Clinical and genetic investigation of 14 families with various forms of short stature syndromes.

Skeletal dysplasias are a heterogeneous group of disorders presenting mild to lethal defects. Several factors, such as genetic, prenatal, and postnatal environmental may contribute to reduced growth. Fourteen families of Pakistani origin, presenting the syndromic form of short stature either in the autosomal recessive or autosomal dominant manner were clinically and genetically investigated to uncover the underlying genetic etiology. Homozygosity mapping, whole exome sequencing, and Sanger sequencing were used to search for the disease-causing gene variants. In total, we have identified 13 sequence variants in 10 different genes. The variants in the HSPG2 and XRCC4 genes were not reported previously in the Pakistani population. This study will expand the mutation spectrum of the identified genes and will help in improved diagnosis of the syndromic form of short stature in the local population.

Sequence Variants in MEGF8 and GJA1 Underlying Syndactyly.

Introduction: Syndactyly is a common congenital limb malformation. It occurs due to embryological failure of digit separation during limb development. Syndactyly often runs in families with an incidence of about one out of every 2,500-3,000 live births. Methods: Here, we have reported two families presenting features of severe forms of syndactyly. The disorder segregated in autosomal recessive in one and in autosomal dominant manner in the second family. Search for the causative variants was carried out using whole-exome sequencing in family A and candidate gene sequencing in family B. Results: Analysis of the sequencing data revealed two novel missense variants, including p.(Cys1925Arg) in MEGF8 in family A and p.(Thr89Ile) in GJA1 in family B. Conclusion: In conclusion, the novel findings, presented here, not only expand the mutation spectrum in the genes MEGF8 and GJA1, but this will also facilitate screening other families carrying similar clinical features in the Pakistani population.

A novel biallelic variant further delineates PRDX3-related autosomal recessive cerebellar ataxia.

Cerebellar ataxias (CAs) comprise a rare group of neurological disorders characterized by extensive phenotypic and genetic heterogeneity. In the last several years, our understanding of the CA etiology has increased significantly and resulted in the discoveries of numerous ataxia-associated genes. Herein, we describe a single affected individual from a consanguineous family segregating a recessive neurodevelopmental disorder. The proband showed features such as global developmental delay, cerebellar atrophy, hypotonia, speech issues, dystonia, and profound hearing impairment. Whole-exome sequencing and Sanger sequencing revealed a biallelic nonsense variant (c.496A > T; p.Lys166*) in the exon 5 of the PRDX3 gene that segregated perfectly within the family. This is the third report that associates the PRDX3 gene variant with cerebellar ataxia. In addition, associated hearing impairment further delineates the PRDX3 associated gene phenotypes.

ADAMTS1, MPDZ, MVD, and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment.

Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.

Further evidence of involvement of TMEM132E in autosomal recessive nonsyndromic hearing impairment.

Autosomal-recessive (AR) nonsyndromic hearing impairment (NSHI) displays a high degree of genetic heterogeneity with >100 genes identified. Recently, TMEM132E, which is highly expressed in inner hair cells, was suggested as a novel ARNSHI gene for DFNB99. A missense variant c.1259G>A: p.(Arg420Gln) in TMEM132E was identified that segregated with ARNSHI in a single Chinese family with two affected members. In the present study, a family of Pakistani origin with prelingual profound sensorineural hearing impairment displaying AR mode of inheritance was investigated via exome and Sanger sequencing. Compound heterozygous variants c.382G>T: p.(Ala128Ser) and c.2204C>T: p.(Pro735Leu) in TMEM132E were observed in affected but not in unaffected family members. TMEM132E variants identified in this and the previously reported ARNSHI family are located in the extracellular domain. In conclusion, we present a second ARNSHI family with TMEM132E variants which strengthens the evidence of the involvement of this gene in the etiology of ARNSHI.

A novel insertion and deletion mutation in the BHLHA9 underlies polydactyly and mesoaxial synostotic syndactyly with phalangeal reduction.

