M. A. Khan

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

BackgroundProteasome 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.MethodsCell 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.ResultsTreatment 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.ConclusionsAltogether, 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.

Homozygosity mapping identified a novel protein truncating mutation (p.Ser100Leufs*24) of the BBS9 gene in a consanguineous Pakistani family with Bardet Biedl syndrome

BackgroundBardet Biedl Syndrome (BBS) is a rare condition of multi-organ dysfunction with characteristic clinical features of retinal degeneration, truncal obesity, postaxial polydactyly, genital anomaly, intellectual disability and renal dysfunction. It is a hetero-genetic disorder and nineteen BBS genes have been discovered so far.MethodsWhole genome SNP genotyping was performed by using CytoScan® 750 K array (Affymetrix). Subsequently, the segregation of the disease locus in the whole family was carried out by genotyping STS markers within the homozygous interval. Finally, the mutation analysis was performed by Sanger DNA sequencing.ResultsIn the present molecular study a consanguineous Pakistani family, with autosomal recessive BBS, was analyzed. The clinical analysis of affected individuals presented with synpolydactyly, obesity, intellectual disability, renal abnormality and retinitis pigmentosa. The presented phenotype was consistent with the major features of BBS syndrome. Homozygosity mapping identified a common homozygous interval within the known BBS9 locus. Sequence analysis of BBS9/PTHB1 gene revealed a single base deletion of c.299delC (p.Ser100Leufs*24) in exon 4. This frame-shift mutation presumably leads to a 122 amino acid truncated protein with complete loss of its C-terminal PTHB1 domain in combination with a partial loss of the N-terminal PTHB1 domain as well. BBS9/PTHB1 gene mutations have been shown to be associated with BBS syndrome and to the best of our knowledge this study reports the first Pakistani family linked to the BBS9 gene.ConclusionOur molecular findings expand the mutational spectrum of BBS9 gene and also explain the genetic heterogeneity of Pakistan families with BBS syndrome. The growing number of mutations in BBS genes in combination with a detailed phenotypical description of patients will be helpful for genotype-phenotype correlation, targeted genetic diagnosis, prenatal screening and carrier testing of familial and non-familial BBS patients.

Molecular genetic analysis of consanguineous families with primary microcephaly identified pathogenic variants in the ASPM gene

Autosomal recessive primary microcephaly is a rare genetic disorder that is characterized by reduced head circumference and a varying degree of intellectual disability. Genetic studies on consanguineous families with primary microcephaly have identified 15 (MCPH) causative genes that include MCPH1, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK6, CENPE, SASS6MFSD2AANKLE2 and CIT (Khan et al.2014; Yamamoto et al.2014; Alakbarzade et al.2015; Morris-Rosendahl and Kaindl 2015; Basit et al.2016). Physiologically, most of these MCPH proteins are involved in cell cycle and its regulation. In the present clinical genetic study, we have present two consanguineous Pakistani families segregating primary microcephaly and intellectual disability. These families were ascertained from the Saraiki ethnic part of Khyber-Pakhtunkhwa province in Pakistan. Whole exome sequencing in one family revealed a novel 1-bp deletion NM_018136.4: c.10013delA (p.Asp3338Valfs*2), while the other family showed a previously reported nonsense mutation NM_018136.4: c.9730C > T (rs199422195 (p.Arg3244*)) in ASPM gene. The novel frame-shift mutation (p.Asp3338Valfs*2) in ASPM presumably truncates the protein synthesis that results in loss of armadillo-type fold domain.