX10 is one of the nine genes known to cause ARO, shown to interact with V-ATPase (vacuolar type H( + )-ATPase), as it plays an important role in bone resorption. Our study highlights the importance of regulatory regions in the 5'-UTR of SNX10 for its expression while also demonstrating the importance of genome sequencing for detecting large deletion of the regulatory region of SNX10.

Clinical, radiological and molecular studies in 24 individuals with Dyggve-Melchior-Clausen dysplasia and Smith-McCort dysplasia from India.

BACKGROUND: Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC types 1 and 2) are rare spondyloepimetaphyseal dysplasias with identical radiological findings. The presence of intellectual disability in DMC and normal intellect in SMC differentiates the two. DMC and SMC1 are allelic and caused by homozygous or compound heterozygous variants in DYM. SMC2 is caused by variations in RAB33B. Both DYM and RAB33B are important in intravesicular transport and function in the Golgi apparatus. METHODS: Detailed clinical phenotyping and skeletal radiography followed by molecular testing were performed in all affected individuals. Next-generation sequencing and Sanger sequencing were used to confirm DYM and RAB33B variants. Sanger sequencing of familial variants was done in all parents. RESULTS: 24 affected individuals from seven centres are described. 18 had DMC and 6 had SMC2. Parental consanguinity was present in 15 of 19 (79%). Height <3 SD and gait abnormalities were seen in 20 and 14 individuals, respectively. The characteristic radiological findings of lacy iliac crests and double-humped vertebral bodies were seen in 96% and 88% of the affected. Radiological findings became attenuated with age. 23 individuals harboured biallelic variants in either DYM or RAB33B. Fourteen different variants were identified, out of which 10 were novel. The most frequently occurring variants in this group were c.719 C>A (3), c.1488_1489del (2), c.1484dup (2) and c.1563+2T>C (2) in DYM and c.400C>T (2) and c.186del (2) in RAB33B. The majority of these have not been reported previously. CONCLUSION: This large cohort from India contributes to the increasing knowledge of clinical and molecular findings in these rare 'Golgipathies'.

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Ectodysplasin pathogenic variants affecting the furin-cleavage site and unusual clinical features define X-linked hypohidrotic ectodermal dysplasia in India.

Hypohidrotic ectodermal dysplasia (HED) is a rare genetic disorder caused by mutational inactivation of a developmental pathway responsible for generation of tissues of ectodermal origin. The X-linked form accounts for the majority of HED cases and is caused by Ectodysplasin (EDA) pathogenic variants. We performed a combined analysis of 29 X-linked hypohidrotic ectodermal dysplasia (XLHED) families (including 12 from our previous studies). In addition to the classical triad of symptoms including loss (or reduction) of ectodermal structures, such as hair, teeth, and sweat glands, we detected additional HED-related clinical features including facial dysmorphism and hyperpigmentation in several patients. Interestingly, global developmental delay was identified as an unusual clinical symptom in many patients. More importantly, we identified 22 causal pathogenic variants that included 15 missense, four small in-dels, and one nonsense, splice site, and large deletion each. Interestingly, we detected 12 unique (India-specific) pathogenic variants. Of the 29 XLHED families analyzed, 11 (38%) harbored pathogenic variant localized to the furin cleavage site. A comparison with HGMD revealed significant differences in the frequency of missense pathogenic variants; involvement of specific exons and/or protein domains and transition/transversion ratios. A significantly higher proportion of missense pathogenic variants (33%) localized to the EDA furin cleavage when compared to HGMD (7%), of which p.R155C, p.R156C, and p.R156H were detected in three families each. Therefore, the first comprehensive analysis of XLHED from India has revealed several unique features including unusual clinical symptoms and high frequency of furin cleavage site pathogenic variants.

Deficiency of TMEM53 causes a previously unknown sclerosing bone disorder by dysregulation of BMP-SMAD signaling.

Bone formation represents a heritable trait regulated by many signals and complex mechanisms. Its abnormalities manifest themselves in various diseases, including sclerosing bone disorder (SBD). Exploration of genes that cause SBD has significantly improved our understanding of the mechanisms that regulate bone formation. Here, we discover a previously unknown type of SBD in four independent families caused by bi-allelic loss-of-function pathogenic variants in TMEM53, which encodes a nuclear envelope transmembrane protein. Tmem53-/- mice recapitulate the human skeletal phenotypes. Analyses of the molecular pathophysiology using the primary cells from the Tmem53-/- mice and the TMEM53 knock-out cell lines indicates that TMEM53 inhibits BMP signaling in osteoblast lineage cells by blocking cytoplasm-nucleus translocation of BMP2-activated Smad proteins. Pathogenic variants in the patients impair the TMEM53-mediated blocking effect, thus leading to overactivated BMP signaling that promotes bone formation and contributes to the SBD phenotype. Our results establish a previously unreported SBD entity (craniotubular dysplasia, Ikegawa type) and contribute to a better understanding of the regulation of BMP signaling and bone formation.

