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

on predisposition to and prognosis of the disease. This work 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 Þ nd 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 signiÞ cance 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 signiÞ cantly 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 signiÞ cantly 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, signiÞ cantly 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 signiÞ cant (χ2 is equal to 12.38, P is equal to 0.002; and χ2 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 Hsp70hom gene polymorphisms is conÞ ned 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 Polymorphic analysis of MHClinked Heat Shock Protein 70 genes: Their susceptibility and prognostic implication in Kangri cancer cases of Kashmiri population


Introduction
[3] The induction of HSP's, occur in wide range of tumors and the mechanism of increased transcription is by either reversal of repression of HSP gene promoter by mutant TP53 (wild type TP53 act as repressor of HSP70 gene) [4][5][6] by increased transcription and stabilization of Heat Shock Factor1 {HSF1} [7] and by positive regulation of HSP genes by protooncogenes like c-myc. [8]The 70Kdal HSP70 family includes three intronless genes, Hsp70-1, Hsp70-2, Hsp 70-hom, that have been mapped within the Class III region of MHC complex on 6p21.3. [9,10]Hsp70-1 and Hsp70-2 genes are 12kb apart and lie 92kb telomeric to C2 gene whileas Hsp70-hom is located 4kb telomeric to Hsp70-1. [9,10]Hsp70-1 and Hsp70-2 encode identical 641 aminoacid proteins [11] whileas the Hsp70-hom encode a 641 aminoacid protein that shares a 90% sequence identity with other Hsp70 proteins. [12]Genetic polymorphism in HSP70 genes may inß uence its anti-apoptotic and immune modulator function and, therefore, may have consequences on predisposition to and prognosis of the disease.This work 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 Þ nd 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 signiÞ cance 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 signiÞ cantly 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 signiÞ cantly 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, signiÞ cantly 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 signiÞ cant (χ 2 is equal to 12.38, P is equal to 0.002; and χ 2 is equal to 12.21, is a case-control study to investigate a potential association of genetic variation of Hsp70-2 and Hsp70-hom genes with the risk to and prognosis of disease (disease outcome) in a cohort of Kangri cancer cases from Kashmir, North India.
Our data indicate that genetic polymorphism in Hsp70-2 and Hsp70-hom genes may represent susceptibility and prognostic indicators.

Patients and controls
The gene and allele frequencies of the Hsp70- Clinico-pathological data of 118 Kangri cancer patients were collected from the department of Medical Oncology and Pathology of SKIMS, Kashmir, India.Control subjects were healthy blood donors from the same population of Kashmir, who have also used Kangri throughout their life but did not develop the cancer.Written informed consent was obtained from all subjects before taking the sample.

DNA extraction
Genomic DNA was extracted from peripheral blood leukocytes by standard procedure.About 5 ml of heparinised blood was mixed with 15 ml of DNA lysis solution (155 mM NH 4 Cl, 10 ml KHCO 3 ,0.1 mM EDTA; PH 8.0).Leucocytes were spun down; suspended in 10 ml of saline EDTA solution (75 mM NaCl, 20 mM EDTA PH 8.0),1 ml of 10% SDS, and incubated with proteinase K at 37°C in a water bath overnight.DNA was subsequently separated from proteins by phenol chloroform iso-amyl alcohol procedure.DNA in the supernatant ß uid was precipitated with ethanol and the pellet was dissolved in 400 µl of DNA storage buffer and stored at 4°C.

Polymorphism analysis of the Hsp70-2 and Hsp70-hom genes
Polymorphism within Hsp70-2 and Hsp70-hom genes has been characterized by Milner and Campbell (1992)   who identiÞ ed a polymorphic Pst1 site at position 1267A greater than G [=1249A greater than G, (GI is equal to 5123454)] of the Hsp70-2 gene and a polymorphic Nco1

Statistical analysis
All statistical analysis was performed using S-PLUS soft-ware.Chi-square test was used to test for a signiÞ cant association between Kangri cancer and Hsp70-2 and Hsp70-hom genotypes (allelic frequencies).Relative risk associated with a particular genotype was estimated by the odds ratio formula. [13]Chi-square test was used to [T2(a,b)] (TNM); [14] lymph node status (N 1 Vs N 0 ) and histopathological Grade [Grade I Vs (Grade II)]. [15]The level of signiÞ cance was set at P less than/equalto 0.05.