Mesoaxial syndactyly is characterized by fusion of the central digits. The disorder segregates in autosomal recessive pattern and mapped on human chromosome 17p13.3. Homozygous missense mutations in the BHLHA9 have been reported to cause mesoaxial synostotic syndactyly with phalangeal reduction (MSSD). In the present study, we have investigated a family segregating mesoaxial synostotic syndactyly with phalangeal reduction (MSSD) in autosomal recessive manner. Genotyping using microsatellite markers followed by Sanger sequencing revealed a homozygous deletion and insertion mutation (NM_001164405: c.252_270delinsGCA; p.(Phe85Glufs*108)) in the BHLHA9 gene in affected individuals of the family. This study reports the first frameshift mutation in the BHLHA9 causing mesoaxial synostotic syndactyly and phalangeal reduction.

Novel homozygous sequence variants in the GDF5 gene underlie acromesomelic dysplasia type-grebe in consanguineous families.

Acromesomelic dysplasia Grebe type (AMDG) is characterized by severe knob like non-functional fingers and short acromesomelic limbs, and is inherited in an autosomal recessive manner. Disease causing sequence variants in the GDF5 (Growth Differentiation Factor 5) gene located on chromosome 20q11.22 are responsible for causing AMDG. In the study, presented here, two consanguineous families with AMDG were clinically and genetically characterized. After establishing linkage in the two families (A and B) to GDF5 gene on chromosome 20q11.22, Sanger DNA sequencing was performed in all available affected and unaffected members. Sequence analysis of the GDF5 gene revealed two novel variants including a duplication (c.157_158dupC, p.Leu53Profs*41) in family A, and a nonsense (p.Trp291*) in family B. Our findings extend the body of evidence that supports the importance of GDF5 in the development of limbs.

Mitral regurgitation as a phenotypic manifestation of nonphotosensitive trichothiodystrophy due to a splice variant in MPLKIP.

BACKGROUND: Nonphotosensitive trichothiodystrophy (TTDN) is a rare autosomal recessive disorder of neuroectodermal origin. The condition is marked by hair abnormalities, intellectual impairment, nail dystrophies and susceptibility to infections but with no UV sensitivity. METHODS: We identified three consanguineous Pakistani families with varied TTDN features and used homozygosity mapping, linkage analysis, and Sanger and exome sequencing in order to identify pathogenic variants. Haplotype analysis was performed and haplotype age estimated. A splicing assay was used to validate the effect of the MPLKIP splice variant on expression. RESULTS: Affected individuals from all families exhibit several TTDN features along with a heart-specific feature, i.e. mitral regurgitation. Exome sequencing in the probands from families ED168 and ED241 identified a homozygous splice mutation c.339 + 1G > A within MPLKIP. The same splice variant co-segregates with TTDN in a third family ED210. The MPLKIP splice variant was not found in public databases, e.g. the Exome Aggregation Consortium, and in unrelated Pakistani controls. Functional analysis of the splice variant confirmed intron retention, which leads to protein truncation and loss of a phosphorylation site. Haplotype analysis identified a 585.1-kb haplotype which includes the MPLKIP variant, supporting the existence of a founder haplotype that is estimated to be 25,900 years old. CONCLUSION: This study extends the allelic and phenotypic spectra of MPLKIP-related TTDN, to include a splice variant that causes cardiomyopathy as part of the TTDN phenotype.

In silico analysis of missense mutations in LPAR6 reveals abnormal phospholipid signaling pathway leading to hypotrichosis.

Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development.

Novel mutations in natriuretic peptide receptor-2 gene underlie acromesomelic dysplasia, type maroteaux.

BACKGROUND: Natriuretic peptides (NPs) are peptide hormones that exert their biological actions by binding to three types of cell surface natriuretic peptide receptors (NPRs). The receptor NPR-B binding C-type natriuretic peptide (CNP) acts locally as a paracrine and/or autocrine regulator in a wide variety of tissues. Mutations in the gene NPR2 have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), an autosomal recessive skeletal disproportionate dwarfism disorder in humans. METHODS: In the study, presented here, genotyping of six consanguineous families of Pakistani origin with AMDM was carried out using polymorphic microsatellite markers, which are closely linked to the gene NPR2 on chromosome 9p21-p12. To screen for mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected and unaffected individuals of the families and sequenced. RESULTS: Sequence analysis of the gene NPR2 identified a novel missence mutation (p.T907M) in five families, and a splice donor site mutation c.2986 + 2 T > G in the other family. CONCLUSION: We have described two novel mutations in the gene NPR2. The presence of the same mutation (p.T907M) and haplotype in five families (A, B, C, D, E) is suggestive of a founder effect.