Ahmed Sadeque

Association of ANRIL polymorphism (rs1333049:C>G) with myocardial infarction and its pharmacogenomic role in hypercholesterolemia

Single nucleotide polymorphisms (SNPs) of non-coding RNA in the INK4 locus (ANRIL) have been found to be associated with myocardial infarction (MI). However, the effect of rs1333049:C>G in INK4 locus in familial hypercholesterolemia patients and on lipid profile of the patients has not been studied in Pakistan. We therefore investigated the association of SNP rs1333049:C>G with MI as well as familial hypercholesterolemia patients and also determined the effect of genotype on lipid levels in a northern Pakistani population. A case-control association study was performed in which 611 individuals (294 patients, 290 healthy controls and 27 patients from hypercholesterolemia families) were genotyped for rs1333049:C>G, using an Allele specific polymerase chain reaction. We found a significant association of rs1333049:C>G with MI (χ(2)=22.3, p<0.001). The frequency of risk genotype CC was significantly different from the healthy controls (p<0.001, χ(2)=22.3). The risk allele C was at a higher frequency in the MI patients as compared to the controls (odds ratio [OR]=1.55 (95% confidence interval [CI]=1.22-1.96), p<0.001). The logistic regression analysis for the genotype distribution resulted in strong association of risk allele C with MI under recessive model (OR=3.17 (95% CI=1.85-5.44) p<0.001). When the data were further analyzed along the lines of gender, a significant association with both males and females was observed. The pleiotropic role of rs1333049 was revealed further when CC genotype hypercholesterolemic individuals on statins were found to have a significantly lower TC, LDL-C and Tg levels as compared to the CG and GG individuals (p<0.05). The current study demonstrates a strong association of the ANRIL SNP (rs1333049) with MI as well as familial hypercholesterolemia patients in a northern Pakistani population and could be used as a useful genetic marker for the screening of MI in the general Pakistani population.

Identification of Novel Mutations in Pakistani Families With Autosomal Recessive Retinitis Pigmentosa

R etinitis pigmentosa (RP) is a major cause of inherited blindness and accounts for 20% of children attending blind schools in Pakistan. Based on the European and American populations, the prevalence is estimated at 1 in 4000 individuals but has been reported to be as high as 1 in 372 in rural areas of South India. Eight loci and 44 genes have been associated with RP (RetNet, http://www.sph.uth.tmc.edu/RetNet). Pakistan is among the countries with the highest prevalence of consanguineous marriages. Consanguineous families are ideally studied by homozygosity mapping to identify the locus harboring the mutated gene causing the disease phenotype. To identify novel mutations and genes causing RP, we studied a panel of 23 consanguineous families with autosomal recessive RP (arRP) from different areas of Pakistan.

A novel pathogenic nonsense triple-nucleotide mutation in the low-density lipoprotein receptor gene and its clinical correlation with familial hypercholesterolemia.

AIM The aim of this study was to determine the genetic basis of familial hypercholesterolemia in a Pakistani family with a history of myocardial infarction and premature coronary artery disease. RESULTS Direct sequencing of the low-density lipoprotein receptor gene resulted in the identification of a novel missense mutation c.264G>C (p.R88S) in exon 3 and a novel nonsense triple-nucleotide polymorphism (TNP) c.887-889GCA>AGC (p.C296X) in exon 6, the latter being probably the disease-causing mutation in this family. Both of these mutations were not present in the probands of 14 familial hypercholesterolemia families, 100 myocardial infarction patients, as well as 150 normolipidemic ethnically matched control individuals. CONCLUSIONS The identification of the novel nonsense TNP is the first report of a nonsense pathogenic TNP in low-density lipoprotein receptor or any other gene and only the fourth report of a pathogenic TNP of any type, which emphasizes the importance of screening for TNPs in patients and in familial studies that might otherwise be missed if only analyzed on single-nucleotide polymorphism arrays.

The genetic spectrum of familial hypercholesterolemia in Pakistan

Background Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the genes coding for the low density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type-9 (PCSK9) or apo-lipoprotein B-100 (APOB). The aim of the present work was to determine the genetic basis of dyslipidemia in 11 unrelated Pakistani families. Methods High resolution melting (HRM), sequencing and restriction fragment length polymorphism (RFLP). Results Probands were screened for the promoter and all coding regions, including intron/exon boundaries, of LDLR and PCSK9 and part of exon 26 of APOB including p.(R3527Q). Two families were identified with previously unreported LDLR mutations (c.1019_1020delinsTG, p.(C340L) and c.1634G>A, p.(G545E)). Both probands had tendon xanthomas or xanthelasma and/or a history of cardiovascular disease. Co-segregation with hypercholesterolemia was demonstrated in both families. In silico studies predicted these variations to be damaging. In two families, novel PCSK9 variations were identified (exon2; c.314G > A, p.(R105Q) and exon3; c.464C>T, p.(P155L)). In silico studies suggested both were likely to be damaging, and family members carrying the p.(105Q) allele had lower total cholesterol levels, suggesting this is a loss-of-function mutation. For c.464C>T p.(P155L) the small number of relatives available precluded any strong inference. Conclusion This report brings to seven the number of different LDLR mutations reported in FH patients from Pakistan and, as expected in this heterogeneous population, no common LDLR mutation has been identified.