Polymorphism in Hsp70-2 and Hsp-hom genes as risk factors for Kangri carcin oma (a) HSP70-2 Polymorphism
The genotype frequencies of Hsp70-2 in patients with Kangri cancer and the control group from the population of Kashmir is shown in Table 1 [Figure 1].The frequency of Hsp70-2A/A allele in patients with Kangri cancer is 0.084 (Vs 0.252 in controls) resulting in a signiÞ cantly negative relative risk (RR is equal to 0.273) associated with this genotype (P less than 0.05).An increase in frequency of Hsp70-2 A/G heterozygote was observed in Kangri cancer cases (0.87) compared to control group (0.736).
The allelic frequency of Hsp70-2 A/G heterozygote was signiÞ cantly more with a relative risk 2.45 fold in Kangri cancer cases (P less than 0.05) compared to control.The allelic frequency of Hsp70-hom genotypes in Kangri cancer patients and control group given in   3], which suggest it rather a very low risk imposing genotypic variant of Hsp70-hom for Kangri cancer.

Discussion
Several studies have shown statistical evidence of association between speciÞ c human leukocyte antigen (HLA) alleles and risk for or protection against various cancers. [16,17]In the present study we report a strong association between speciÞ c Hsp70-2 and Hsp70-hom allelic variants and risk for or protection against Kangri cancers in Kashmiri population.

Comparison of Hsp70-2 allele frequencies in patients
with Kangri cancer and control subjects from same population of Kashmir indicated a signiÞ cant decrease of  Conversely, a high relative risk was found in Kangri cancer cases that carry Hsp70-2 G allele in heterozygous or homozygous state [Table 1].Our Þ nding is in agreement with the two separate reports from Tunisia, where also statistically signiÞ cant breast cancer cases carry Hsp70-2 G allele in homozygous state (0.25) (Vs 0.02 in control) (RR =16.3, P = 0.0001 [18] and 0.280 Vs 0.05 in control (RR = 7.12; P is = 0.0001). [19]The Hsp70-2 G allele in homozygous form has been also reported to impart risk in cancers other than Breast like Non-Hodgkins lymphoma (RR = 18.2;2] Similarly, a striking difference in the frequency of Hsp70-hom genotypes was found in Kangri cancer patients of Kashmir when compared with controls [Table 2].Hsp70-hom C/C genotype in Kangri cancer cases (0.50) (Vs 0.29 in controls) was found to impart signiÞ cantly high relative risk to Kangri cancer (RR = 2.47, P ≤ 0.05) and the risk decreases signiÞ cantly as the allelic frequency of Hsp70-homG allele increases in homozygous or heterozygous state (RR = 0.52, P ≤ 0.05).This is contrary to what has been reported from Tunisia where allelic frequency of Hsp70-hom G allele in homozygous or heterozygous is more in breast cancer cases (0.13) compared to controls (0.05) (RR = 3.4,P = 0.01). [18]More interestingly, Hsp70- The polymorphic Pst1 site in Hsp70-2, although a synonymous variation, has functional signiÞ cance as per recent reports which show the inß uence of synonymous gene variation on the expression levels and enzyme activity, possibly, by affecting the secondary structure of mRNA, its stability and the timing of co-translational folding that alters the substrate or inhibitor binding sites. [23,24]NcoI Hsp70hom polymorphism corresponds to Met greater than Thr substitution at amino acid 493, which from a part of peptide binding domain of Hsp70-hom protein. [12]Based on the Hsp-70 structural model, a Met to Thr substitution at amino acid suggests low assurance of reproducibility. [25]In summary

FORM IV
site at position 2437(C greater than G) [=1630 C greater than G,(G1I is equal to 27436929)] of the Hsp70-hom gene.The position 1267 of the Hsp70-2 gene lies in the coding region, but corresponds to a silent mutation.The polymorphic nucleotide 2437 of the Hsp70-hom gene corresponds to a Met greater than Thr aminoacid substitution.The coding sequence of this Hsp70-2 and Hsp70-hom genes were amplified from g-DNA using sequence speciÞ c oligo-nucleotide primers.For Hsp70-2, the 5' primer: 5' ACC CTG GAG CCC GTG GAG AA was used in combination with the 3' primer: 5' CAC CCG CCC GCC CCG TAG G.For Hsp70-hom, the 5' primer: GGA CAA GTC TGA GAA GGT ACA G-3' was used in combination with the 3' primer: 5' GTA ACT TAG ATT CAG GTC TGG 3'.The PCR mixture contained to a Þ nal concentration of 500 nano-grams of g-DNA, 200 µM of dNTP's, 1.5 mM MgCl 2 , 1X Taq polymerase buffer, 10 picomoles/ul of each primer and 1U of Taq polymerase.AmpliÞ cation was accomplished by initial incubation at 94°C for Þ ve minutes followed by 35 cycles of incubation at 94°C for 30 sec; annealing at 62°C for 35sec and 58°C for 45sec respectively for Hsp70-2 and Hsp-hom; and extension at 72°C for 55 sec.followed by Þ nal incubation at 72°C for seven minutes.To assess the polymorphism of the Hsp70-2 at position 1267 and that of Hsp-hom at position 2437, the corresponding PCR products were digested with Pst1 ) respectively.The presence of Pst1 in Hsp70-2 gene was indicated by the cleavage of the 189bp ampliÞ ed PCR product to yield fragments of 116bp and 73bp products.The two allelic forms of Hsp70-2 corresponding to the presence and absence of Pst1 site are referred to as Hsp70-2 A/A and Hsp70-2 G/G allele respectively.Similarly, the presence of Nco1 site in Hsp70-hom gene was indicated by the cleavage of 878bp ampliÞ ed PCR product to yield fragments of 551bp and 327bp.The two allelic forms of Hsp70-hom corresponding to presence and absence of Nco1 site are referred to as Hsp70-hom C/C and Hsp70-homG/G allele respectively.

