JAK2 and Beyond: Mutational Study of JAK2V617 in Myeloproliferative Disorders and Haematological Malignancies in Kashmiri population.

BACKGROUND: Janus Tyrosine Kinase-2 (JAK2 V617F), a novel point mutation affecting the MPD'S is a somatic gain-of-function mutation. It alters a highly conserved amino acid valine in the negative regulatory JH2 domain to phenylalanine predicted to dysregulate kinase activity. AIM: To evaluate the prevalence and clinical significance of JAK2 V617F mutation in various MPD's as well as in hematological malignancies. SUBJECTS AND METHODS: JAK2 mutation was assessed in 90 patients with myeloproliferative disorders and 47 leukemic patients. In addition, peripheral blood samples from 90 healthy donors were also collected as control. We used a highly sensitive Allele-Specific polymerase chain reaction (AS-PCR) for the detection and confirmed the mutation further by direct sequencing. RESULTS: Our results showed significant differences between various disorders with respect to either the proportion of positivity or that of mutant alleles. JAK2-V617F was detected in 67/90 MPD patients and 02/17 for AML,01/11 for ALL-L1,02/12 for ALL-L2 and 02/07 for CML and 90 healthy controls. CONCLUSION: From the above findings it is evident that the JAK2 V617F mutation is widespread not only in MPD's but also in hematological malignancies, which might as well lead to the new classification of MPD'S. Our data also suggest that different genetic events may lead to JAK-STAT pathway activation in different malignancies.

5'-CpG island promoter hypermethylation of the CAV-1 gene in breast cancer patients of Kashmir.

BACKGROUND: Caveolin-1 (CAV-1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signalling. Aberrant promoter methylation of CAV-1 is associated with inactivation of expression. We previously observed CAV-1 mutations in breast cancers and therefore devised this study to examine the hypermethylation status of the promoter region of CAV-1 with reference to breast cancer progression and development. METHODS: Hypermethylation status of CAV-1 was analyzed by methylation specific PCR. Loss of expression of the CAV-1 gene was further evaluated by semi-quantitative rt-PCR. RESULTS: 28/130 (21.5%) breast cancer cases showed promoter hypermethylation with reduced CAV-1 expression levels when compared with adjacent normal breast tissue. CAV-1 gene hypermethylation was significantly related to menopausal status, histopathological grade and age. CONCLUSION: The rationale of our study is that CAV-1 gene is transcriptionally repressed in breast cancer cells due to hypermethylation. Our results reveal that promoter hypermethylation and loss of expression of the CAV-1 gene is an important alternative mechanism for inactivation of CAV-1 leading to complete gene silencing.

TP53 - molecular soldier's mutations in bladder cancer in the Kashmiri population.

PURPOSE: We made a preliminary attempt to study mutations in exons 5-8 (the DNA binding domain) of the tumor suppressor gene TP53, in urinary bladder cancer patients from Kashmir. Further the relation of clinicopathological characteristics with mutation status was asessed. MATERIALS AND METHODS: The study population consisted of 60 patients diagnosed with transitional cell carcinomas who underwent transurethral resection and /or radical cystectomy. Mutations in 5-8 exons of TP53 gene were detected by means of single strand conformation polymorphism (SSCP). All samples which showed different migration bands in SSCP were confirmed by DNA sequencing. RESULTS: 19 of 60 (31.6%) bladder cancers had mutations of the TP53 gene (11 transitions and 8 transversions), three were G→A transitions, two G→T transversions, three A→C transversions, five C→T transitions and six A→T transversions. Predominantly missense mutations (66%) were detected but no deletions or insertions were found. Statistically significant associations (< 0.05) were noted with higher tumor stage (T2 or higher), recurrence and large tumor size (> 3 cm). No correlation was found between smoking and tumor grade and the presence of TP53 mutations. CONCLUSIONS: Mutation of the TP53 gene is one of the commonest genetic changes in human bladder cancer, also in a high risk ethnic Kashmiri population.

A Rare Case of FAP in Kashmir Valley.

