Characterization of pathogenic mutations in 21-hydroxylase gene of Pakistani patients with congenital adrenal hyperplasia and their family members--a preliminary report

Objective: To characterize specific mutations within the 21-hydroxylase gene (CYP21-B) using ARMS-PCR assay in patients with congenital adrenal hyperplasia (CAH) and to compare it with that reported in other populations. Subjects and Methods: Five families, having an index case with CAH diagnosed on the basis of clinical and biochemical findings volunteered to give blood samples for analysis. A strategy, based on ARMS-PCR (Amplified Refractory Mutation System) was employed for the detection of mutations in 21-hydroxylase gene. The products of ARMS-PCR were resolved on agarose gels and the PCR products were visualized over ultra violet illumination. Results: Twenty-six specimens were analyzed for common point mutations in the 21-hydroxlase genes at the nucleotide positions 659, 1004 and 1688. Seven samples belonged to index cases with CAH. Of these 7, the assigned sex was male in 5 and female in 2 cases. However, genotypic sex was 3 males and 4 females. The mean age was 8 months in 5 cases while the median 17-OH Progesterone levels was 273.2 ng/ml. Vomiting, precocious puberty and ambiguous genitalia were the presenting features in 2, 1 and 4 cases respectively. Analysis for mutation at 659, 100 and 1688 was performed on 7 index cases and the family members of 5 index cases. The mutation analysis for the family members of index case 6 and 7 was not performed due to non-availability of their blood specimens. Index case No. 1, 4 and 7 showed homozygosity for splice mutations at nucleotide position 659, intron 2 with a sequence change of A to G, while the index case No. 2 and 6 showed heterozygosity for the same mutation. No mutation was found at 659, 1004 or 1688 in index case No. 3 and 4 at the analyzed nucleotide position. Nineteen family members of Case Nos. 1-5 were also analyzed for the same mutations. (Family No. 1, 2, 3, 4 and 5 included 3, 2, 7, 4 and 5 members respectively). These included 8 males and 11 females. All were asymptomatic. Both the parents of index case 1 and 4 were heterozygous at 659 while the father of index case No. 2 was heterozygous at 659 and mother was normal. Conculsion: Our results demonstrated the A to G transition at nucleotide 659 causing aberrant splicing, reported for some other populations as the most commonly identified point mutations. All cases were appropriately assigned to paternal or maternal chromosomes

Association of Epstein Barr Virus (EBV) with nasopharyngeal carcinoma (NPC)

Aim: To observe the frequency of nasopharyngeal carcinoma (NPC) and its association with Epstein Barr Virus (EBV) infection. Setting: This study included consecutive cases of nasopharyngeal carcinoma, which were diagnosed in the Department of Pathology at the Aga Khan University Hospital, Karachi in the period of two years (1996-97). Methods: These tumors were initially evaluated on H&E stained sections. The tumors showing evidence of keratinization were excluded from the study. The Epstein Barr Virus was detected with the help of Polymerase chain reaction in formalin fixed, paraffin embedded tissue sections. Results: During the study period, seventeen cases of nasopharyngeal carcinoma were diagnosed which comprised 0.3% of all malignant tumors. The age ranged from 5 years to 70 years with male to female ratio of 2.4:1. The NPC was more prevalent in adults (71%) as compared to children (29%) under 15 years. Six cases (35%) exhibited positive signal for Epstein Barr Virus. Conclusion: Nasopharyngeal carcinoma is an infrequent tumor. The prevalence of Epstein Barr virus infection in nasopharyngeal carcinoma is quite low as compared to other regions of the world

Genetic diversity of beta-thalassemia mutations in Pakistani population

Background: beta-thalassemia is one of the most common inherited single gene disorder in Pakistan. It is characterized by reduced or absent beta-globin gene expression resulting in abnormal maturation and survival of red blood cells. Due to high prevalence of this disease in the local population, it has become important for the health care providers to encourage people to utilize laboratory facilities for carrier and prenatal genetic testing. Objective: To study the frequency of beta-thalassemia mutations in Pakistani population. Setting: A tertiary care teaching hospital. Methods: Blood samples of 72 couples and chorionic villus (CV) biopsy specimen collected at the Aga Khan University Hospital, Karachi were tested by Amplified Refractory Mutation Systems (ARMS) for the 12 most common mutations in the beta-globin gene. Results: Out of 72 chorionic villus biopsy specimen analyzed, 17 (23%) had mutations in both alleles of the beta-globin gene. Homozygosity was identified in 6 CV samples, whereas 11 CV specimens were diagnosed as double heterozygous. Almost 60% of the CV biopsies showed mutations in one allele and were diagnosed as carriers. IVSI-5 (G-C) was the most common mutation identified in this study. It was found in 53% of the subjects and was represented equally in all the ethnic groups except Pathans. Several regional and ethnic differences were observed in the distribution of common mutations, for example in Pathan families Fr 8-9 (+G) mutation was most prevalent. In addition, variation in the distribution of mutations was also observed between the Northern and the Southern regions. Conculsion: This study indicates that in Pakistan, the five most common mutations are IVS1-5 (G-C), IVS1-1 (G-T), Fr 41-42 (-TTCT) Fr 8-9 (+G) and deletion 619 bp. An important factor contributing to high incidence of thalassemia is the unawareness among people about the available diagnostic facilities for the prenatal diagnosis in Pakistan. Strict implementation of collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing beta-thalassemia in Pakistan

