Asmat Ullah

A novel deletion mutation in the DSG4 gene underlies autosomal recessive hypotrichosis with variable phenotype in two unrelated consanguineous families.

Autosomal recessive hypotrichosis is a rare human hereditary disorder presenting as sparse scalp hair or as woolly hair occurring on various parts of the body. Various forms of isolated hypotrichosis have been reported to date. Mutations in at least 11 genes have been reported to cause hypotrichosis.

A novel missense variant in the PNPLA1 gene underlies congenital ichthyosis in three consanguineous families

detected serpentine vessels which were similar to the previously reported spermatozoa-like vessels, except for the lacking of a dotted vessel at one of their extremities (which is typical of the latter). Additionally, we observed other less common features, including further subtypes of curved vessels (convoluted and chalice-shaped), linear irregular blurry vessels and dotted vessels. Interestingly, most of the curved vessels were fairly focused; this is likely due to the epidermal thinning commonly present in ZPCB, which makes dermal vessels closer to the surface and, consequently, more sharp and bright. According to our findings, the detection of focal/diffuse orange-yellowish structureless areas and fairly focused curved vessels (including serpentine, convoluted and chalice-shaped) might assist the clinical diagnosis of ZPCB, distinguishing it from its main differential diagnoses, namely erythroplasia of Queyrat, which has been reported to show scattered glomerular vessels, psoriasis, typically displaying regular dotted/glomerular vessels, and seborrheic dermatitis and non-specific balanoposthitis, usually showing linear irregular unspecific blurry vessels (EE, GS, personal observations).

A Novel Heterozygous Intragenic Sequence Variant in DLX6 Probably Underlies First Case of Autosomal Dominant Split-Hand/Foot Malformation Type 1

Split-hand and foot malformation (SHFM; MIM 183600) is a rare human genetic limb malformation. It is characterized by missing digital rays in the hands and feet. SHFMs vary in severity from mild abnormalities affecting a single limb to acute malformations involving all 4 limbs. It is inherited, as part of both a syndromic and nonsyndromic disorder, in an autosomal recessive, autosomal dominant, and X-linked patterns. So far, 9 loci of hand and foot malformation have been mapped on human chromosomes. The present study describes a family with 2 affected individuals segregating SHFM in an autosomal dominant fashion. Sanger sequencing of the genes involved in SHFM was performed to identify the disease-causing variant. Sequence analysis revealed the first heterozygous missense variant (c.632T>A, p.Val211Glu) in the distal-less homeobox 6 (DLX6) gene, located in chromosome 7q21, causing SHFM in the present family. This study supports the evidence of DLX6 as an SHFM-causing gene.

GLI1 inactivation is associated with developmental phenotypes overlapping with Ellis-van Creveld syndrome

GLI1, GLI2 and GLI3 form a family of transcription factors which regulate development by mediating the action of Hedgehog (Hh) morphogens. Accordingly, inactivating variants in GLI2 and GLI3 are found in several developmental disorders. In contrast, loss-of-function mutations in GLI1 have remained elusive, maintaining enigmatic the role of this gene in the human embryo. We describe eight patients from three independent families having biallelic truncating variants in GLI1 and developmental defects overlapping with Ellis-van Creveld syndrome (EvC), a disease caused by diminished Hh signaling. Two families had mutations in the last exon of the gene and a third family was identified with an N-terminal stop gain variant predicted to be degraded by the NMD-pathway. Analysis of fibroblasts from one of the patients with homozygous C-terminal truncation of GLI1 demonstrated that the corresponding mutant GLI1 protein is fabricated by patient cells and becomes upregulated in response to Hh signaling. However, the transcriptional activity of the truncated GLI1 factor was found to be severely impaired by cell culture and in vivo assays, indicating that the balance between GLI repressors and activators is altered in affected subjects. Consistent with this, reduced expression of the GLI target PTCH1 was observed in patient fibroblasts after chemical induction of the Hh pathway. We conclude that GLI1 inactivation is associated with a phenotypic spectrum extending from isolated postaxial polydactyly to an EvC-like condition.