Familial adenomatous polyposis (FAP) is the commonest form of inherited form of CRC. It comprises of about 5% of all the colorectal cancers (CRCs). FAP patients have a family history of CRC that suggests a genetic contribution, common exposures among family members, or a combination of both. This case report gives a glimpse of the phenotypic manifestation of FAP and the underlying molecular mechanism which leads to FAP, in addition it also sheds a light on the management of FAP in early stages of life.

Mutational and promoter hypermethylation status of FHIT gene in breast cancer patients of Kashmir.

OBJECTIVE: Fragile histidine triad (FHIT) gene located at chromosome 3p14.2 is a putative tumor suppressor gene involved in the pathogenesis of breast cancer. Both genetic and epigenetic alterations in FHIT have been implicated in breast carcinoma. In the present study, our main aim was to study the impact of these two kinds of alterations of FHIT gene in breast cancer patients of Kashmir. METHODS: We screened a total of 130 breast cancer patients of Kashmir by PCR-SSCP followed by direct sequencing and methylation specific PCR. RESULTS: Mutational screening of FHIT gene revealed significant amount of mutations [40.7% (53/130)] in five hot spot exons (exon 5-9), FHIT promoter was found to be hypermethylated in 59 of 130 [45.3%] breast cancer patients in our population. CONCLUSION: In the present study we have shown a significant association between the mutational and hypermethylation profile of FHIT gene. Hence, we provide the first evidence to our knowledge that the significant association of FHIT mutation and hypermethylation leads to the complete inactivation of FHIT gene in patients with breast cancer. Silencing of the FHIT gene by promoter hypermethylation occurs in breast carcinomas, especially those with the significant amount of mutations.

Squamous cell carcinoma of rectum presenting in a man: a case report.

BACKGROUND: Primary squamous cell carcinomas of the colorectum are very uncommon. Until now, to the best of our knowledge, only 114 cases of squamous cell carcinoma in the colorectum exist in the reported literature. Here we report a case of squamous cell carcinoma of the rectum in the ethnic Kashmiri population in northern India. CASE PRESENTATION: The case of a 60-year-old male patient (Asian) with a pure squamous cell carcinoma of the rectum is presented here. The patient underwent a curative surgery with concomitant chemotherapy. Two years after the initial curative resection of the tumor he is still alive. CONCLUSION: The prognosis for squamous cell carcinoma of the colorectum is worse than for that of adenocarcinoma, because of the delayed diagnosis. The etiopathogenicity of squamous cell carcinoma of the colorectum is discussed. Surgical resection of the lesion seems to be the treatment of choice. Chemotherapy also helps in improvement of the prognosis.

Mutational profile of the CAV-1 gene in breast cancer cases in the ethnic Kashmiri population.

The role of caveolae and the caveolin proteins in cancer has been the subject of extensive research. It has been suggested that Caveolin-1 may contribute to certain steps of carcinogenesis in various types of cancer. Therefore in our study we focused on the potential clinical relevance of Caveolin-1 in 130 malignant breast tissue specimens along with their adjacent normal tissues. Using allele specific PCR we were able to rule out the mutation status of all the samples and then we did the conventional PCR-SSCP and sequencing of the mutated samples along with the normal adjacent tissues. Caveolin-1 was identified in a screen for genes involved in breast cancer progression and we demonstrated 29.2% mutational status in our Kashmiri ethnic population. We were able to detect 38 mutations out of which 22 were missense, 4 were nonsense, and 12 were frame shifts. Ten novel Cav-1 mutations (missense and frameshift). We conclude that the gene encoding CAV-1 plays an important role in the promotion of mammary tumorigenesis in Kashmir.

Caveolin-1 promotes mammary tumorigenesis: mutational profile of the Kashmiri population.

