Do Gamma Rays of Cancer Radiotherapy Effect on the Sequence of Collagenase Gene?

1. Department of biological Applications, Nuclear Research Center, Atomic Energy Authority, Egypt. 2. Department of Zoology, Faculty of Science, Benha University, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Flowcytometry for anti HLA-G was carried out in Hematology Unit in Clinical and Chemical Pathology Department of Sohag University Hospital.
HLA Typing:-All patients and controls were subjected to molecular HLA-DRB1 typing. 2ml EDTA-anticoagulated blood samples were collected from patients and controls. Samples stored by freezing to -80C o then thawed in 37 C o in the incubator in the day of the run. Samples were processed for detection of HLA-DRB1 alleles by Sequence Specific oligonucleotide PCR (SSO-PCR) in following steps : DNA extraction using the spin column technique (QIAamp DNA Blood Mini kits; Qiagen, Hilden, Germany) was performed according to the manufacturer's guidelines; polymerase chain reaction (PCR) amplification of (exon 2) of the HLA-DRB1 alleles (INNO-LiPA HLA-DRB1 Amplification Plus kit; Fuji Rebio Europe, Ghent, Belgium) were performed and the amplicons were chemically denatured to form single-stranded DNA. Reverse dot-blotting was performed on a nylon membrane (INNO-LiPA HLA-DRB1 Plus strips, Fuji Rebio Europe), which contains an array of immobilized, sequence-specific oligonucleotide (SSO) probes. The biotin-labeled amplicons were then bound (hybridized) to these SSO probes, which contain a complementary target sequence and, thus, were captured onto the membrane strip; 3) visualization of the results was achieved by incubating with an enzyme conjugate (streptavidin and alkaline phosphatase), which binds to the biotin of the PCR product, followed by the addition of a substrate. The bands with the captured PCR product turned blue. Interpretation was achieved by using standard tables with the help of specialized software: Gene frequencies of the HLA-DRB1 alleles of both study groups were calculated by direct count. For comparison between the groups, we used 2 analysis by using 2×2 contingency tables and Fisher's exact test when appropriate; values less than 0.05 were considered statistically significant. For estimating risks, we employed odds ratio (OR) with a 95% confidence interval (95% CI).

Results:-
The polymorphism of HLA-DRB1, genes was investigated in twenty normal individuals and forty leukemic patients. The clinical data of the examined subjects were presented in table (1) Comparison of the results between the normal population and acute leukemia patients revealed that there is no difference between anti HLA-G antibodies in both patients and control groups as in table (2) figure (1,2 and), there is an allelic association between certain HLA-DRB1, and leukemia disease. Our results showed that in leukemic patients and in normal individuals, the difference between DRB1*13 was significant (p = 0.006 and p = 0.016 respectively) (protective nature), but there were moderate difference among DRB1*06 (p = 0.098), DRB1*14 (p = 0.098), (also, protective nature) and DRB1 *15 (p = 0.098) (Risk factor). However, we did not observe any significant difference for the others HLA-DRB1.
As regard gender the HLA-DRB1*10 allele was not detected in any of the male patients (P = 0.091). With no significance difference between males and females in other HLA-DRB1, illustrated in figure (3).According to the immunophenotyping of leukemia, the HLA-DRB1*01 allele was not detected in any of the AML patients in relation to ALL patients (P = 0.109). With no significance difference between AML and ALL in other HLA-DRB1.The HLA-DRB1*14 allele was not detected in any of the controls in relation to ALL patients with moderate significance (P = 0.057) (relatively risk factor). The HLA-DRB1*13 show high frequency in controls in relative to ALL patient with moderate significance (P = 0.054) (Relatively Protective). With no significance difference between ALL and controls in other HLA-DRB1 as demonstrated in table -3 and 4. HLA-DRB1*06allele was not detected in any of the controls in relation to AML patients that significant difference (P = 0.044) (risk factor). The HLA-DRB1*13 show high frequency in controls in relative to AML patient (P = 0.006) (Protective nature). With no significance difference between AML and controls in other HLA-DRB1.

