👤 Ashfaq Ahmad

Several studies have suggested that chemokine receptors are important mediators of inflammatory response in rheumatoid arthritis (RA). B cells are also known to play an important role in RA pathology. C-X-C chemokine receptor type 3 (CXCR3) is considered a potential therapeutic target in different inflammatory diseases; however, the mechanism remains unclear. Here, we evaluated the potentially protective effect of AMG487, a selective CXCR3 antagonist, in collagen-induced arthritis (CIA) mouse model. CIA mice were treated with AMG487 (5 mg/kg) every 48 h, from day 21 until day 41. We then investigated the effect of AMG487 on NF-κB p65-, NOS2-, MCP-1-, TNF-α-, IFN-γ, IL-4-, and IL-27-producing CD19 Show less

Neuroinflammation has been recognized as an important factor in the pathogenesis of Alzheimer's disease (AD). One of the most recognized pathways in mediating neuroinflammation is the prostaglandin E2-EP1 receptor pathway. Here, we examined the efficacy of the selective EP1 antagonist ONO-8713 in limiting amyloid-β (Aβ), lesion volumes, and behavioral indexes in AD mouse models after ischemic stroke. Transgenic APP/PS1, 3xTgAD, and wildtype (WT) mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO) and sham surgeries. Functional outcomes, memory, anatomical outcomes, and Aβ concentrations were assessed 14 days after surgery. pdMCAO resulted in significant deterioration in functional and anatomical outcomes in the transgenic mice compared with the WT mice. No relevant differences were observed in the behavioral tests when comparing the ONO-8713 and vehicle-treated groups. Significantly lower cavitation (p = 0.0373) and percent tissue loss (p = 0.0247) were observed in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 mice. However, the percent tissue injury was significantly higher in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 group (p = 0.0373). Percent tissue loss was also significantly lower in the 3xTgAD + ONO-8713 mice than in the WT + ONO-8713 mice (p = 0.0185). ONO-8713 treatment also attenuated cortical microgliosis in APP/PS1 mice as compared with the vehicle (p = 0.0079); however, no differences were observed in astrogliosis across the groups. Finally, APP/PS1 mice presented with characteristic Aβ load in the cortex while 3xTgAD mice exhibited very low Aβ levels. In conclusion, under the experimental conditions, EP1 receptor antagonist ONO-8713 showed modest benefits in anatomical outcomes after stroke, mainly in APP/PS1 mice. Show less

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene. A second copy, SMN2, is similar to SMN1 but produces ∼10% SMN protein because of a single-point mutation that causes splicing defects. Chronic low levels of SMN cause accumulation of co-transcriptional R-loops and DNA damage leading to genomic instability and neurodegeneration in SMA. Severity of SMA disease correlates inversely with SMN levels. SMN2 is a promising target to produce higher levels of SMN by enhancing its expression. Mechanisms that regulate expression of SMN genes are largely unknown. We report that zinc finger protein ZPR1 binds to RNA polymerase II, interacts in vivo with SMN locus and upregulates SMN2 expression in SMA mice and patient cells. Modulation of ZPR1 levels directly correlates and influences SMN2 expression levels in SMA patient cells. ZPR1 overexpression in vivo results in a systemic increase of SMN levels and rescues severe to moderate disease in SMA mice. ZPR1-dependent rescue improves growth and motor function and increases the lifespan of male and female SMA mice. ZPR1 reduces neurodegeneration in SMA mice and prevents degeneration of cultured primary spinal cord neurons derived from SMA mice. Further, we show that the low levels of ZPR1 associated with SMA pathogenesis cause accumulation of co-transcriptional RNA-DNA hybrids (R-loops) and DNA damage leading to genomic instability in SMA mice and patient cells. Complementation with ZPR1 elevates senataxin levels, reduces R-loop accumulation and rescues DNA damage in SMA mice, motor neurons and patient cells. In conclusion, ZPR1 is critical for preventing accumulation of co-transcriptional R-loops and DNA damage to avert genomic instability and neurodegeneration in SMA. ZPR1 enhances SMN2 expression and leads to SMN-dependent rescue of SMA. ZPR1 represents a protective modifier and a therapeutic target for developing a new method for the treatment of SMA. Show less