BACKGROUND: The role of caveolae and the caveolin proteins in cancer has been the subject of extensive research. It has been suggested that caveolin-1 (Cav-1) may contribute to certain steps of carcinogenesis. In the present study we focused on its potential clinical relevance in mammary malignancies. METHODS: We investigated 130 breast cancer samples along with adjacent normal tissues using allele specific PCR for the mutation status and then conventional PCR-SSCP and sequencing of mutated samples along with the normal adjacent tissues. RESULTS: Caveolin-1 was identified in a screen for genes involved in breast cancer progression and we demonstrated 29.2% mutational status in our Kashmiri ethnic population. We were able to detect 38 mutations out of which 22 were missense, 4 were nonsense, and 12 were frame shifts amongst these 38 we were also able to detect ten novel Cav-1 mutations (missense and frameshift mutations). CONCLUSION: We conclude that our study suggests that the gene encoding Cav-1 plays an important role in the promotion of mammary tumorigenesis and are associated with the development and progression of breast cancer.

Genetic aberrations of the K-ras proto-oncogene in bladder cancer in Kashmiri population.

PURPOSE: To assess the frequency of specific point mutations in the K-ras gene in a group of Kashmiri patients with bladder cancer. MATERIALS AND METHODS: We analyzed the incidence of K-ras exon 1 gene mutations in tumors and surgical margins in 60 patients with transitional cell carcinoma of varied clinical stages and histological grades using the polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. RESULTS: A significant correlation was found between the K-ras, the lymph node status, and tumor recurrence (P < 0.05). Also, smokers and patients with higher tumor grade showed a significantly higher relative risk of developing K-ras mutations than the normal ones. CONCLUSION: K-ras exon 1 gene mutations were found with low frequency in the bladder cancer tumors from Kashmir valley, which suggests that K-ras gene might be involved in a sub-set of bladder tumors, but it needs further investigation on a larger cohort sample to authenticate the current findings.

NAD(P)H:quinone oxidoreductase 1 (NQO1) Pro187Ser polymorphism and colorectal cancer predisposition in the ethnic Kashmiri population.

The C609T single nucleotide polymorphism of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene has been identified as an important risk parameter for the susceptibility to colorectal cancer. We here carried out a case-control study and examined the genotype distribution of NQO1 C609T (Pro189Ser) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach, to investigate the possible role of this SNP as a risk factor in colorectal cancer development in Kasmir, India. We investigated the genotype distribution in 86 CRC cases in comparison with 160 healthy subjects and also focused on clinicopathological variables in the CRC cases. The observed genotype frequencies in cases and controls were significantly different [OR=1.64; 95%CI=0.94-2.86]. We also found a significant association between the Ser/Ser variant form with age group, smoking status, tumor location, nodal status/ higher tumor grade and with exposure to pesticides. Therefore, we suggest that the NQO1 C609T SNP is involved either in susceptibility or development of CRC in the ethnic Kashmiri population.

SMAD4--molecular gladiator of the TGF-beta signaling is trampled upon by mutational insufficiency in colorectal carcinoma of Kashmiri population: an analysis with relation to KRAS proto-oncogene.

BACKGROUND: The development and progression of colorectal cancer has been extensively studied and the genes responsible have been well characterized. However the correlation between the SMAD4 gene mutations with KRAS mutant status has not been explored by many studies so far. Here, in this study we aimed to investigate the role of SMAD4 gene aberrations in the pathogenesis of CRC in Kashmir valley and to correlate it with various clinicopathological variables and KRAS mutant genotype. METHODS: We examined the paired tumor and normal tissue specimens of 86 CRC patients for the occurrence of aberrations in MCR region of SMAD4 and exon 1 of KRAS by PCR-SSCP and/or PCR-Direct sequencing. RESULTS: The overall mutation rate of mutation cluster region (MCR) region of SMAD4 gene among 86 patients was 18.6% (16 of 86). 68.75% (11/16) of the SMAD4 gene mutants were found to have mutations in KRAS gene as well. The association between the KRAS mutant genotype with SMAD4 mutants was found to be significant (P = or < 0.05). Further more, we found a significant association of tumor location, tumor grade, node status, occupational exposure to pesticides and bleeding PR/Constipation with the mutation status of the SMAD4 gene (P = or < 0.05). CONCLUSION: Our study suggests that SMAD4 gene aberrations are the common event in CRC development but play a differential role in the progression of CRC in higher tumor grade (C+D) and its association with the KRAS mutant status suggest that these two molecules together are responsible for the progression of the tumor to higher/advanced stage.