Homozygous variant, p.(Arg643Trp) in VAC14 causes striatonigral degeneration: report of a novel variant and review of VAC14-related disorders.

VAC14-related disorders include two distinct phenotypes, striatonigral degeneration [MIM# 617054] and Yunis-Varon syndrome. Striatonigral degeneration is a recently described childhood onset dystonia caused by pathogenic variants in VAC14. It is characterized by a period of apparent normalcy followed by abrupt onset neuroregression, dystonia, involuntary movements and degenerative brain lesions involving caudate nucleus, putamen and substantia nigra. Yunis-Varon syndrome is a well described severe condition characterised by skeletal findings and dysmorphism along with neuronal degeneration. Pathogenic variants in FIG4 have been previously reported to cause Yunis-Varon syndrome. Recently, loss of function variants in VAC14 were also reported in an individual affected with Yunis-Varon syndrome. Total seven individuals from four families are reported to have VAC14-related disorders till date. Here, we report another individual with clinical and radiological features suggestive of striatonigral degeneration with homozygous missense variant in VAC14. The patient fibroblasts showed extensive vacuolization, characteristic of VAC14-related disorders. We also review the phenotype and genotype associated with these disorders.

The homozygous variant c.797G>A/p.(Cys266Tyr) in PISD is associated with a Spondyloepimetaphyseal dysplasia with large epiphyses and disturbed mitochondrial function.

Spondyloepimetaphyseal dysplasias (SEMD) are a group of genetically heterogeneous skeletal disorders characterized by abnormal vertebral bodies and epimetaphyseal abnormalities. We investigated two families with a new SEMD type with one proband each. They showed mild facial dysmorphism, flat vertebral bodies (platyspondyly), large epiphyses, metaphyseal dysplasia, and hallux valgus as common clinical features. By trio-exome sequencing, the homozygous missense variant c.797G>A/p.(Cys266Tyr) in PISD was found in both affected individuals. Based on exome data analyses for homozygous regions, the two patients shared a single homozygous block on chromosome 22 including PISD, indicating their remote consanguinity. PISD encodes phosphatidylserine (PS) decarboxylase that is localized in the inner mitochondrial membrane and catalyzes the decarboxylation of PS to phosphatidylethanolamine (PE) in mammalian cells. PE occurs at high abundance in mitochondrial membranes. Patient-derived fibroblasts showed fragmented mitochondrial morphology. Treatment of patient cells with MG-132 or staurosporine to induce activation of the intrinsic apoptosis pathway revealed significantly decreased cell viability with increased caspase-3 and caspase-7 activation. Remarkably, ethanolamine (Etn) supplementation largely restored cell viability and enhanced apoptosis in MG-132-stressed patient cells. Our data demonstrate that the biallelic hypomorphic PISD variant p.(Cys266Tyr) is associated with a novel SEMD form, which may be treatable with Etn administration.

Phenotyping and genotyping of skeletal dysplasias: Evolution of a center and a decade of experience in India.

Genetic heterogeneity, high burden and the paucity of genetic testing for rare diseases challenge genomic healthcare for these disorders in India. Here we report our experience over the past decade, of establishing the genomic evaluation of skeletal dysplasia at a tertiary university hospital in India. Research or clinical genomic testing was carried out by Sanger sequencing and next-generation sequencing. Close national and international collaborations aided phenotyping and genotyping. We report 508 families (557 affected individuals) with the definitive molecular diagnosis of skeletal dysplasia. Dysostoses multiplex (n = 196), genetic inflammatory/rheumatoid-like osteoarthropathies (n = 114) and osteogenesis imperfecta and decreased bone density (n = 58) were the most common diagnoses. We enumerate the processes, clinical diagnoses and causal variants in the cohort with 48 novel variants in 21 genes. We summarize scientific contributions of the center to the description of clinical and mutation profiles and discovery of new phenotypes and genetic etiology. Our study illustrates the establishment and application of genomic testing tools for genetic disorders of skeleton in a large cohort. We believe this could be a model to emulate for other developing genetic centers.

Diagnostic strategies and genotype-phenotype correlation in a large Indian cohort of osteogenesis imperfecta.

Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous disorder. Although differential diagnosis is greatly facilitated by next generation sequencing, its availability can vary considerably. In this study, we compared targeted gene panel or exome sequencing with clinical scoring and grouping in a cohort of 50 OI index patients recruited by a single Indian clinical center in an unselected fashion. In 48 patients we observed a total of 24 novel mutations and 24 known OI mutations, of which several were recurrent. In one patient neither gene panel nor exome sequencing revealed any significant mutation and another patient harbored a class III COL1A1 intronic variant. The percentage of autosomal recessive forms due to mutations in BMP1, FKBP10, LEPRE1, SERPINF1, and WNT1 was unusually high (48%). Grouping according to phenotypic and radiographic features revealed four individuals with Bruck syndrome due to FKBP10 mutations, three patients with hypertrophic callus caused by IFITM5 mutations, and twenty with pronounced bone bowing, of which eight carried WNT1 mutations. There was a clear correlation between genotype and phenotype severity: IFITM5=LEPRE1>WNT1>SERPINF1>COL1A1 (qualitative)>BMP1>FKBP10>COL1A2 (qualitative)>COL1A1 (quantitative)>COL1A2 (quantitative). In one patient we found heterozygous variants in COL1A1 and COL1A2 inherited from parents without an obvious bone phenotype indicating that both variants might contribute to the phenotype. Our findings demonstrate the clinical utility of gene panel testing for OI, but in cases with contractures, hypertrophic callus formation, or - to some extent - extensive bowing single gene analysis might still be more cost-effective.

Additional three patients with Smith-McCort dysplasia due to novel RAB33B mutations.

Smith-McCort dysplasia (SMC OMIM 615222) and Dyggve-Melchior-Clausen dysplasia (DMC OMIM 223800) are allelic skeletal dysplasias caused by homozygous or compound heterozygous mutations in DYM (OMIM 607461). Both disorders share the same skeletal phenotypes characterized by spondylo-epi-metaphyseal dysplasia with distinctive lacy ilia. The difference rests on the presence or absence of intellectual disability, that is, intellectual disability in DMC and normal cognition in SMC. However, genetic heterogeneity was suspected in SMC. Recently, RAB33B (OMIM 605950) has been identified as the second gene for SMC. Nevertheless, only two affected families have been reported so far. Here we present three SMC patients with four novel pathogenic variants in RAB33B, including homozygosity for c.211C>T (p.R71*), homozygosity for c.365T>C (p.F122S), and compound heterozygosity for c.48delCGGGGCAG (p.G17Vfs*58) and c.490C>T (p.Q164*). We also summarize the clinical, radiological, and mutation profile of RAB33B after literature mining. This report ascertains the pathogenic relationship between RAB33B and SMC. © 2017 Wiley Periodicals, Inc.

Metatropic Dysplasia with a Novel Mutation in TRPV4.

BACKGROUND: Metatropic dysplasia is a skeletal dysplasia characterized by rhizomelia, severe kyphoscoliosis and a coccygeal tail. CASE CHARACTERISTICS: A 12 day-old male neonate had facial dysmorphism, short limbs and coccygeal tail and showed radiological features of metatropic dysplasia. OBSERVATION: A novel heterozygous variant was observed in TRPV4 gene. MESSAGE: We report a novel mutation in an Indian neonate with metatropic dysplasia.

Spectrum of SMPD1 mutations in Asian-Indian patients with acid sphingomyelinase (ASM)-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc.

A novel multiple joint dislocation syndrome associated with a homozygous nonsense variant in the EXOC6B gene.

We report two brothers from a consanguineous couple with spondyloepimetaphyseal dysplasia (SEMD), multiple joint dislocations at birth, severe joint laxity, scoliosis, gracile metacarpals and metatarsals, delayed bone age and poorly ossified carpal and tarsal bones, probably representing a yet uncharacterized SEMD with laxity and dislocations. This condition has clinical overlap with autosomal dominantly inherited SEMD with joint laxity, leptodactylic type caused by recurrent missense variants in the kinesin family member 22 gene (KIF22). Single-nucleotide polymorphism array analysis and whole-exome sequencing in the two affected siblings revealed a shared homozygous nonsense variant [c.906T>A/p.(Tyr302*)] in EXOC6B as the most likely cause. EXOC6B encodes a component of the exocyst complex required for tethering secretory vesicles to the plasma membrane. As transport of vesicles from the golgi apparatus to the plasma membrane occurs through kinesin motor proteins along microtubule tracks, the function of EXOC6B is linked to KIF22 suggesting a common pathogenic mechanism in skeletal dysplasias with joint laxity and dislocations.

Clinical and mutation profile of multicentric osteolysis nodulosis and arthropathy.

​Multicentric osteolysis nodulosis and arthropathy (MONA) is an infrequently described autosomal recessive skeletal dysplasia characterized by progressive osteolysis and arthropathy. Inactivating mutations in MMP2, encoding matrix metalloproteinase-2, are known to cause this disorder. Fifteen families with mutations in MMP2 have been reported in literature. In this study we screened thirteen individuals from eleven families for MMP2 mutations and identified eight mutations (five novel and three known variants). We characterize the clinical, radiographic and molecular findings in all individuals with molecularly proven MONA from the present cohort and previous reports, and provide a comprehensive review of the MMP2 related disorders.