Identification of a novel homozygous TRAPPC9 gene mutation causing non-syndromic intellectual disability, speech disorder, and secondary microcephaly.

TRAPPC9 gene mutations have been linked recently to autosomal recessive mental retardation 13 (MRT13; MIM#613192) with only eight families reported world-wide. We assessed patients from two consanguineous pedigrees of Pakistani descent with non-syndromic intellectual disability and postnatal microcephaly through whole exome sequencing (WES) and cosegregation analysis. Here we report six further patients from two pedigrees with homozygous TRAPPC9 gene mutations, the novel nonsense mutation c.2065G>T (p.E689*) and the previously identified nonsense mutation c.1423C>T (p.R475*). We provide an overview of previously reported clinical features and highlight common symptoms and variability of MRT13. Common findings are intellectual disability and absent speech, and frequently microcephaly, motor delay and pathological findings on MRI including diminished cerebral white matter volume are present. Mutations in TRAPPC9 should be considered in non-syndromic autosomal recessive intellectual disability with severe speech disorder.

A missense mutation in the PISA domain of HsSAS-6 causes autosomal recessive primary microcephaly in a large consanguineous Pakistani family.

Asymmetric cell division is essential for normal human brain development. Mutations in several genes encoding centrosomal proteins that participate in accurate cell division have been reported to cause autosomal recessive primary microcephaly (MCPH). By homozygosity mapping including three affected individuals from a consanguineous MCPH family from Pakistan, we delineated a critical region of 18.53 Mb on Chromosome 1p21.3-1p13.1. This region contains the gene encoding HsSAS-6, a centrosomal protein primordial for seeding the formation of new centrioles during the cell cycle. Both next-generation and Sanger sequencing revealed a homozygous c.185T>C missense mutation in the HsSAS-6 gene, resulting in a p.Ile62Thr substitution within a highly conserved region of the PISA domain of HsSAS-6. This variant is neither present in any single-nucleotide polymorphism or exome sequencing databases nor in a Pakistani control cohort. Experiments in tissue culture cells revealed that the Ile62Thr mutant of HsSAS-6 is substantially less efficient than the wild-type protein in sustaining centriole formation. Together, our findings demonstrate a dramatic impact of the mutation p.Ile62Thr on HsSAS-6 function and add this component to the list of genes mutated in primary microcephaly.

Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells.

BACKGROUND: Proteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade(®), a proteasome inhibitor that has been approved by the food and drug administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML. METHODS: Cell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose. RESULTS: Treatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells. CONCLUSIONS: Altogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML.

Identification of a novel protein truncating mutation p.Asp98* in XPC associated with xeroderma pigmentosum in a consanguineous Pakistani family.

BACKGROUND: Xeroderma pigmentosum (XP) is a rare genetic disorder, which is characterized by hyper-sensitivity to solar ultraviolet (UV) radiation. Clinical consequences of sun exposure are skin lesions and an increased risk of developing skin cancer. Genetic studies have identified eight genes associated with xeroderma pigmentosum. The proteins encoded by these genes are mainly involved in DNA repair mechanisms. METHODS: Molecular genetic characterization of patients with xeroderma pigmentosum involved positional cloning methods such as homozygosity mapping and subsequent candidate gene analysis. Mutation screening was performed through Sanger DNA sequencing. RESULTS AND DISCUSSION: In this case study, we report a novel protein truncating mutation in XPC associated with autosomal recessive xeroderma pigmentosum in a consanguineous Pakistani family. Genetic mapping revealed a novel single base insertion of a thymine nucleotide NM_004628.4: c.291dupT (c.291_292insT) in the second exon of XPC. The identified mutation leads to a premature stop codon (TGA) at amino acid position 98 (p.Asp98*) and thus presumably results in a truncated protein. The Xeroderma pigmentosum, complementation group C (XPC) is located on 3p25.1 and encodes a protein involved in nucleotide excision repair. The identified mutation presumably truncates all functional domains of the XPC protein, which likely results in the loss of protein function. CONCLUSION: The study expands the knowledge of the mutational spectrum of XPC and is valuable for genetic counseling of affected individuals and their families.

Molecular outcomes, clinical consequences, and genetic diagnosis of Oculocutaneous Albinism in Pakistani population.

Nonsyndromic oculocutaneous Albinism (nsOCA) is clinically characterized by the loss of pigmentation in the skin, hair, and iris. OCA is amongst the most common causes of vision impairment in children. To date, pathogenic variants in six genes have been identified in individuals with nsOCA. Here, we determined the identities, frequencies, and clinical consequences of OCA alleles in 94 previously unreported Pakistani families. Combination of Sanger and Exome sequencing revealed 38 alleles, including 22 novel variants, segregating with nsOCA phenotype in 80 families. Variants of TYR and OCA2 genes were the most common cause of nsOCA, occurring in 43 and 30 families, respectively. Twenty-two novel variants include nine missense, four splice site, two non-sense, one insertion and six gross deletions. In vitro studies revealed retention of OCA proteins harboring novel missense alleles in the endoplasmic reticulum (ER) of transfected cells. Exon-trapping assays with constructs containing splice site alleles revealed errors in splicing. As eight alleles account for approximately 56% (95% CI: 46.52-65.24%) of nsOCA cases, primarily enrolled from Punjab province of Pakistan, hierarchical strategies for variant detection would be feasible and cost-efficient genetic tests for OCA in families with similar origin. Thus, we developed Tetra-primer ARMS assays for rapid, reliable, reproducible and economical screening of most of these common alleles.