Þ
nd any signiÞ cant association of high frequency alleles in Kangri cancer cases with various clinico-pathological parameters of prognostic signiÞ cance.The parameters included were Age; less than 50years Vs greater than/ equal to50 years; Clinical tumor Stage [T1(a,b)] Vs Hsp70-2 G/G allele was 0.042 in Kangri cancer cases (Vs 0.010 in control) with RR is equal to 4.15.The overall frequency of Hsp70-2 G/G allele in homozygous or heterozygous condition was 0.477 in Kangri cancer cases (Vs 0.378 in control).These results indicate that the RR of Kangri cancer associated with Hsp70-2 polymorphism is conÞ ned to Hsp70-2 G allele in homozygous or heterozygous state while as Hsp70-2 A allele in homozygous condition is rather a protective allele for Kangri cancer in our population.(b) Hsp70-hom polymorphism

HSP70's (Hsp70- 2 A
/G or G/G and Hsp70-hom CC in our population) when compared with various clinicopathological attributes of Kangri cancer patients showed statistically signiÞ cant association of Hsp70-2 A/G or G/G genotype with advanced Clinical Tumor Stage [T2 (a,b)] (P ≤ 0.05) and histopathological Grade II (P ≤ 0.05), features that reß ect poor prognosis [Table 3].However, Hsp70-homCC genotype, though found at high frequency among Kangri cancer cases, was signiÞ cantly associated with early Clinical tumor stage T1 (a,b) (P ≤ 0.05) and histopathological grade I (P ≤ 0.05) [Table
the high frequency of Hsp70-2A/G or G/G and Hsp70-homCC haplotype in Kangri cancer cases compared to controls suggest them as susceptibility genotypic variants for Kangri cancer to our population.Further, the signiÞ cant association of high frequency allelic variants of HSP70 genes in Kangri tumors with various clinicopathological parameters suggests them as prognostic indicators as well.The high frequency, especially, of Hsp70-2A/G or G/G genotype in Kangri tumors of patients with such clinico-pathological features as advanced Clinical tumor stage [T2 (a,b)] and histopathological grade II, is a Þ nding which assumes signiÞ cance in view of the fact that these features reß ect poor prognosis.The study suggests Hsp70-2A/G or G/G and Hsp70-homCC genotypes as attractive susceptibility markers and independent prognostic indicators in Kangri cancer patients of Kashmiri population.Nevertheless, these observations need further investigation, a larger cross section of the Kangri cancer patients and relevant control.

Table 2 : Genotype frequencies of Hsp70-hom in control subjects and in patients with Kangri cancer
P< 0.05 Hsp= Heat shock protein; f = genotype frequencies; χ 2 = Chi-square; OR= odds ratio; $ NS= Not signiÞ cant; The Chi-square test was used whether signiÞ cant differences (P-value) in genotype frequencies were observed when patient group was compared with control subjects $ NS; χ 2 =12.21,P = 0.002

Table 3 : Association of high frequency Hsp70-2A/G or G/G and Hsp70-homC/C genotypes with various clinico-pathological features of Kangri cancer patients of Kashmir
rd -4 th class (based on P-values) that Institute of Immunohaematology (ICMR), 13 th Floor, NMS Building, KEM Hospital, Parel, Mumbai -400012, India.6. Names and addresses of individuals who own the newspaper and partners or shareholders holding More than one per cent of the total capital.: Indian Society of Human Genetics I, hereby declare that the particulars given above are true to the best of my knowledge and belief.