Background The objective of this study was to investigate the association of polymorphisms in four genes, tumor necrosis factor-α (TNFA), patatin-like phospholipase domain containing 3 (PNPLA3), adiponectin (ADIPOQ) and apolipoprotein C3 (APOC3), with obesity and non-alcoholic fatty liver disease (NAFLD) in Asian Indian adolescents. Methods In this case-control study, 218 Asian Indian adolescents with overweight/obesity and 86 lean healthy adults without fatty liver were enrolled. Hepatic steatosis was assessed and graded by ultrasonography (USG). Serum insulin, lipids, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TNF-α, adiponectin and apolipoprotein C3 were measured and genotyping was done. Frequencies of variant and wild genotypes in all adolescents and in the subgroups without steatosis, with grade 1 steatosis and with grade 2 or 3 steatosis were compared to those in the controls. The frequencies were also compared in the overweight adolescents with grade 2 or 3 steatosis and without steatosis. Results Variant genotypes of polymorphisms -863 C > A and -1031 T > C of the TNFA gene, 455 T > C of the APOC3 gene and the wild type of +276 G > T of the ADIPOQ gene were associated with obesity with odds ratios (OR, 95% confidence interval [CI]) of 2.5 (1.5-4.4), 2.5 (1.5-4.2), 2.0 (1.1-3.6) and 2.5 (1.4-5.0), respectively. Polymorphisms 455 T > C of APOC3 and rs738409 C > G of PNPLA3 were associated with NAFLD. Fasting insulin and triglycerides (TG) were higher in the adolescents with homozygous variant polymorphisms -1031 T > C of TNFA and 455 T > C of APOC3 genes, respectively. Conclusions Several polymorphisms were noted to have a significant association with obesity and NAFLD in Asian Indian adolescents. Show less

Genetic and epidemiologic studies have shown that lipid genes and high-density lipoproteins (HDLs) are implicated in age-related macular degeneration (AMD). We studied circulating lipid levels in relationship to AMD in a large European dataset. Pooled analysis of cross-sectional data. Individuals (N = 30 953) aged 50 years or older participating in the European Eye Epidemiology (E3) consortium and 1530 individuals from the Rotterdam Study with lipid subfraction data. AMD features were graded on fundus photographs using the Rotterdam classification. Routine blood lipid measurements, genetics, medication, and potential confounders were extracted from the E3 database. In a subgroup of the Rotterdam Study, lipid subfractions were identified by the Nightingale biomarker platform. Random-intercepts mixed-effects models incorporating confounders and study site as a random effect were used to estimate associations. AMD features and stage; lipid measurements. HDL was associated with an increased risk of AMD (odds ratio [OR], 1.21 per 1-mmol/l increase; 95% confidence interval [CI], 1.14-1.29), whereas triglycerides were associated with a decreased risk (OR, 0.94 per 1-mmol/l increase; 95% CI, 0.91-0.97). Both were associated with drusen size. Higher HDL raised the odds of larger drusen, whereas higher triglycerides decreases the odds. LDL cholesterol reached statistical significance only in the association with early AMD (P = 0.045). Regarding lipid subfractions, the concentration of extra-large HDL particles showed the most prominent association with AMD (OR, 1.24; 95% CI, 1.10-1.40). The cholesteryl ester transfer protein risk variant (rs17231506) for AMD was in line with increased HDL levels (P = 7.7 × 10 Our study suggested that HDL cholesterol is associated with increased risk of AMD and that triglycerides are negatively associated. Both show the strongest association with early AMD and drusen. Extra-large HDL subfractions seem to be drivers in the relationship with AMD, and variants in lipid genes play a more ambiguous role in this association. Whether systemic lipids directly influence AMD or represent lipid metabolism in the retina remains to be answered. Show less

Mutations in the genes encoding leptin (LEP), the leptin receptor (LEPR), and the melanocortin 4 receptor (MC4R) are known to cause severe early-onset childhood obesity. The aim of the current study was to examine the prevalence of damaging LEP, LEPR, and MC4R mutations in Pakistani families having a recessive heritance of early-onset obesity. Using targeted resequencing, the presence of rare mutations in LEP, LEPR, and MC4R, was investigated in individuals from 25 families suspected of having autosomal recessive early-onset obesity. Segregation patterns of variants were assessed based on chip-based genotyping. Homozygous LEPR variants were identified in two probands. One carried a deletion (c.3260AG) resulting in the frameshift mutation p.Ser1090Trpfs*6, and the second carried a substitution (c.2675C > G) resulting in the missense mutation p.Pro892Arg. Both mutations were located within regions of homozygosity shared only among affected individuals. Both probands displayed early-onset obesity, hyperphagia and diabetes. No mutations were found in LEP and MC4R. The current study highlights the implication of LEPR mutations in cases of severe early-onset obesity in consanguineous Pakistani families. Through targeted resequencing, we identified novel damaging mutations, and our approach may therefore be utilized in clinical testing or diagnosis of known forms of monogenic obesity with the aim of optimizing obesity treatment. Show less

