Assessment of WT1 expression as a marker of treatment outcome in karyotype normal acute myeloid leukemia patients in Pakistan

Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Follow-up monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. This pilot study evaluated newly diagnosed and post-induction or consolidation chemotherapy of AML patients who were registered with the Oncology Clinics of the Aga Khan University Hospital, Karachi. High WT1 burden [> 5000 copies/ml] in 2 patients was indicative of early recurrence of the disease along with shorter disease-free and overall survival. Low WT1 expression [< 200 copies/ml] in 2 patients after induction and consolidation therapy, respectively, was suggestive of better prognosis

BRCA1 status in Pakistani breast cancer patients with moderate family history

To determine BRCA1 status in breast carcinoma patients of Pakistani origin. Observational study. The Oncology Clinics of the Aga Khan University Hospital, Karachi, between May 2005 and December 2009. Fifty three breast cancer patients based on clinical and laboratory diagnosis were recruited for this study. Moderate family history was defined as having a close relative [mother, daughter, sister] diagnosed with breast cancer under 45 years. Peripheral blood samples were collected from each patient in a 5 ml tube containing EDTA as anticoagulant. Subsequent to DNA extraction, mutational analysis of BRCA1 exons 2, 5, 6, 16, 20 and 22 was carried out using single strand conformation polymorphism [SSCP] assay while protein truncation test [PTT] was used to examine mutations in exon 11. All BRCA1 sequence variants were confirmed by DNA sequencing. Twenty-three patients were diagnosed with early onset breast cancer, 30 patients had moderate family history. At the time of diagnosis, the median age of enrolled patients was 39 years [range 24-65 years]. Out of 53 patients, analyzed by SSCP assay, mobility shift was detected in exon 6, 16 and 20 of three patients, whereas one patient was tested positive for mutation in exon 11 by PTT assays. All patients with BRCA1 mutations were further confirmed by DNA sequencing analysis. In exon 16 c.4837A > G was confirmed, which is a common polymorphism reported in several populations including Asians. Moreover, mutations in exon 6 [c.271T > G], exon 20 [c.5231 delG] and exon 11 [c.1123 T > G] were reported first time in the Pakistani population. Several BRCA1 mutations were observed in Pakistani breast cancer patients with moderate family history. Therefore, mutation-based genetic counselling for patients with moderate family history can facilitate management, if one first or second degree relative or early onset disease is apparent

Phylogenetic analysis of hepatitis B virus in Pakistan

To identify the distribution pattern of Hepatitis B Virus [HBV] genotype in a group of patients and to study its phylogenetic divergence. The clinics of Gastroenterology Unit, Ziauddin University, from January to December 2006. Two hundred and one HBV infected patients were genotyped for this study. All HbsAg positive individuals, either healthy carriers or suffering from conditions such as acute or chronic hepatitis, cirrhosis and hepatocellular carcinoma were included. Hepatitis B patients co-infected with other hepatic viruses were excluded. Hepatitis B virus DNA was extracted from serum, and subjected to a nested PCR, using the primers type-specific for genotype detection. Phylogenetic analysis was performed in the pre-S1 through S genes of HBV. The divergence was studied through 15 sequences of 967bp submitted to the DBJ/EMBL/GenBank databases accessible under accession number EF584640 through EF584654. Out of 201 patients tested, 156 were males and 45 were females. Genotype D was the predominant type found in 128 [64%] patients followed by A in 47 [23%] and mixed A/D in 26 [13%]. Phylogenetic analysis confirmed the dominance of genotype D and subtype ayw2. There was dominance of genotype D subtype ayw2. It had a close resemblance with HBV strains that circulate in Iran, India and Japan

Screening for cystic fibrosis: the importance of using the correct tools

Cystic Fibrosis [CF] is a potentially lethal genetic disorder. The most frequent mutation worldwide in the Cystic Fibrosis Transmembrane Conductance Regulator [CFTR] gene is designated as the Delta F508 mutation. This mutation was found in only 33% of Pakistani patients studied. Since the common Pakistani mutations remain to be identified, appropriate screening tools are required to identify disease. Sweat chloride determinations remain the gold standard for diagnosing CF. This study was done to emphasize the importance of using the correct tests. The study was conducted at the Aga Khan University Hospital. The CFTR delta F508 mutation was tested on blood samples from patients suspected with CF. Sweat chloride analysis using pilocarpine iontopharesis was done with a positive value of greater than 60 meq/L. 57 pediatric samples were screened for the delta F508 mutation and were positive in only 10.6% of all patients tested. 12/57 [21%] had a preliminary sweat test. 6/12 [50%] of these patients had an abnormal sweat test and 3/6 patients with an abnormal sweat chloride [50%] had deltaF508 mutations - 2/6 [33%] were homozygotes and 1 was a compound heterozygote. Since 79% did not have a sweat test, it was difficult to assess whether this subset of patients had cystic fibrosis with a CFTR mutation other than the delta F508 tested or no CF. Sweat chloride analysis is critical to distinguish CF from other causes of severe pulmonary and pancreatic insufficiencies and to define patients requiring further analysis

