Shahid M. Baba

Role of CYP2E1 genotypes in susceptibility to colorectal cancer in the Kashmiri population

Cytochrome P450 2E1 (CYP2E1) is a key enzyme involved in the metabolic activation of procarcinogens such as N-nitrosoamines and low-molecular-weight organic compounds. The main aim of this study was to determine whether CYP450 2E1 polymorphisms are associated with the risk of colorectal cancer (CRC). We investigated the genotype distribution of the CYP2E1 gene RsaI and a 96-base pair (bp) insertion in 86 CRC cases in comparison with 160 healthy subjects. We found the frequency of the CYP2E1 RsaI genotype to be 53.5 per cent (46/86) for c1/c1, 17.4 per cent (15/86) for c1/c2 and 29.1 per cent (25/86) for c2/c2, and the CYP2E1 98-bp insertion frequencies to be 63.9 per cent (55/86) for non-insertion (i/i), 22.1 per cent (19/86) for heterozygous insertion (i/I) and 36.0 per cent (12/86) for homozygous insertion (I/I) among CRC cases. We also found the CYP2E1 RsaI c2/c2 and CYP2E1 98-bp heterozygous i/I genotypes to be significantly associated with an increased risk of CRC (p = 0.01). We suggest that CYP2E1 polymorphisms are involved in the susceptibility to developing CRC in the ethnic Kashmiri population.

Phytohemagglutinin-Induced Peripheral Blood Cytogenetics: A Valid Means for Diagnosis and Imatinib Therapy Monitoring of Chronic Phase Chronic Myeloid Leukemia Patients

Background: Conventional cytogenetic studies have been viewed as the standard follow-up method for Chronic Myeloid Leukemia patients on Imatinib. However, this approach is beset with high probability of poor metaphase index. In this study, we evaluated the application of Phytohemagglutinin (PHA)-induced peripheral blood culture based cytogenetic analysis (Karyotyping) in diagnosis and Imatinib treatment monitoring of the Chronic Phase CML patients. Methods: The patient samples were subjected to the PHA-induced peripheral blood culture based cytogenetic technique (Karyotyping) to establish their baseline cytogenetic status followed by their follow up Karyotyping twice at the end of 3 and 6 months of treatment. The simultaneous quantitative PCR (q-PCR) assay for BCR-ABL fusion gene transcript on the samples corresponding to their baseline as well as the follow up cytogenetic status was also carried out to authenticate the cytogenetic findings. Results: Complete Cytogenetic Response (CCR) and Partial Cytogenetic Response (PCR) was initially observed in 09 (30%) and 16 (53.3%) respectively of 30 CML patients with 05 (16.6%) patients showing no such response at the end of 3 months. At 6 months, 25 (83.3%) and 02 (6.6%) showed CCR and PCR respectively with 03 (10%) of patients without any response. The findings completely correlated with the hematological response, the q-PCR assay as well as the overall disease condition observed in the patients. Conclusion: As acquiring bone-marrow sample involves morbid consequences for patients and metaphases yielded thereby are difficult to analyze, PHA-induced peripheral blood Karyotyping was explored as an alternative. It was found to have significant potential in serving as a valid tool in the diagnosis and assessment of follow up response to Imatinib mesylate treatment of patients with chronic phase CML.

p15ink4b Loss of Expression by Promoter Hypermethylation Adds to Leukemogenesis and Confers a Poor Prognosis in Acute Promyelocytic Leukemia Patients.

Purpose The p15Ink4b gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India). Materials and Methods p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysiswas carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA. Conclusion Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.

RAD51 G135C gene polymorphism and risk of colorectal cancer in Kashmir.

RAD51 – a DNA double-strand breaks repair gene plays an important role in homologous recombination, a process frequently involved in cancer transformation. The aim of this study was to compare the distribution of the genotype of the RAD51 G135C polymorphism between colorectal cancer (CRC) patients and controls. We also tested the association between the G135C polymorphism of the RAD51 gene and the risk of CRC, and various clinicopathological parameters. Polymorphism was evaluated by restriction fragment length polymorphism PCR in 100 CRC patients and 120 age-matched and sex-matched controls. There was a significant association between RAD51 genotypes and CRC cases (P<0.05). Also, the GC genotype was associated with an increased risk of CRC (odds ratio >3.84). Our results suggest that the G135C polymorphism of the RAD51 gene is associated with an increased risk of CRC in our population.

Identification of Unique Pattern of CFTR Gene Mutations in Cystic Fibrosis in an Ethnic Kashmiri Population (North India)

Background: Cystic Fibrosis (CF) one of the most common severe autosomal recessive disorders is caused by mutations in CFTR gene. The mutation distributions vary widely between different geographical and ethnic groups. In view of ethnic nature of Kashmiri population (North India), we aim at looking for the 3 common mutations Δ508, 3849+10 kb, C>T and W1282X in CF suspected cases. Method: The mutations were evaluated in 150 highly suspected children with CF, proven by clinical features. ARMS-PCR was used for mutation detection of Δ508 and W1282X while as 3849+10 kb, C>T was assessed by indigenously developed ARMS-PCR and results were confirmed by RFLP. Results: Of the 150 suspected CF cases, one of the three mutations was found in 60 out of the 300 alleles genotyped. Δ508 mutation was found in 36 of 150 (24%) cases, 3849+10 kb, C>T in 24 of 150(16%) cases while as no mutation was observed in W1282X. Interestingly 08 of 09 samples with normal sweat chloride were detected positive for 3849+10 kb, C>T mutation. Conclusion: In this report, frequency of the Δ508 mutation in Kashmiri children with CF is less as compared to the Western Countries. Interestingly, we identified 3849+10 kb, C>T mutation as unique in population under study with much higher frequency as compared to rest of the world. Further we found intron 19, 3849+10 kb, C>T mutation serves as marker in those CF cases having sweat chloride negative.