An early intervention using biomarkers to predict acute myocardial infarction (AMI) will effectively reduce global heart attack incidence, particularly among high-risk patients with type 2 diabetes mellitus (T2DM). This study attempted to identify potential biomarkers by detecting changes in the levels of plasma proteins in T2DM patients following onset of AMI in comparison with those without AMI. Volunteer T2DM patients without AMI (control; n=10) and T2DM patients with AMI (n=10) were recruited. Plasma samples from these patients were evaluated via two-dimensional gel electrophoresis (2DE) to screen for proteins with level changes between the two groups. The abundance of spots on gel images was analyzed using Progenesis SameSpots and subjected to false discovery rate (FDR) analysis. Protein spots with statistically significant changes of at least 1.5 fold were selected for mass spectrometry (MS) analysis. Due to strong cardiac connections, tetranectin and titin were evaluated by enzymelinked immunosorbent assay (ELISA). The adjusted P-values and fold changes between the two groups resulted in identification of 34 protein spots with significantly altered abundance. Upon MS analysis, 17 plasma proteins were identified: tetranectin, titin, clusterin, haptoglobin, myosin-13, zinc fnger protein 445, DNA repair protein RAD50, serum albumin, apolipoprotein A-IV, caspase-6, aminoacyl tRNA synthase complex-interacting multifunctional protein 1, serotransferrin, retinol-binding protein 4, transthyretin, alpha-1-antitrypsin, apolipoprotein A-I and serum amyloid A. Comparable patterns of changes in tetranectin and titin between the control and AMI groups were confirmed using ELISA. In summary, tetranectin and titin in plasma appeared to be closely associated with the onset of AMI among T2DM patients and can be used as potential biomarkers for prediction of a cardiac event, though this requires validation in a prospective cohort study. Show less

Mitogen-activated protein kinases (MAPKs) are the main regulators of cellular proliferation, growth, and survival in physiological or pathological conditions. Aberrant MAPK signaling plays a pivotal role in carcinogenesis, which leads to development and progression of human cancer. Dual-specificity phosphatase 6 (DUSP6), a member of the MAPK phosphatase family, interacts with specifically targeted extracellular signal-regulated kinase 1/2 via negative feedback regulation in the MAPK pathway of mammalian cells. This phosphatase functions in a dual manner, pro-oncogenic or tumor-suppressive, depending on the type of cancer. To date, the tumor-suppressive role of DUSP6 has been demonstrated in pancreatic cancer, non-small cell lung cancer, esophageal squamous cell and nasopharyngeal carcinoma, and ovarian cancer. Its pro-oncogenic role has been observed in human glioblastoma, thyroid carcinoma, breast cancer, and acute myeloid carcinoma. Both roles of DUSP6 have been documented in malignant melanoma depending on the histological subtype of the cancer. Loss- or gain-of-function effects of DUSP6 in these cancers highlights the significance of this phosphatase in carcinogenesis. Development of methods that use the DUSP6 gene as a therapeutic target for cancer treatment or as a prognostic factor for diagnosis and evaluation of cancer treatment outcome has great potential. This review focuses on molecular characteristics of the DUSP6 gene and its role in cancers in the purview of development, progression, and cancer treatment outcome. Show less

