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The phenotypic spectrum associated with heterozygous mutations in cartilage oligomeric matrix protein gene (COMP) range from a mild form of multiple epiphyseal dysplasia (MED) to pseudoachondroplasia (PSACH). However, the phenotypic effect from biallelic COMP variants is unclear. We investigated a large consanguineous Pakistani family with a severe form of PSACH in 2 individuals. Another 14 family members presented with a mild PSACH phenotype similar to MED. Using exome sequencing and subsequent segregation analysis, we identified homozygosity for a COMP missense variant [c.1423G>A; p.(D475N)] in the 2 severely affected individuals, whereas family members with the mild PSACH phenotype were heterozygous. Our observations show for the first time that a biallelic COMP variant may be associated with pronounced and widespread skeletal malformations suggesting an additive effect of the 2 mutated alleles., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Spinocerebellar ataxias comprise a large and heterogeneous group of disorders that may present with isolated ataxia, or ataxia in combination with other neurologic or non-neurologic symptoms. Monoallelic or biallelic GRID2 mutations were recently reported in rare cases with cerebellar syndrome and variable degree of ataxia, ocular symptoms, hypotonia and developmental delay., Case Presentation: We report on a consanguineous family with autosomal recessive childhood onset of slowly progressive cerebellar ataxia and delayed psychomotor development in three siblings. MRI of an adult and affected family member revealed slightly widened cerebral and cerebellar sulci, suggesting generalized brain atrophy, and mild cerebellar atrophy. Using whole exome sequencing we identified a novel homozygous missense variant [c.2128C > T, p.(Arg710Trp)] in GRID2 that segregates with the disease. The missense variant is located in a conserved region encoding the extracellular serine-binding domain of the GluD2 protein and predicts a change in conformation of the protein., Conclusion: The widespread supratentorial brain abnormalities, absence of oculomotor symptoms, increased peripheral muscle tone and the novel missense mutation add to the clinical and genetic variability in GRID2 associated cerebellar syndrome. The neuroradiological findings in our family indicate a generalized neurodegenerative process to be taken into account in other families segregating complex clinical features and GRID2 mutations.

Spinocerebellar ataxias (SCA) comprise a heterogeneous group of inherited neurological disorders characterized by a range of symptoms from both cerebellar and extra cerebellar structures. We investigated the cause of autosomal recessive, congenital SCA in six affected family members from a large consanguineous family. Using whole-exome sequencing, we identified a homozygous ITPR1 missense variant [c.5360T>C; p.(L1787P)] segregating in all affected individuals. Heterozygous carriers were asymptomatic despite cerebellar hypoplasia. Variants in the ITPTR1 gene have previously been associated exclusively with autosomal dominant SCA15 and SCA29 with slow or no progression. The L1787 residue is highly conserved and the leucine to proline substitution has a predicted destabilizing effect on the protein structure. Additionally, the L1787P variant is located in a domain separated from previously described and dominant-acting missense variants consistent with a distinct effect on IP3R1 tetramer structure and function. Taken together, we show for the first time that a biallelic ITPR1 missense variant may cause an autosomal recessive and infantile onset SCA29, albeit with subclinical cerebellar hypoplasia in carriers. Our findings add to the genetic complexity of SCA29 and broaden the correlations between ITPR1 variants and their clinical expression.

Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a rare congenital disorder, in which heterozygous missense variants in the Enteric Smooth Muscle actin γ-2 (ACTG2) gene have been recently identified. To investigate the mechanism by which ACTG2 variants lead to MMIHS, we screened a cohort of eleven MMIHS patients, eight sporadic and three familial cases, and performed immunohistochemistry, molecular modeling and molecular dynamics (MD) simulations, and in vitro assays. In all sporadic cases, a heterozygous missense variant in ACTG2 was identified. ACTG2 expression was detected in all intestinal layers where smooth muscle cells are present in different stages of human development. No histopathological abnormalities were found in the patients. Using molecular modeling and MD simulations, we predicted that ACTG2 variants lead to significant changes to the protein function. This was confirmed by in vitro studies, which showed that the identified variants not only impair ACTG2 polymerization, but also contribute to reduced cell contractility. Taken together, our results confirm the involvement of ACTG2 in sporadic MMIHS, and bring new insights to MMIHS pathogenesis., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Familial visceral myopathy (FVM) is a rare heritable and heterogeneous condition due to impaired smooth muscle function. We identified a family segregating 11 individuals with a spectrum of visceral symptoms involving the small intestine, colon, biliary tract, urinary tract and uterus. Whole-exome sequencing revealed a novel heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in ACTG2, encoding smooth muscle actin γ-2, in affected family members. Variants in ACTG2 were recently identified in FVM with intestinal pseudo-obstruction as well as with the congenital megacystics-microcolon-intestinal hypoperistalsis syndrome. In our family, eight affected members presented with severe complications from the biliary and/or the urinary tracts in addition to gastrointestinal pseudo-obstructions. Furthermore, all affected mothers had a history of assisted deliveries owing to poor progress during labor and weak uterine contractions. The variable involvement of multiple smooth muscle-dependent organs in our family, including the biliary tract and the uterus, add to the phenotypic spectrum associated with ACTG2 missense variants.

Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of disorders characterized by progressive spasticity and weakness of the lower limbs. Autosomal dominant and 'pure' forms of HSP account for ∼80% of cases in Western societies of whom 10% carry atlastin-1 (ATL1) gene mutations. We report on a large consanguineous family segregating six members with early onset HSP. The pedigree was compatible with both autosomal dominant and autosomal recessive inheritance. Whole-exome sequencing and segregation analysis revealed a homozygous novel missense variant c.353G>A, p.(Arg118Gln) in ATL1 in all six affected family members. Seven heterozygous carriers, five females and two males, showed no clinical signs of HSP with the exception of sub-clinically reduced vibration sensation in one adult female. Our combined findings show that homozygosity for the ATL1 missense variant remains the only plausible cause of HSP, whereas heterozygous carriers are asymptomatic. This apparent autosomal recessive inheritance adds to the clinical complexity of spastic paraplegia 3A and calls for caution using directed genetic screening in HSP.

Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T>C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.

Cenani-Lenz syndrome (CLS) is a rare autosomal recessive developmental disorder of the limbs. The disorder is characterized by complete syndactyly with metacarpal fusions and/or oligodactyly sometimes accompanied by radioulnar synostosis. The clinical expression is variable and kidney agenesis/hypoplasia, craniofacial dysmorphism and teeth abnormalities are frequent features as well as lower limb involvement. CLS was recently associated with mutations in the low-density lipoprotein receptor-related protein 4 (LRP4) gene and dysregulated canonical WNT signaling. We have identified a large consanguineous Pakistani pedigree with 9 members affected by CLS. The affected individuals present with a consistent expression of the syndrome restricted to the limbs and kidneys. Symptoms from the lower limb are mild or absent and there were no radioulnar synostosis or craniofacial involvement. Genetic analysis using autozygosity mapping and sequencing revealed homozygosity for a novel missense mutation c.2858T > C (p.L953P) in the LRP4 gene. The mutation is located in a region encoding the highly conserved low-density lipoprotein receptor repeat class B domain of LRP4. Our findings add to the genotype-phenotype correlations in CLS and support kidney anomalies as a frequent associated feature., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Background: Anonychia/hyponychia congenita is a rare autosomal recessive developmental disorder characterized by the absence (anonychia) or hypoplasia (hyponuchia) of finger- and/or toenails frequently caused by mutations in the R-spondin 4 (RSPO4) gene., Methods: Three hypo/anonychia consanguineous Pakistani families were ascertained and genotyped using microsatellite markers spanning the RSPO4 locus on chromosome 20p13. Mutation screening of the RSPO4 gene was carried out by direct sequencing of the entire coding region and all intron-exon boundaries., Results: Mutations in the RSPO4 gene were identified in all families including a novel missense mutation c.178C>T (p.R60W) and two recurrent variants c.353G>A (p.C118Y) and c.3G>A (p.M1I). The c.3G>A variant was identified in unaffected family members and a control sample in a homozygous state., Conclusions: This study raises to 17 the number of known RSPO4 mutations and further expands the molecular repertoire causing hypo/anonychia. The c.353G>A emerges as a recurrent change with a possible founder effect in the Pakistani population. Our findings suggest that c.3G>A is not sufficient to cause the disorder and could be considered a polymorphism.