Novel heterozygous frameshift mutation in distal‐less homeobox 5 underlies isolated split hand/foot malformation type 1

Split hand/foot malformation (SHFM), also known as ectrodactyly, is a genetically heterogeneous group of limb malformations involving the central rays of the autopod with deep median clefts in the hands and feet, fusion of the digits, and aplasia/hypoplasia of phalanges and metacarpal/metatarsal bones. In most of the reported cases, SHFM is an isolated malformation, but in a few cases other anomalies such as intellectual disability and hearing loss have been found to be associated with SHFM. To date, seven loci for SHFM (SHFM1–6; split-hand/foot malformation with long bone deficiency-3 [SHFLD3]) have been mapped on different human chromosomes. For these loci, six genes have been identified. In most cases, SHFM1 is inherited in an autosomal dominant fashion, but an autosomal recessive form of SHFM1 caused by a homozygous missense mutation in distalless homeobox 5 (DLX5) has also been reported. Intra-familial and inter-familial variations of clinical features with reduced penetrance, variable expression and sex-related segregation distortion have been found in patients with autosomal dominant SHFM1. In this report, we investigated a large family of Pakistani origin with four affected individuals with SHFM (Fig. 1a). Clinical features of these members are presented in Figure 1(b–h). The study was carried out according to the protocol approved by the Institutional Review Board (IRB) of the Quaid-i-Azam University, Islamabad, Pakistan. All participants or their legal guardians provided written informed consent. Genomic DNA was extracted from lymphocytes. DLX5, mapped on chromosome 7q21.2–21.3, was previously reported as the causative factor for the SHFM type 1 phenotype. Therefore the same gene was sequenced using gene-specific primers in all affected and unaffected members of the family. Sequence analysis identified a heterozygous 4 bp duplication at nucleotide position 482 (c.482_485dupACCT, p.Ala163Profs*55) in exon 2 of DLX5 in affected members (Fig. 1i–k). The sequence variant was associated with disease phenotype in the family. Non-pathogenic nature of the variant was excluded by sequencing 200 ethnically matched individuals. Previously, one homozygous variant (c.533 A>C, p.Gln178Pro) and two heterozygous variants (c.558G>T, p.Gln186His; c.G115 T, p.Glu39*) in DLX5 causing SHFM type 1 have been reported. In addition, four chromosomal aberrations including a deletion, duplication, translocation and an inversion at chromosome 7q21.2–21.3, encompassing DLX5 and DLX6 and their enhancer sequences, were reported to cause SHFM type 1 in patients with an autosomal dominant pattern of inheritance. DLX5, homologous to Drosophila distal-less, is a member of the DLX family, and is expressed in most developing skeletal elements. DLX5, encoding 289 amino acid proteins (homeobox DLX-5, UniProtKB/Swiss-Prot accession Q53Y73), spans 24.71 kb of genomic DNA on chromosome 7q21.3. Homeobox protein DLX-5 is a transcriptional factor with a 60 amino acids DNA-binding domain (137–196 amino acids) involved in patterning the embryonic anterior–posterior body axis. This protein acts as an immediate early bone morphogenetic protein-responsive transcriptional activator essential for osteoblast differentiation. The two missense mutations (p.Gln178Pro,p.Gln186His) reported previously, are located in the homeodomain affecting highly conserved amino acid residues. The p.Gln186His variant results in reduction of the level of DLX5 transcript. The frameshift mutation (p.Ala163Profs*55) in DLX5, found in the present study, is also localized in the homeodomain of DLX5. Change of codon CTC to CTA by the 4 bp duplication (c.482_485dupACCT) specifies the same amino acid, thus the first amino acid at the position of duplication remains unchanged and frameshift starts at a later amino acid (p.Ala163Profs*55). The mutation is predicted to affect gene function by altering cellular localization, spatial structure of the protein or the recognition site in the target genes. DLX5/6 play important role in embryonic development due to their expression in the median apical ectodermal ridge (AER) of embryonic limb buds. In the mouse, double knockout of DLX5/6 causes SHFM, while knocking out DLX5/6 in either a single or double heterozygous manner failed to result in SHFM. Humans heterozygous for DLX5/6 are more sensitive than the mouse with the same genotype, due to the dosage effect. On the basis of the aforementioned reports, it is clear that the heterozygous mutation identified in DLX5, in the present family, is the causative variant. Correspondence: Wasim Ahmad PhD, Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan. Email: wahmad@qau.edu.pk Received 18 January 2016; revision received 19 March 2016; accepted 4 April 2016.