Molecular and Cytogenetic Evaluation of Gender in Patients Born with Ambiguous Genitalia from Different Regions of the Valley of Kashmir,North India

Ambiguous Genitalia, a rare genetic disorder is caused by defects in the process of fetal sexual determination and differentiation where a newborn needs prompt evaluation to detect lethal conditions and gender assignment. This genetic screening is taken up with an aim to evaluate the prevalence of the patients born with Ambiguous Genitalia existing in our population. We evaluated the patients for Ambiguous Genitalia with Polymerase Chain Reaction (PCR) using SRY gene and cytogenetic analysis for detecting X and Y chromosome was performed by conventional Karyotyping. Of the 50 Ambiguous Genitalia cases, 21 (42.0 %) were detected positive for SRY gene with 46, XY Karyotype and were recognized as males while as rest of cases 29 of 50 (58%) were confirmed as females by negative SRY gene coupled with 46, XX Karyotype. All the 21 cases positive for SRY gene were cytogenetically evaluated with 46, XY except two cases, one of testicular feminization syndrome and another case of AIS where a Karyotype revealed 46, XY but were negative for SRY gene. 09 of the 50 cases (18%) had undetermined sex and among these 05 (55.5%) were found to be negative for SRY gene with 46, XX Karyotype except one case with 47, XX which was an Edward’s syndrome. 04 of the 09 cases (45.5%) were detected as males with positive SRY gene. Overall 04 cases (08.0 %) that were socially recognized as female for two decades were confirmed to be carrying Y chromosome as depicted by positive SRY gene with 46, XY Karyotype. We found that newborn with Ambiguous Genitalia exist in our region in a good number and the cytogenetic method coupled with molecular analysis by PCR are essential to unfold the sex and help in proper assignment of gender in intersex disorders.

Pathogenetic implication of fusion genes in acute promyelocytic leukemia and their diagnostic utility

Acute promyelocytic leukemia (APL) has been recognized as a discrete subset of hematopoietic malignancies constituting approximately 10% of acute myeloid leukemia cases. The hallmark reciprocal chromosomal translocation t(15;17) involving fusion between the retinoic acid receptor (RARα) gene and promyelocytic leukemia (PML) gene is a characteristic feature in APL which consequently results in the emergence of PML‐RARα chimeric gene. This gene has been substantiated to be responsible for cellular transformation and is a prime target of all‐trans‐retinoic acid (ATRA) as well as arsenic‐trioxide (ATO) therapy. Since this initial discovery, about 10 diverse translocation partner genes of RARα have been reported that result in variant APL forms strongly suggesting that disruption of RARα underlies its pathogenesis. The nature of the fusion partner has a significant bearing upon disease characteristics including sensitivity to retinoids and ATO and thereby underpins the need for rapid and accurate diagnosis and also demands a highly specific treatment approach. In this article we laid emphasis on the rearrangement of the RARα gene and its different fusion partners resulting in variant forms of APL, their implication in underlying molecular pathogenesis of APL and also the different diagnostic modalities that should be employed for their rapid and accurate diagnosis.

PHA-Induced Peripheral Blood Cytogenetics and Molecular Analysis: a Valid Diagnostic and Follow-up Modality for Acute Promyelocytic Leukemia Patients Treated with ATRA and/or Arsenic Tri-oxide.

BACKGROUND Acute promyelocytic leukemia (APML) is characterized by the reciprocal translocation t(15;17) (q22;q12) resulting in the PML-RARα fusion gene. A dual diagnostic and follow up approach was applied including cytogenetic demonstration of the t(15;17) translocation and detection of PML-RARα chimeric transcripts by molecular means. PURPOSE Conventional cytogenetics involving bone marrow is beset with high probability of poor metaphase index and was substituted with phytohemagglutinin (PHA)-induced peripheral blood culture based cytogenetic analysis as a diagnostic and follow up modality in APML patients of Kashmir (North India). Both qualitative (RT-PCR) and quantitative (Q-PCR) tests were simultaneously carried out to authenticate the modified cytogenetics. MATERIALS AND METHODS Patient samples were subjected to the said techniques to establish their baseline as well as follow-up status. RESULTS Initial cytogenetics revealed 30 patients (81%) positive for t(15;17) whereas 7 (19%) had either cryptic translocation or were negative for t(15;17). Two cases had chromosome 16q deletion and no hallmark translocation t(15;17). Q-PCR status for PML-RARα was found to be positive for all patients. All the APML patients were reassessed at the end of consolidation phase and during maintenance phase of chemotherapy where 6 patients had molecular relapse, wherein 4 also demonstrated cytogenetic relapse. CONCLUSIONS It was found that PHA-induced peripheral blood cytogenetics along with molecular analysis could prove a reliable modality in the diagnosis and assessment of follow up response of APML patients.