Discussion:-
The first study on HLA in human leukemia demonstrated an increased frequency of HLA-A2 in ALL in 1967 .From that date many previous studies have demonstrated some significant differences in HLA allele frequencies in leukemic patients and normal subjects which had reported by Diase et al (2015) and others (14,15,16). In our study, significant positive association with the disease, in patients compared to controls, was found for two alleles: HLA-DRB1*013.Also there were moderate difference among DRB1*06 DRB1*14, and *15. It is suggested that HLA-DRB1*13, and HLA-DRB1*15 alleles may play a protective genetic factor against leukemia while the HLA-DRB1*06 and -DRB1*14 allele could be a presumptive predisposing factor. Regarding anti-HLA-G antibodies analysis, no significant association was found between patients and control groups. Also, our study shows that HLA-DRB1*10 allele was not detected in any of the male patients with moderate significance with no significance difference between males and females in other HLA-DRB1 or anti-HLA-G antibodies. Our results are consistent with Du et al (2013), that demonstrated that the allele frequencies of DRB1*15 obviously decreased in patients with leukemia so, it can be considered as genetic indicators for resistance of leukemia. But they demonstrated that the DRB1*07 have a protective role too (17). Diase et al (2015) had reported that HLA-DRB1*13, which showed a decrease in patients, should be protective against ALL (14). The findings that were demonstrated also by Dorak et al (2013),who reported that the frequency of the HLA-DRB1*13 allele was lower in male patients of leukemia (15).

1814
Also Bosen et al (2013); reported that the frequency of the HLA-DRB1*13 allele was lower the female ALL patients compared to controls (7). On the other hand, Chaing et al (2012) had found that there is significant positive association with the disease, in patients compared to controls, was found for three alleles: HLA-DRB1*13 and HLA-DRB*01 (10). Also there were moderate difference among DRB1*04, *03 and *15.It is suggested that HLA-DRB1*13 (on contrast to our results), *03 and *04 alleles may play a presumptive predisposing factor while the HLA-DRB*01 and *15 (on contrast to our results) alleles could be a protective genetic factor against leukemia (16). A significant increase in the frequency of the HLA-DRB1*04 allele in the overall and ALL patients, was reported in many studies although there is no significant difference between patient and controls (17).We found that the HLA-DRB1*14 allele was not detected in any of the controls in relation to ALL patients with moderate significance (relatively risk factor). There is no significance difference between ALL and controls in other HLA-DRB1 or anti-HLA-G antibodies. Our results are consistent with Ehernberg et al (2014); who found, significant decrease in HLA-DRB1*04 (in contrast to be previously reported by Bosen et al (2013), and an increase in allelic distribution of HLA-DRB1*03 in ALL patients in relative to controls which show no significant difference in our study (7,18).
In our study, The HLA-DRB1*06 allele was not detected in any of the controls in relation to AML patients that significant difference) (risk factor). The HLA-DRB1*13 show high frequency in controls in relative to AML patient (protective nature). There is no significance difference between AML and controls in other HLA-DRB1 or anti-HLA-G antibodies.
Our findings are inconsistent with Elansary et al (2015), who reported that high significant increase allelic distribution in HLA-DRB1*11 and moderate decrease in HLADRB1*07 in AML patients in relative to controls which show no significant difference in our study (19). Bosen et al (2013), observed that the frequencies of the HLADRB1*15 alleles in patients with AML were significantly higher in AML patients compared with healthy controls, Du et a ( 2013 ), observed significant increase in allelic distribution of HLA-DRB1*07 in AML patients and suggests that it is susceptible to the disease in contrast to Ehernberg al (2014) observations. These results not consistent with Chaing et al (2012) that demonstrated that allelic distribution of the HLA-DRB1*13 is significantly high among AML and overall acute leukemia patients group in relation to controls (7, 10, 17 and18). Few studies had been performed on HLA-G expression in different types of leukemia patients, however, data are limited and conclusions remain controversial and discordant (19).Regarding anti-HLA-G antibodies analysis, no significant association was found between patients and control groups. Also our results are inconsistent with the results of other authors who have not found membrane bound or cytoplasmic HLA-G expression (7).A group of previous studies addressed that no cell surface HLA-G was expressed in various types of hematopoietic diseases, such as AML, ALL, CLL and CML. In contrast to these previous studies, B-CLL samples express cell surface HLA-G antigen in a variable proportion of leukemic tumor cells. Methodology difference address this controversy (20).The discrepancy between the results of different studies, including our results, may be attributed to the differences in population race and geographical distribution. However, further larger studies are required to support our findings.

Conclusion:-
Some HLA alleles are associated with an increased susceptibility while others are protective. Although, some of the results from the present study support these earlier findings. The discrepancy between the results of different studies, including our results, may be attributed to the differences in population race and geographical distribution, environmental, occupational factors.

Conflicts of interest:-
The authors had no conflicts of interest to declare in relation to this article.