Virological and clinical characteristics of hepatitis delta virus in South Asia

<p>Abstract</p> <p>Background & Aims</p> <p>There is a paucity of data on the impact of hepatitis D virus (HDV) in patients with hepatitis B virus (HBV) infection from South Asia. We studied the impact of HDV co-infection on virological and clinical characteristics.</p> <p>Methods</p> <p>We collected data of 480 patients with HBsAg positive and a detectable HBV DNA PCR, who presented to the Aga Khan University, Karachi and Isra University in Hyderabad, Pakistan in the last 5 years. HDV co-infection was diagnosed on the basis of anti-HDV. ALT, HBeAg, HBeAb and HBV DNA PCR quantitative levels were checked in all patients. We divided all patients into two groups based on anti-HDV, and compared their biochemical, serological & virological labs and clinical spectrum. Clinical spectrum of disease included asymptomatic carrier (AC), chronic active hepatitis (CAH), immuno-tolerant phase (IP), and compensated cirrhosis (CC).</p> <p>Results</p> <p>HDV co-infection was found in 169 (35.2%). There were 164 (34.6%) HBeAg positive and 316 (65.4%) HBeAg negative patients. Mean ALT level was 66 ± 73 IU. 233 (48.5%) had raised ALT. HBV DNA level was ≥ 10e5 in 103(21.5%) patients. Overall, among HBV/HDV co-infection, 146/169 (86.4%) had suppressed HBV DNA PCR as compared to 231/311 (74.3%) patients with HBV mono-infection; p-value = 0.002. Among HBeAg negative patients 71/128(55.5%) had raised ALT levels among HBV/HDV co-infection as compared to 71/188 (37.8%) with HBV mono-infection (p-value = 0.002); levels of HBV DNA were equal in two groups; there were 27/128 (21%) patients with CC among HBV/HDV co-infection as compared to 23 (12%) in HBV mono-infection (p-value = 0.009); there were less AC (p-value = 0.009) and more CAH (p-value = 0.009) among HBV/HDV co-infection patients. Among HBeAg positive patients, serum ALT, HBV DNA levels and the spectrum of HBV were similar in the two groups.</p> <p>Conclusions</p> <p>HBV/HDV co-infection results in the suppression of HBV DNA. A fair proportion of HBV/HDV co-infected patients with HBeAg negative have active hepatitis B infection and cirrhosis as compared to those with mono-infection.</p

Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

Background: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2). We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH. Methods: A cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay. Results: Overall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6%) chromosomes. The most frequent mutation was I2 splice (27%) followed by Ile173Asn (26%), Arg 357 Trp (19%), Gln319stop, 16% and Leu308InsT (12%), whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state. Conclusion: Our study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined

A comparison of four fibrosis indexes in chronic HCV: Development of new fibrosis-cirrhosis index (FCI)

<p>Abstract</p> <p>Background</p> <p>Hepatitis C can lead to liver fibrosis and cirrhosis. We compared readily available non-invasive fibrosis indexes for the fibrosis progression discrimination to find a better combination of existing non-invasive markers.</p> <p>Methods</p> <p>We studied 157 HCV infected patients who underwent liver biopsy. In order to differentiate HCV fibrosis progression, readily available AAR, APRI, FI and FIB-4 serum indexes were tested in the patients. We derived a new fibrosis-cirrhosis index (FCI) comprised of ALP, bilirubin, serum albumin and platelet count. FCI = [(ALP × Bilirubin) / (Albumin × Platelet count)].</p> <p>Results</p> <p>Already established serum indexes AAR, APRI, FI and FIB-4 were able to stage liver fibrosis with correlation coefficient indexes 0.130, 0.444, 0.578 and 0.494, respectively. Our new fibrosis cirrhosis index FCI significantly correlated with the histological fibrosis stages F0-F1, F2-F3 and F4 (r = 0.818, p < 0.05) with AUROCs 0.932 and 0.996, respectively. The sensitivity and PPV of FCI at a cutoff value < 0.130 for predicting fibrosis stage F0-F1 was 81% and 82%, respectively with AUROC 0.932. Corresponding value of FCI at a cutoff value ≥1.25 for the prediction of cirrhosis was 86% and 100%.</p> <p>Conclusions</p> <p>The fibrosis-cirrhosis index (FCI) accurately predicted fibrosis stages in HCV infected patients and seems more efficient than frequently used serum indexes.</p

Cyclooxygenase-2 Polymorphisms and Breast Cancer associated risk in Pakistani patients

Prostaglandins produced by Cyclooxygenase-2 enzyme have been implicated to have a role in breast carcinogenesis. Several single nucleotide polymorphisms (SNPs) linked to COX-2 enzyme are reported to modulate its expression. The aim of the present study was to examine association of these SNPs to breast cancer risk in Pakistani patients. Methods: In this case-control study, three sequence variants rs689465, rs689466, rs20417 in the promoter region of COX-2 were screened to evaluate the association with breast cancer risk. A total of 150 breast cancer patients and 101 healthy control genomic DNA were genotyped for rs689456, rs689466, rs20417 and their genotypes distribution in cases and control were compared using Pearson chi square test. Risk association was analyzed through odd ratio calculated by logistic regression. Results: A screening analysis of COX-2 SNPs in 101 healthy controls showed distribution of Minor allelic frequency distribution of SNPs as follows : rs689465 (0.12), rs689466 (0.15), rs20417 (0.23). Further analyses revealed that their observed genotype frequencies were consistent with Hardy Weinberg equilibrium and strong linkage disequilibrium was identified between rs20417, rs689465 and rs689466. The Combined allele variants analysis showed that Haplotype rs68965G- 689466A-20417C (OR 2.909; CI 95 %1.3776.327; P = 0.007) was significantly associated with breast cancer. Conclusions: Our results indicate no strong association between three most frequent COX-2 SNPs rs689465 rs689466, rs20417 studied with breast cancer risk in the single locus analysis. However, our data suggested that combined COX-2 SNP haplotype have a role in breast cancer associated risk in Pakistani patients