Biallelic loss of function variants in FUZ result in an orofaciodigital syndrome.

Orofaciodigital syndrome is a distinctive subtype of skeletal ciliopathies. Disease-causing variants in the genes encoding the CPLANE complex result in a wide variety of skeletal dysplasia with disturbed ciliary functions. The phenotypic spectrum includes orofaciodigital syndrome and short rib polydactyly syndrome. FUZ, as a part of the CPLANE complex, is involved in intraflagellar vesicular trafficking within primary cilia. Previously, the variants, c.98_111+9del and c.851G>T in FUZ were identified in two individuals with a skeletal ciliopathy, manifesting digital anomalies (polydactyly, syndactyly), orofacial cleft, short ribs and cardiac defects. Here, we present two novel variants, c.601G>A and c.625_636del in biallelic state, in two additional subjects exhibiting phenotypic overlap with the previously reported cases. Our findings underscore the association between biallelic loss of function variants in FUZ and skeletal ciliopathy akin to orofaciodigital syndrome.

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.

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.

Early and severe tricuspid valve dysplasia in a fetus with cardiospondylocarpofacial syndrome due to a variant c.616T>G p.(Tyr206Asp) in MAP3K7.

Cardiospondylocarpofacial syndrome (CSCF; MIM#157800) is a rare condition caused by monoallelic variants in the MAP3K7 gene. The characteristic features of CSCF include growth retardation, facial dysmorphism, carpal-tarsal fusion, dorsal spine synostosis, deafness, inner ear malformation, cardiac septal defect and valve dysplasia. We present here a 20-week-old fetus with cardiospondylocarpofacial syndrome arising from a de novo variant c.616T>G p.(Tyr206Asp) in the MAP3K7 (NM_145331.3) gene with early and severe tricuspid valve dysplasia as a prenatal manifestation. Fetal echocardiography revealed tricuspid regurgitation with valve prolapse. Fetus had facial dysmorphism and dilated right atrium and right ventricle with tricuspid valve dysplasia on perinatal evaluation. To the best of our knowledge, this is the first report mentioning the prenatal manifestation of cardiospondylocarpofacial syndrome.

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'.

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.

Steel syndrome: Report of three patients, including monozygotic twins and review of clinical and mutation profiles.

Steel syndrome (MIM# 615155) is an autosomal recessive skeletal disorder, characterized by dislocations of the hips and radial heads, carpal coalition, short stature, facial dysmorphism, and scoliosis. Until date 47 patients have been reported. However, disease causing variants have been identified only in twenty Puerto Rican and nine non-Puerto Rican families. Here we report two monozygotic twins and a boy from two families with novel missense variants, c.295G > A p.(Ala99 Thr), c.3056C > A p.(Pro1019His) and c.2521G > A p.(Gly841Arg) in COL27A1. We describe for the first time, cleft palate and delayed carpal bone ossification as features of Steel syndrome. We reviewed clinical features in all mutation-proven Steel syndrome patients. Short stature and dislocation/subluxation of hip joint are consistently observed. Other features include dislocated radial heads, scoliosis, lordosis, carpal coalition, facial dysmorphism, hearing loss, bilateral fifth finger clinodactyly, knee deformities and developmental delay. Seven missense variants and eight null variants are reported in COL27A1 until date. We also looked into the genotype-phenotype correlation in Puerto Rican and non-Puerto Rican patients.

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.

Pseudoachondroplasia: Phenotype and genotype in 11 Indian patients.

Pseudoachondroplasia (PSACH) is an autosomal dominant disorder characterized by rhizomelic short-limbed skeletal dysplasia. The primary clinical and radiographic features include disproportionate dwarfism, joint laxity and hyperextensibility, exaggerated lumbar lordosis, and late ossification of the epiphyses. Identification of disease-causing variants in heterozygous state in COMP establishes the molecular diagnosis of PSACH. We examined 11 families with clinical features suggestive of PSACH. In nine families, we used Sanger sequencing of exons 8-19 of COMP (NM_000095.2) and in two families exome sequencing was used for confirming the diagnosis. We identified 10 de novo variants, including five known variants (c.925G>A, c.976G>A, c.1201G>T, c.1417_1419del, and c.1511G>A) and five variants (c.874T>C, c.1201G>C, c.1309G>A, c.1416_1421delCGACAA, and c.1445A>T) which are not reported outside Indian ethnicity. We hereby report the largest series of individuals with molecular diagnosis of PSACH from India and reiterate the well-known genotype-phenotype corelation in PSACH.

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.

Mutation Spectrum of Tuberous Sclerosis Complex Patients in Indian Population.

Tuberous sclerosis complex (TSC) is a multiorgan disorder characterized by formation of hamartomas and broad phenotypic spectrum including seizures, mental retardation, renal dysfunction, skin manifestations and brain tubers. It is inherited in an autosomal dominant pattern, caused due to mutation in either TSC1 or TSC2 genes. Seizures are one of the major presenting symptoms of TSC that helps in early diagnosis. The present study describes the mutation spectrum in TSC1 and TSC2 genes in TSC patients and their association with neurocognitive-behavioral phenotypes. Ninety-eight TSC patients were enrolled for TSC genetic testing after detailed clinical and neurobehavioral assessment. Large genomic rearrangement testing was performed by multiplex ligation-dependent probe amplification (MLPA) technique for all cases and Sanger sequencing was performed for MLPA negative cases. Large rearrangements were identified in approximately 1% in TSC1 and 14.3% in TSC2 genes. The present study observed the presence of duplications in two (2%) cases, both involving TSC2/PKD1 contiguous genes which to the best of our knowledge is reported for the first time. 8.1% of small variants were identified in the TSC1 gene and 85.7% in TSC2 gene, out of which 23 were novel variations and no variants were found in six (6.1%) cases. This study provides a representative picture of the distribution of variants in the TSC1 and TSC2 genes in Indian population along with the detailed assessment of neurological symptoms. This is the largest cohort study from India providing an overview of comprehensive clinical and molecular spectrum.