Melanoma is an aggressive skin cancer that can rapidly metastasize to become fatal, if not diagnosed early. Despite recent therapeutic advances, management of melanoma remains difficult. Therefore, novel molecular targets and strategies are required to manage this neoplasm. This study was undertaken to determine the role of the sirtuin SIRT6 in melanoma. Employing a panel of human melanoma cells and normal human melanocytes, we found significant SIRT6 mRNA and protein upregulation in melanoma cells. Further, using a tissue microarray coupled with quantitative Vectra analysis, we demonstrated significant SIRT6 overexpression in human melanoma tissues. Lentiviral short hairpin RNA-mediated knockdown of SIRT6 in A375 and Hs 294T human melanoma cells significantly decreased cell growth, viability, and colony formation, induced G1-phase arrest and increased senescence-associated beta-galactosidase staining. As autophagy is important in melanoma and is associated with SIRT6, we used a qPCR array to study SIRT6 knockdown in A375 cells. We found significant modulation in several genes and/or proteins (decreases in AKT1, ATG12, ATG3, ATG7, BAK1, BCL2L1, CLN3, CTSB, CTSS, DRAM2, HSP90AA1, IRGM, NPC1, SQSTM1, TNF, and BECN1; increases in GAA, ATG10). Our data suggests that increased SIRT6 expression may contribute to melanoma development and/or progression, potentially via senescence-and autophagy-related pathways. Show less

Spinal muscular atrophy (SMA) is caused by the low levels of survival motor neuron (SMN) protein and is characterized by motor neuron degeneration and muscle atrophy. Respiratory failure causes death in SMA but the underlying molecular mechanism is unknown. The zinc finger protein ZPR1 interacts with SMN. ZPR1 is down regulated in SMA patients. We report that ZPR1 functions downstream of SMN to regulate HoxA5 levels in phrenic motor neurons that control respiration. Spatiotemporal inactivation of Zpr1 gene in motor neurons down-regulates HoxA5 and causes defects in the function of phrenic motor neurons that results in respiratory failure and perinatal lethality in mice. Modulation in ZPR1 levels directly correlates and influences levels of HoxA5 transcription. In SMA mice, SMN-deficiency causes down-regulation of ZPR1 and HoxA5 that result in degeneration of phrenic motor neurons. Identification of ZPR1 and HoxA5 as potential targets provides a paradigm for developing strategies to treat respiratory distress in SMA. Show less

Coronary artery disease (CAD) is one of the largest causes of death worldwide yet the traditional risk factors, although useful in identifying people at high risk, lack the desired predictive accuracy. Techniques like quantitative plasma proteomics holds immense potential to identify newer markers and this study (conducted in three phases) was aimed to identify differentially expressed proteins in stable CAD patients. In the first (discovery) phase, plasma from CAD cases (angiographically proven) and controls were subjected to iTRAQ based proteomic analysis. Proteins found to be differentially expressed were then validated in the second and third (verification and validation) phases in larger number of (n = 546) samples. After multivariate logistic regression adjusting for confounding factors (age, diet, etc.), four proteins involved in the reverse cholesterol pathway (Apo A1, ApoA4, Apo C1 and albumin) along with diabetes and hypertension were found to be significantly associated with CAD and could account for approximately 88% of the cases as revealed by ROC analysis. The maximum odds ratio was found to be 6.70 for albumin (p < 0.0001), followed by Apo AI (5.07, p < 0.0001), Apo CI (4.03, p = 0.001), and Apo AIV (2.63, p = 0.003). Down-regulation of apolipoproteins and albumin implicates the impairment of reverse cholesterol pathway in CAD. Show less

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA. Show less

Type I hyperlipoproteinemia (T1HLP) is a rare, autosomal recessive disorder characterized by extreme hypertriglyceridemia that fails to respond to lipid-lowering agents, predisposing to frequent attacks of acute pancreatitis. Mutations in lipoprotein lipase (LPL), apolipoprotein CII (APOC2), lipase maturation factor 1 (LMF1), glycosyl-phosphatidylinositol anchored high-density lipoprotein-binding protein 1 (GPIHBP1), and apolipoprotein AV (APOA5) cause T1HLP, but we lack data on phenotypic variations among the different genetic subtypes. To study genotype-phenotype relationships among subtypes of T1HLP patients. Genetic screening for mutations in LPL, APOC2, GPIHBP1, LMF1, and APOA5. Tertiary referral center. Ten patients (7 female, 3 male) with chylomicronemia, serum triglyceride levels about 2000 mg/dL, and no secondary causes of hypertriglyceridemia. Genotyping and phenotypic features. Four patients harbored homozygous or compound heterozygous mutations in LPL, 3 had homozygous mutations in GPIHBP1, and 1 had a heterozygous APOA5 mutation. We failed to fully identify the genetic etiology in 2 cases: 1 had a heterozygous LPL mutation only and another did not have any mutations. We identified 2 interesting phenotypic features: the patient with heterozygous APOA5 mutation normalized triglyceride levels with weight loss and fish oil therapy, and all 7 female patients were anemic. Our data suggest the possibility of novel loci for T1HLP. We observed that heterozygous APOA5 mutation can cause T1HLP but such patients may unexpectedly respond to therapy, and females with T1HLP suffer from anemia. Further studies of larger cohorts may elucidate more phenotype-genotypes relationships among T1HLP subtypes. Show less