Promoter methylation profile of GSTP1 and RASSF1A in benign hyperplasia and metastatic prostate cancer patients in a Kashmiri population.

Promoter hypermethylation is a marginal approach to inactivating tumor suppressor genes in cancer. DNA hypermethylation is a well-recognized epigenetic malfunction observed in several malignancies, most predominantly in prostate cancer. Aberrant DNA methylation patterns are considered to be the earliest somatic genome changes in prostate cancer. The function of promoter hypermethylation in malignant transformation of the prostate has been widely studied, from its presence in benign hyperplasia (BHP) to development and to the advanced stages of tumor formation. In the present study, we examined the promoter hypermethylation status of the glutathione S-transferase P1 (GSTP1) and RASSF1A genes in 45 BHP samples, 50 proven prostate tumor samples and 80 normal samples. Hypermethylated GSTP1 was found in 29/50 (58.0%) prostate carcinoma cases and 12/45 (26.6%) BHP cases. The RASSF1A gene was methylated in 17/50 (34.0%) prostate cancer samples and 7/45 (15.5%) BHP samples. On the basis of these findings, we propose that the epigenetic regulation of the GSTP1 and RASSF1A genes through promoter hypermethylation may play a crucial role in the progression of prostate cancer, and has probable involvement in BHP.

ACE I/D Polymorphism in Hypertensive Patients of Kashmiri Population.

BACKGROUND: The angiotensin-converting enzyme (ACE) gene in humans has an insertion-deletion (I/D) polymorphic state in intron 16 on chromosome 17q23. This polymorphism has been widely investigated in different diseases. In this study we aimed to investigate the ACE I/D genotype frequency in hypertensive cases in Kashmiri population. MATERIALS AND METHODS: We designed a case control study, where 52 hypertensive cases were studied for ACE I/D polymorphism against 150 age/sex matched controls taken from general population. The polymorphisms of ACE gene were investigated using polymerase chain reaction for detection of ACE I/D genotype. Fisher's Chi square test was used for calculation of P value and OR. RESULTS: We found the frequency of ACE DD genotype to be 46.15% (24/52), II 23.07% (12/52) and DI 30.77% (16/52) in 52 hypertensive cases. CONCLUSIONS: The ACE I/D genotype is positively associated with hypertension in our population.

A Case-Control Study of TP53 R72P Polymorphism in the Breast Cancer Patients of Ethnic Kashmiri Population.

BACKGROUND: TP53 R72P polymorphism has been proposed as a risk factor for breast cancer and is more likely to differ among different ethnic populations. We carried out the study to determine the role of R72P polymorphism in breast cancer patients of Kashmir, an ethnic population by PCR-RFLP. METHODS: To evaluate the role of this polymorphism in our ethnic Kashmiri population, we devised our study to study its role in breast cancer patients and healthy controls. The contribution of TP53 R72P polymorphism in 130 breast cancer patients and 220 female healthy controls was assessed using a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). RESULTS: We observed that women with PP genotype have increased risk for developing breast cancer. We found Pro/Pro genotype statistically significantly associated with dwelling, lymph node metastases, histopathological grade, and menopausal status. Pro/Pro genotype in cases and controls was observed and it was found that it is significantly associated with the breast cancer. CONCLUSIONS: Our findings suggest that TP53 R72P polymorphism is a risk factor in breast cancer. Furthermore, these results suggest Pro72 allele is associated with higher risk for breast cancer patients. Women with PP genotype have increased risk for developing breast cancer.

Molecular gate keepers succumb to gene aberrations in colorectal cancer in Kashmiri population, revealing a high incidence area.