Biallelic deep intronic variant c.5457+81T>A in TRIP11 causes loss of function and results in achondrogenesis 1A.

Biallelic loss of function variants in TRIP11 encoding for the Golgi microtubule-associated protein 210 (GMAP-210) causes the lethal chondrodysplasia achondrogenesis type 1A (ACG1A). Loss of TRIP11 activity has been shown to impair Golgi structure, vesicular transport, and results in loss of IFT20 anchorage to the Golgi that is vital for ciliary trafficking and ciliogenesis. Here, we report four fetuses, two each from two families, who were ascertained antenatally with ACG1A. Affected fetuses in both families are homozygous for the deep intronic TRIP11 variant, c.5457+81T>A, which was found in a shared region of homozygosity. This variant was found to cause aberrant transcript splicing and the retention of 77 base pairs of intron 18. The TRIP11 messenger RNA and protein levels were drastically reduced in fibroblast cells derived from one of the affected fetuses. Using immunofluorescence we also detected highly compacted Golgi apparatus in affected fibroblasts. Further, we observed a significant reduction in the frequency of ciliated cells and in the length of primary cilia in subject-derived cell lines, not reported so far in patient cells with TRIP11 null or hypomorphic variants. Our findings illustrate how pathogenic variants in intronic regions of TRIP11 can impact transcript splicing, expression, and activity, resulting in ACG1A.

High diagnostic yield in skeletal ciliopathies using massively parallel genome sequencing, structural variant screening and RNA analyses.

Skeletal ciliopathies are a heterogenous group of disorders with overlapping clinical and radiographic features including bone dysplasia and internal abnormalities. To date, pathogenic variants in at least 30 genes, coding for different structural cilia proteins, are reported to cause skeletal ciliopathies. Here, we summarize genetic and phenotypic features of 34 affected individuals from 29 families with skeletal ciliopathies. Molecular diagnostic testing was performed using massively parallel sequencing (MPS) in combination with copy number variant (CNV) analyses and in silico filtering for variants in known skeletal ciliopathy genes. We identified biallelic disease-causing variants in seven genes: DYNC2H1, KIAA0753, WDR19, C2CD3, TTC21B, EVC, and EVC2. Four variants located in non-canonical splice sites of DYNC2H1, EVC, and KIAA0753 led to aberrant splicing that was shown by sequencing of cDNA. Furthermore, CNV analyses showed an intragenic deletion of DYNC2H1 in one individual and a 6.7 Mb de novo deletion on chromosome 1q24q25 in another. In five unsolved cases, MPS was performed in family setting. In one proband we identified a de novo variant in PRKACA and in another we found a homozygous intragenic deletion of IFT74, removing the first coding exon and leading to expression of a shorter message predicted to result in loss of 40 amino acids at the N-terminus. These findings establish IFT74 as a new skeletal ciliopathy gene. In conclusion, combined single nucleotide variant, CNV and cDNA analyses lead to a high yield of genetic diagnoses (90%) in a cohort of patients with skeletal ciliopathies.

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.

Hedgehog acyl-transferase-related multiple congenital anomalies: Report of an additional family and delineation of the syndrome.

This study includes previous reports of four affected individuals from two unrelated families with hedgehog acyl-transferase (HHAT)-related multiple congenital anomaly syndrome. Microcephaly, small cerebellar vermis, holoprosencephaly, agenesis of corpus callosum, intellectual disability, short stature, skeletal dysplasia, microphthalmia-anophthalmia, and sex reversal constitute the phenotypic spectrum of this condition with variable expression. We report an additional family with three affected conceptuses: two abortuses and one living proband. We did proband-parents trio exome sequencing and identified a biallelic in-frame deletion c.365_367del; (p.Thr122del) in exon 5 of HHAT. With this report, we delineate the phenotype and allelic heterogeneity of the HHAT-related multiple congenital anomaly syndrome.

Clinically relevant variants in a large cohort of Indian patients with Marfan syndrome and related disorders identified by next-generation sequencing.

Marfan syndrome and related disorders are a group of heritable connective tissue disorders and share many clinical features that involve cardiovascular, skeletal, craniofacial, ocular, and cutaneous abnormalities. The majority of affected individuals have aortopathies associated with early mortality and morbidity. Implementation of targeted gene panel next-generation sequencing in these individuals is a powerful tool to obtain a genetic diagnosis. Here, we report on clinical and genetic spectrum of 53 families from India with a total of 83 patients who had a clinical diagnosis suggestive of Marfan syndrome or related disorders. We obtained a molecular diagnosis in 45/53 (85%) index patients, in which 36/53 (68%) had rare variants in FBN1 (Marfan syndrome; 63 patients in total), seven (13.3%) in TGFBR1/TGFBR2 (Loeys-Dietz syndrome; nine patients in total) and two patients (3.7%) in SKI (Shprintzen-Goldberg syndrome). 21 of 41 rare variants (51.2%) were novel. We did not detect a disease-associated variant in 8 (15%) index patients, and none of them met the Ghent Marfan diagnostic criteria. We found the homozygous FBN1 variant p.(Arg954His) in a boy with typical features of Marfan syndrome. Our study is the first reporting on the spectrum of variants in FBN1, TGFBR1, TGFBR2, and SKI in Indian individuals.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

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.