Increased dietary ratios of ω6/ω3 polyunsaturated fatty acids have been implicated in the pathogenesis of Crohn's disease (CD), but epidemiologic data are limited. We investigated whether variants of genes that control polyunsaturated fatty acid metabolism (CYP4F3, FADS1, and FADS2), along with the dietary ratio of ω6/ω3, confers susceptibility to CD. Based on data from 182 children newly diagnosed with CD and 250 controls, we found that children who consumed a higher dietary ratio of ω6/ω3 were susceptible for CD if they were also carriers of specific variants of CYP4F3 and FADS2 genes. Our findings implicate diet-gene interactions in the pathogenesis of CD. Show less

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation. Show less

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. Vision loss in DR principally occurs due to breakdown of the blood-retinal barrier (BRB), leading to macular edema, retinal detachment and inner retinal and vitreous hemorrhage. Several growth factors have been shown to play crucial role in the development of these vascular changes; however, the cellular and molecular mechanisms of DR are not yet fully revealed. In the current study we investigated the role of bone morphogenetic protein-2 (BMP2) in DR. We examined the changes in the protein levels of BMP2 in human vitreous and retina in addition to the mouse retina of streptozotocin-induced diabetes. To detect the source of BMP2 during diabetes, human retinal endothelial cells (hRECs) were subjected to high glucose (HG) for 5 days and levels of BMP2 protein were analyzed in conditioned media of these cells relative to control. We also evaluated the effect of BMP2 on the levels of VEGF in cultured rat Müller cells (rMC1). In addition, we tested the pro-inflammatory effects of BMP2 by examining its effect on leukocyte adhesion to cultured hRECs, and levels of adhesion molecules and cytokines production. Finally, the effect of different concentrations of BMP2 on permeability of confluent monolayer of hRECs was evaluated using FITC-Dextran flux permeability assay and by measuring Transcellular Electrical Resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS). Our results show, for the first time, the up-regulation of BMP2 in diabetic human and mouse retinas in addition to its detection in vitreous of patients with proliferative DR (72 ± 7 pg/ml). In vitro, hRECs showed upregulation of BMP2 in HG conditions suggesting that these cells are a potential source of BMP2 in diabetic conditions. Furthermore, BMP2 induced VEGF secretion by Müller cells in-vitro; and showed a dose response in increasing permeability of cultured hRECs. Meanwhile, BMP2 pro-inflammatory effects were recognized by its ability to induce leukocyte adhesion to the hRECs, intercellular adhesion molecule-1 (ICAM-1) and upregulation of interleukin-6 and 8 (IL-6 and IL-8). These results show that BMP2 could be a contributing growth factor to the development of microvascular dysfunction during DR via enhancing both pro-angiogenic and inflammatory pathways. Our findings suggest BMP2 as a potential therapeutic target to prevent/treat DR. Show less

Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved. A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls. A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM. Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling. Show less

Numerous obesity loci have been identified using genome-wide association studies. A UK study indicated that physical activity may attenuate the cumulative effect of 12 of these loci, but replication studies are lacking. Therefore, we tested whether the aggregate effect of these loci is diminished in adults of European ancestry reporting high levels of physical activity. Twelve obesity-susceptibility loci were genotyped or imputed in 111,421 participants. A genetic risk score (GRS) was calculated by summing the BMI-associated alleles of each genetic variant. Physical activity was assessed using self-administered questionnaires. Multiplicative interactions between the GRS and physical activity on BMI were tested in linear and logistic regression models in each cohort, with adjustment for age, age(2), sex, study center (for multicenter studies), and the marginal terms for physical activity and the GRS. These results were combined using meta-analysis weighted by cohort sample size. The meta-analysis yielded a statistically significant GRS × physical activity interaction effect estimate (Pinteraction  = 0.015). However, a statistically significant interaction effect was only apparent in North American cohorts (n = 39,810, Pinteraction  = 0.014 vs. n = 71,611, Pinteraction  = 0.275 for Europeans). In secondary analyses, both the FTO rs1121980 (Pinteraction  = 0.003) and the SEC16B rs10913469 (Pinteraction  = 0.025) variants showed evidence of SNP × physical activity interactions. This meta-analysis of 111,421 individuals provides further support for an interaction between physical activity and a GRS in obesity disposition, although these findings hinge on the inclusion of cohorts from North America, indicating that these results are either population-specific or non-causal. Show less