BACKGROUND/AIM: Colorectal cancer (CRC) is one of the leading malignancies worldwide and has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The aim of this study was to identify p53 and K-ras gene mutations in CRC patients in a Kashmiri population, and to assess whether these mutations are linked with clinicopathological parameters. MATERIALS AND METHODS: Paired tumor and normal tissue samples from a consecutive series of 53 patients undergoing resective surgery for CRC were prospectively studied for p53 and K-ras gene mutations by PCR/single strand conformation polymorphism (SSCP). RESULTS: Less than half (45%, 19/42) of the patients presented mutations in the p53 gene. Twenty eight mutations were found in the p53 gene, which comprised of 23 substitutions (17 transitions + 6 transversions), and five insertions. The 23 substitutions constituted 18 missense mutations, two nonsense mutations, and three silent mutations. Of the 28 mutations (7.14%) observed in this study, 2 were not previously reported for CRC samples and were identified as novel p53 mutations. A few patients (22.64%, 12/53) presented with mutations in K-ras, constituting 13 missense mutations, out of which 11 were G-->A transitions, one was a G-->C transversion, and one a G-->T transversion. More than half (61.5%) of the mutations occurred in codon 12 whereas a few (38.5%) occurred in codon 13. One tumor contained missense mutations in both codons. Comparison of the mutation profiles of our patients with those of other ethnic populations and regions reflected both differences and similarities, indicating co-exposure to a unique set of risk factors. CONCLUSION: Mutations of the p53 and K-ras genes are some of the most common genetic changes in the development of human CRC. The high frequency of p53 gene mutations implicates p53 as a predominant factor for CRC in the high-risk ethnic Kashmiri population.

Polymorphic analysis of MHClinked Heat Shock Protein 70 genes: Their susceptibility and prognostic implication in Kangri cancer cases of Kashmiri population.

Kangri cancer is a unique thermally-induced squamous cell carcinoma (SCC) of skin that develops due to persistent use of Kangri (a brazier), used by Kashmiri people, to combat the chilling cold during winter months. We designed a large scale case-control study to characterize the frequency of two polymorphisms within the MHC class III-linked HSP70genes, Hsp70-2 and Hsp70-hom, in order to find any association of these genotypic variants for predisposition to and clinical outcome of Kangri cancer patients from Kashmir valley in North India. Polymerase Chain Reaction and restriction enzymes were utilized to characterize the frequency of two polymorphisms with in Hsp70-2 and Hsp70-hom genes in 118 Kangri carcinoma cases and 95 healthy controls from the same population of Kashmir. Association of high frequency allelic variants of Hsp70genes with various clinicopathological features of prognostic significance was assessed by Chi-square test using SPSS software. In this study, allelic frequency of Hsp70-2 A/G heterozygote (0.87) (P = 0.012) was found to be significantly high in Kangri cancer cases compared to control (0.736) with a Relative Risk of 2.45 fold. Conversely, the allelic frequency of Hsp70-2 A/A allele in homozygous condition was significantly low in Kangri cancer cases and worked out to be 0.084 (Vs 0.252 in control) with P is equal to 0.001, implicating it as a protective allele against Kangri cancer in subjects with this genotype. Similarly, significantly high frequency of 0.50 (Vs 0.29 in control) of Hsp70-homC/C allele was found in homozygous condition in Kangri cancer cases suggestive of a positive relative risk associated with this genotype (RR is equal to 2.47) (P is equal to 0.002). The overall allele frequency data analysis of Hsp70-2 and Hsp70-hom genes was significant (chi(2) is equal to 12.38, P is equal to 0.002; and chi(2) is equal to 12.21, P is equal to 0.002). The study also reveals considerable association of high frequency alleles of HSP70 genes, especially of Hsp70-2 A/G or G/G in Kangri tumors with clinico-pathological features of poor prognosis. These results indicate that the relative risk of Kangri cancer associated with Hsp70-2 and Hsp70- hom gene polymorphisms is confined to Hsp70-2 A/G or G/G and Hsp70homC/C haplotype in our population. The study, therefore, suggests Hsp70-2 A/G or G/G and Hsp70homC/C genotypes as potential susceptibility markers and independent prognostic indicators in Kangri carcinoma patients in Kashmiri population.