Medullary carcinoma of breast with a novel germline mutation 1123T>G in exon11 of brca1

Breast cancer, the most common malignancy in females, has an estimated 5-10% hereditary predisposition. BRCA1 is a tumor suppressor gene and is known to be responsible for breast cancer and breast-ovarian cancers running in families. In breast caner patients, several mutations in BRCA1 have been reported throughout the gene. This report describes identification of a mutation in BRCA1 gene using protein truncation [PTT] assay in a patient with medullary carcinoma of breast who also had a family history of breast cancer. Following DNA sequencing, the mutation was confirmed as substitution of thymine at position 1123 with guanine of exon 11 [1123 T>G]. This mutation can be added to the pool of known BRCA1 mutations in Pakistani population, which will help in developing a local screening panel of BRCA1 mutations

Polymorphism of HLa-DR and HLA-DQ in rheumatoid arthritis patients and clinical response to methotrexate - a hospital-based study

To investigate the frequency and distribution of DRB1 and DQB1 alleles in Patients with rheumatoid arthritis [RA] and analyze the relationship between clinical response to methotrexate [MTX] and the HLA-DR and HLA-DQ genotypes in these patients. In this case-control study, the HLA-DRB1 and HLA-DQB1 polymorphism in 91 RA patients and 91 healthy controls was done using polymerase chain reaction and sequence specific primers. There was no statistical difference in frequencies of HLA-DRB1*03, DRB1*04, DRB1*07, DRB1*10, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15 and DRB1*16 genotypes between patients and controls. However, DRB1*01 was found to be significantly more common [p=0.015] in RA patients compared to controls. HLA-DRB1*15 was more common in patients [43.5%] compared to controls [30.8%] but results were not significant. HLA-DRB1*08 and DRB1*09 were present in negligible number in patients as well as controls while HLA-DRB1*12 was conspicuously absent in controls. Similarly, DQB1*06 was also significantly more common [p = 0.01] among the patients compared to healthy control subjects, while there was no statistical difference in the frequencies of DQB1*02, DQB1*03, DQB1*04 and DQB1*05 among the cases and the controls. RA susceptibility in most patients appeared to be associated with the HLA-DRB1*01/DQB1*06 genotype. Regarding association between HLA-DR or HLA-DQ genotype and clinical response to methotrexate [MTX], the data showed that with the exception of HLA-DRB1*03, there appears to be no association between the particular subtypes of HLA-DR and HLA-DQ. HLA-DRB1*03 was significantly more common among non-responders to MTX alluding to the possibility that another genes responsible for MTX metabolism, might be in linkage disequilibrium with HLA-DRB1*03 in the Pakistani population, thereby making such individuals non-responsive to MTX-therapy. RA susceptibility in most Pakistani patients is associated with the HLA-DRB1*01/DQB1*06 genotype. HLA-DRB1*03 was found to be significantly more common among non-responders to MTX treatment suggesting that Pakistani patients with this genotype are less likely to benefit from MTX

Monitoring of HBe antigen and alanine aminotransferase levels in hepatitis B virus DNA positive patients

A retrospective study was conducted on specimens collected during 2002-2004, in which the data obtained from 413 individuals were analyzed for HBV-DNA, ALT and HBe antigen. Two hundred patients were found negative for HBV-DNA and were not included in the study. The remaining 213 patients were selected on the basis of their HBV-DNA detection. HBe antigen was assayed using micro particle enzyme immunoassay [MEIA] technique, manufactured by [Abbott, USA]. Out of 213 patients, 167 [78%] were male. The mean age of the sample population was 25.4 years [range 2-80 years]. Highest ALT value observed was 1192 IU/L whereas mean ALT was 47. 6 IU/L. HBe antigen was reactive in 193 [91%] cases. The absence of HBe antigen in HBV-DNA positive patients may have been due to the presence of precore mutations because HBV exhibits a higher mutational frequency than most DNA viruses. Patients with HBe antigen positive disease were younger than the HBe antigen negative patients [p < 0.05] and their mean ALT was also higher. However, it did not reach upto significant level but a direct significant association was observed between ALT levels and HBe antigen titres at p<0.05