Spinal muscular atrophy (SMA) is caused by mutation of the Survival Motor Neurons 1 (SMN1) gene and is characterized by degeneration of spinal motor neurons. The severity of SMA is primarily influenced by the copy number of the SMN2 gene. Additional modifier genes that lie outside the SMA locus exist and one gene that could modify SMA is the Zinc Finger Protein (ZPR1) gene. To test the significance of ZPR1 downregulation in SMA, we examined the effect of reduced ZPR1 expression in mice with mild and severe SMA. We report that the reduced ZPR1 expression causes increase in the loss of motor neurons, hypermyelination in phrenic nerves, increase in respiratory distress and disease severity and reduces the lifespan of SMA mice. The deficiency of SMN-containing sub-nuclear bodies correlates with the severity of SMA. ZPR1 is required for the accumulation of SMN in sub-nuclear bodies. Further, we report that ZPR1 overexpression increases levels of SMN and promotes accumulation of SMN in sub-nuclear bodies in SMA patient fibroblasts. ZPR1 stimulates neurite growth and rescues axonal growth defects in SMN-deficient spinal cord neurons from SMA mice. These data suggest that the severity of disease correlates negatively with ZPR1 levels and ZPR1 may be a protective modifier of SMA. Show less

Hypotrichosis is a human hereditary hair loss disorder in which affected individuals show sparse to complete absence of hair on scalp and/or on different body parts. To date, at least eight isolated autosomal recessive and dominant forms of hypotrichosis loci have been mapped on different human chromosomes, and the corresponding genes have been identified. Detailed clinical and molecular studies were undertaken of the hereditary hypotrichosis observed in the two consanguineous families (A and B) presented here. Human genome scan, using >500 highly polymorphic microsatellite markers, identified equal evidence of linkage of the hypotrichosis phenotype on chromosomes 12q21.2-q22 and 16q21-q23.1 in both the families. The novel hypotrichosis locus on chromosome 12q21.2-q22 spans 16.3 cM (17.62 Mb), flanked by markers D12S326 and D12S101. At this locus, maximum multipoint logarithm of the odds ratio (LOD) scores of 3.68 and 3.31 were obtained in families A and B, respectively. The second hypotrichosis locus on chromosome 16q21-q23.1, identified in the two families, spans 5.58 cM (8.28 Mb) and is flanked by markers D16S3031 and D16S512. Maximum multipoint LOD scores of 3.17 and 3.31 were obtained with markers mapped at this locus in families A and B, respectively. DNA sequence analysis of six candidate genes (PLEKHG7, SLC6A15, VEZT, DUSP6, KERA and KITLG), located in the linkage interval on chromosome 12q21.2-q22, failed to detect potential sequence variants in the affected individuals of the two families. However, DNA sequence analysis of CDH3 gene, located on chromosome 16q21-q23.1, detected a single base pair homozygous insertion (c.1024₁₀₂₅insG and p.342insGfsX345) in exon 9 in family A and deletion of four base pair (c.1859₁₈₆₂delCTCT and p.620delSfsX629) in exon 13 in family B. We described for the first time digenic inheritance of an autosomal recessive hypotrichosis phenotype in two unlinked loci on chromosomes 12q21.2-q22 and 16q21-q23.1 in two unrelated consanguineous Pakistani families. Show less