A Mutations in the gene encoding the ABCA12 protein are associated with different subtypes of autosomal recessive congenital ichthyosis (ARCI), including Harlequin ichthyosis (HI), lamellar ichthyosis (LI) and non-bullous congenital ichthyosiform erythroderma (NCIE). Disruption of ABCA12 lead to perturbed lipid transport in lamellar granules and a defective intercellular lipid layer of the stratum corneum. We have identified a large consanguineous Pakistani family affected by NCIE. Autozygosity mapping showed that affected individuals are homozygous for the ABCA12 gene region. Subsequent mutation screening revealed a homozygous c.4676G>T transition in all five affected family members. The mutation results in a novel p.G1559V substitution within the first nucleotide binding domain of ABCA12. The combined results support that an ABCA12 missense mutation, despite its location in a functional domain, may be associated with a mild ichthyosis phenotype. Furthermore, our findings increase the mutational spectrum in ABCA12 associated with ARCI of diagnostic and prognostic importance.

Heterozygous mutations in the ribosomal protein S19 (RPS19) gene are associated with Diamond-Blackfan anemia (DBA). The mechanism by which RPS19 mediates anemia are still unclear, as well as the regulation of RPS19 expression. We show herein that RPS19 binds specifically to the 5' untranslated region of its own mRNA with an equilibrium binding constant (K(D)) of 4.1+/-1.9 nM. We investigated the mRNA binding properties of two mutant RPS19 proteins (W52R and R62W) identified in DBA patients. We observed a significant increase in K(D) for both proteins (16.1+/-2.1 and 14.5+/-4.9 nM, respectively), indicating a reduced RNA binding capability (p<0.05). We suggest that the binding of RPS19 to its mRNA has a regulatory function and hypothesize that the weaker RNA binding of mutant rRPS19 may have implications for the pathophysiological mechanisms in DBA., (Copyright 2010 Elsevier Inc. All rights reserved.)

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Wnt signalling is one of a few pathways that are crucial for controlling genetic programs during embryonic development as well as in adult tissues. WNT10A is expressed in the skin and epidermis and it has shown to be critical for the development of ectodermal appendages. A nonsense mutation in WNT10A was recently identified in odonto-onycho-dermal dysplasia (OODD; MIM 257980), a rare syndrome characterised by severe hypodontia, nail dystrophy, smooth tongue, dry skin, keratoderma and hyperhydrosis of palms and soles. We identified a large consanguineous Pakistani pedigree comprising six individuals affected by a complete OODD syndrome. Autozygosity mapping using SNP array analysis showed that the affected individuals are homozygous for the WNT10A gene region. Subsequent mutation screening showed a homozygous c.392C>T transition in exon 3 of WNT10A, which predicts a p.A131V substitution in a conserved alpha-helix domain. We report here on the first inherited missense mutation in WNT10A with associated ectodermal features.

Isolated hypodontia, or congenital absence of one to six permanent teeth (OMIM 300606), is a common condition that affects about 20% of individuals worldwide. We identified two extended Pakistani pedigrees segregating X-linked hypodontia with variable expressivity. Affected males show no other associated anomalies, and obligate carrier females have normal dentition. We analyzed the families with polymorphic markers in the ectodysplasin A (EDA) gene region and obtained significant linkage to the phenotype in each pedigree (Z(max) 3.29 and 2.65, respectively, at theta = 0.00). Sequence analysis of the coding regions of EDA revealed a novel missense mutation c.1091T>C resulting in a methionine to threonine substitution (p.M364T) in the tumor necrosis factor (TNF) homology domain. Met364 is a highly conserved residue located on the outer surface of the EDA protein. From our findings, we suggest that the mutation disturbs but does not destroy the EDA structure, resulting in the partial and unusually mild ED phenotype restricted to hypodontia.

Aplasia of lacrimal and salivary glands (ALSG) is an autosomal dominant congenital anomaly characterized by aplasia, atresia or hypoplasia of the lacrimal and salivary systems. Affected individuals present with irritable eyes and dryness of the mouth with variable expressivity. Mutations in FGF10 were recently described in ALSG and in lacrimo-auriculo-dento-digital (LADD) syndrome which are overlapping clinical entities. We present here two families with ALSG associated with missense mutations (R80S and G138E, respectively) affecting highly conserved residues in FGF10. The clinical features of these patients further broaden the knowledge of FGF10-related phenotypes.