First direct evidence of involvement of a homozygous loss-of-function variant in the EPS15L1 gene underlying split-hand/split-foot malformation

Split‐hand/split‐foot malformation (SHFM) is a severe form of congenital limb deformity characterized by the absence of 1 or more digits and/or variable degree of median clefts of hands and feet. The present study describes an investigation of a consanguineous family of Pakistani origin segregating SHFM in an autosomal recessive manner. Human genome scan using SNP markers followed by whole exome sequencing revealed a frameshift deletion (c.409delA, p.Ser137Alafs*19) in the EPS15L1 gene located on chromosome 19p13.11. This is the first biallelic variant identified in the EPS15L1 gene underlying SHFM. Our findings report the first direct involvement of EPS15L1 gene in the development of human limbs.

Exome sequencing reveals a novel homozygous splice site variant in the WNT1 gene underlying osteogenesis imperfecta type 3

BackgroundOsteogenesis imperfecta (OI) is a heritable bone fragility disorder usually caused by dominant variants in COL1A1 or COL1A2 genes. Over the last few years, 17 genes including 12 autosomal recessive and five autosomal dominant forms of OI, involved in various aspects of bone formation, have been identified.MethodsWhole-exome sequencing followed by conventional Sanger sequencing was performed in a single affected individual (IV-3) in a family.ResultsHere, we report the clinical and genetic characterization of OI type 3 in a consanguineous family with four affected members. Clinical examinations revealed low bone density, short stature, severe vertebral compression fractures, and multiple long bone fractures in the affected members. Exome sequencing revealed a biallelic pathogenic splice acceptor site variant (c.359-3C>G) in a wingless-type mouse mammary tumor virus integration site family 1 (WNT1) gene located on chromosome 12q13.12.ConclusionWe report a biallelic splice site variant underlying OI type 3 and the first case from the Pakistani population.

Exome Sequencing Revealed a Novel Splice Site Variant in the ALX1 Gene Underlying Frontonasal Dysplasia

Frontonasal dysplasia (FND) is a heterogeneous group of disorders characterized by hypertelorism, telecanthus, broad nasal root, wide prominent nasal bridge, short and wide nasal ridge, broad columella and smooth philtrum. To date one X‐linked and three autosomal recessive forms of FND have been reported in different ethnic groups. We sought to identify the gene responsible for FND in a consanguineous Pakistani family segregating the disorder in autosomal recessive pattern. Genome‐wide homozygosity mapping using 250KNsp array revealed five homozygous regions in the selected affected individuals. Exome sequencing found a novel splice acceptor site variant (c.661‐1G>C: NM_006982.2) in ALX1. Sanger sequencing confirmed the correct segregation of the pathogenic variant in the whole family. Our study concludes that the splice site variant identified in the ALX1 gene causes mild form of FND.

A novel homozygous variant in the SMOC1 gene underlying Waardenburg anophthalmia syndrome

ABSTRACT Background: Waardenburg anophthalmia syndrome (WAS), also known as ophthalmo-acromelic syndrome or anophthalmia-syndactyly, is a rare congenital disorder that segregates in an autosomal recessive pattern. Clinical features of the syndrome include malformation of the eyes and the skeleton. Mostly, WAS is caused by mutations in the SMOC-1 gene. Materials and methods: The present report describes a large consanguineous family of Pakistani origin segregating Waardenburg anophthalmia syndrome in an autosomal recessive pattern. Genotyping followed by Sanger sequencing was performed to search for a candidate gene. Results: SNP genotyping using AffymetrixGeneChip Human Mapping 250K Nsp array established a single homozygous region among affected members on chromosome 14q23.1-q24.3 harboring the SMOC1 gene. Sequencing of the gene revealed a novel homozygous missense mutation (c.812G>A; p.Cys271Tyr) in the family. Conclusion: This is the first report of Waardenburg anophthalmia syndrome caused by a SMOC1 variant in a Pakistani population. The mutation identified in the present investigation extends the body of evidence implicating the gene SMOC-1 in causing WAS.

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.