Aberrant mitogen/extracellular signal-regulated kinase 5 (MEK5)-extracellular signal-regulated protein kinase 5 (ERK5)-mediated signalling has been implicated in a number of tumour types including prostate cancer (PCa). The molecular basis of ERK5-driven carcinogenesis and its clinical relevance remain to be fully characterised. Modulation of ERK5 expression or function in human PCa PC3 and PC3-ERK5 (stably transfected with ERK5) cells was performed using siRNA-mediated knockdown or the MEK inhibitor PD18435 respectively. In vitro significance of ERK5 signalling was assessed by assays for proliferation, motility, invasion and invadopodia. Expression of matrix metalloproteinases/tissue inhibitors of metalloproteases was determined by Q-RT-PCR. Extracellular signal-regulated protein kinase 5 expression in primary and metastatic PCa was examined using immunohistochemistry. Reduction of ERK5 expression or signalling significantly inhibited the motility and invasive capability of PC3 cells. Extracellular signal-regulated protein kinase 5-mediated signalling significantly promoted formation of in vivo metastasis in an orthotopic PCa model (P<0.05). Invadopodia formation was also enhanced by forced ERK5 expression in PC3 cells. Furthermore, in metastatic PCa, nuclear ERK5 immunoreactivity was significantly upregulated when compared with benign prostatic hyperplasia and primary PCa (P=0.013 and P<0.0001, respectively). Our in vitro, in vivo and clinical data support an important role for the MEK5-ERK5 signalling pathway in invasive PCa, which represents a potential target for therapy in primary and metastatic PCa. Show less

Evidence is sparse about the genetic determinants of major lipids in Pakistanis. Variants (n=45 000) across 2000 genes were assessed in 3200 Pakistanis and compared with 2450 Germans using the same gene array and similar lipid assays. We also did a meta-analysis of selected lipid-related variants in Europeans. Pakistani genetic architecture was distinct from that of several ethnic groups represented in international reference samples. Forty-one variants at 14 loci were significantly associated with levels of HDL-C, triglyceride, or LDL-C. The most significant lipid-related variants identified among Pakistanis corresponded to genes previously shown to be relevant to Europeans, such as CETP associated with HDL-C levels (rs711752; P<10(-13)), APOA5/ZNF259 (rs651821; P<10(-13)) and GCKR (rs1260326; P<10(-13)) with triglyceride levels; and CELSR2 variants with LDL-C levels (rs646776; P<10(-9)). For Pakistanis, these 41 variants explained 6.2%, 7.1%, and 0.9% of the variation in HDL-C, triglyceride, and LDL-C, respectively. Compared with Europeans, the allele frequency of rs662799 in APOA5 among Pakistanis was higher and its impact on triglyceride concentration was greater (P-value for difference <10(-4)). Several lipid-related genetic variants are common to Pakistanis and Europeans, though they explain only a modest proportion of population variation in lipid concentration. Allelic frequencies and effect sizes of lipid-related variants can differ between Pakistanis and Europeans. Show less

Nonsyndromic deafness locus (DFNB48) segregating as an autosomal recessive trait has been mapped to the long arm of chromosome 15 in bands q23-q25.1 in five large Pakistani families. The deafness phenotype in one of these five families (PKDF245) is linked to D15S1005 with a lod score of 8.6 at theta=0, and there is a critical linkage interval of approximately 7 cM on the Marshfield human genetic map, bounded by microsatellite markers D15S216 (70.73 cM) and D15S1041 (77.69 cM). MYO9A, NR2E3, BBS4, and TMC3 are among the candidate genes in the DFNB48 region. The identification of another novel nonsyndromic recessive deafness locus demonstrates the high degree of locus heterogeneity for hearing impairment, particularly in the Pakistani population. Show less

Multiple hereditary exostoses (HME) is an autosomal dominant developmental disorder exhibiting multiple osteocartilaginous bone tumors that generally arise near the ends of growing long bones. Here, we report two large consanguineous families from Pakistan, who display the typical features of HME. Affected individuals also show a previously unreported feature--bilateral overriding of single toes. Analysis using microsatellite markers for each of the known EXT loci, EXT1, EXT2, and EXT3 showed linkage to EXT1. In the first family, mutation analysis of the EXT1 gene revealed that affected individuals were heterozygous for an in-frame G-to-C transversion at the conserved splice donor site in intron 1. This mutation is predicted to disrupt splicing of the first intron and produce a frameshift that leads to a premature termination codon. In the second family, an insertion of an A in exon 8 is predicted to produce a frameshift at codon 555 followed by a premature termination, a further 10 codons downstream. In both families, an increased number of affected male subjects were observed. In affected females in family 2, phenotypic variability and incomplete penetrance were noted. Show less