HLA Genotyping in Patients with End-Stage Renal Disease Waiting For Cadaveric Renal Transplantation in Federation of Bosnia and Herzegovina

(1)

_______________________________________________________________________________________________________________________________

Open Access Maced J Med Sci. 2017 Mar 15; 5(1):1-5. 1 ID Design 2012/DOOEL Skopje, Republic of Macedonia

Open Access Macedonian Journal of Medical Sciences. 2017 Mar 15; 5(1):1-5.

https://doi.org/10.3889/oamjms.2017.015 eISSN: 1857-9655

Basic Science

HLA Genotyping in Patients with End-Stage Renal Disease Waiting For Cadaveric Renal Transplantation in Federation of Bosnia and Herzegovina

Elma Fejzić¹, Jasenko Karamehić², Izet Eminović³, Damir Suljević³, Andi Alijagić^3*, Semir Bećirević⁴, Amela Šahović¹, Sanela Šišić¹

1Institute of Transfusion Medicine of Federation of Bosnia and Herzegovina, Sarajevo, Bosnia and Herzegovina;

2Department of Immunology, Clinical Center University of Sarajevo, Sarajevo, Bosnia and Herzegovina; ³Department of Biochemistry and Physiology, Faculty of Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina; ⁴Institute of Pathology, Clinical Center University of Tuzla, Tuzla, Bosnia and Herzegovina

Citation: Fejzić E, Karamehić J, Eminović I, Suljević D, Alijagić A, Bećirević S, Šahović A, Šišić S. HLA Genotyping in Patients with End-Stage Renal Disease Waiting For Cadaveric Renal Transplantation in Federation of Bosnia and Herzegovina. Open Access Maced J Med Sci. 2017 Mar 15; 5(1):1-5.

https://doi.org/10.3889/oamjms.2017.015

Keywords: Major Histocompatibility Complex (MHC);

Human Leukocyte Antigens (HLA); Allele groups;

Genotype; Haplotype.

*Correspondence: Andi Alijagic. Trg solidarnosti 21, 71000 Sarajevo, Bosnia and Herzegovina. Tel.:

0038761630937. E-mail: [email protected] Received: 08-Oct-2016; Revised: 18-Nov-2016;

Accepted: 24-Dec-2016; Online first: 30-Jan-2017 Copyright: © 2017 Elma Fejzić, Jasenko Karamehić, Izet Eminović, Damir Suljević, Andi Alijagić, Semir Bećirević, Amela Šahović, Sanela Šišić. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Funding: This research did not receive any financial support.

Competing Interests: The authors have declared that no competing interests exist.

Abstract

AIM: The research was conducted by genotyping two Human Leukocyte Antigen (HLA) gene classes. The main objective of this research was to investigate distribution and frequency of the allelic groups, genotypes and haplotypes in the gene loci of HLA class I (HLA-A*, -B*, -C*) and HLA class II (HLA-DRB1*, -DQB1*) in patients included in the program of cadaveric renal transplantation.

MATERIAL AND METHODS: Our study covered 186 blood samples of patients who are registered on the list for cadaveric renal transplantation in Federation of Bosnia and Herzegovina and included 59 control, healthy unrelated individuals. For the HLA typing, we have used three different methods:

micro lymphocyte cytotoxicity test (MLCT), Polymerase Chain Reaction (PCR) – Sequence Specific Primers (SSP) and PCR – Sequence-Specific Oligonucleotides (SSO) or Luminex technology. All patients and cadaveric donors were tested using the three methods because the system is polymorphic.

RESULTS: Analysis of the results of genotyping HLA class I gene loci identified dominant HLA- A*02, HLA-B*35, HLA-C*07 allelic groups. Analysis of the HLA class II gene loci genotyping showed that HLA-DRB1*11 and HLA-DQB1*03 loci had the highest incidence in HLA class II.

CONCLUSION: Based on our results and previous research, there were no observed differences between allelic frequencies and genotypes of healthy people and people with ESRD. Differences between allelic groups occurred, but they were not statistically significant, except HLA-C*01 (p = 0.020).

Introduction

The main problem with renal transplantation is the lack of suitable dead donors. The improvements in molecular genotyping methods, diagnostics and therapy for maintaining transplants, upgrade the transplantation process, however, the number of dead donors is not increasing, while transplantation lists and the need for dead transplants continues to grow each year [1]. Kidney transplants from live donors produce better results, including faster rehabilitation and better success rate. Another advantage is the

increase in the number of available organs. Higher tissue HLA matching between donors and recipients contributes to the longer survival of transplanted organs [2]. Many transplant centres are limited to living organ donors only. When the donors are divided into groups by degree of HLA compatibility with the organ recipient, a better result in the survival of transplant has been achieved in the cases of higher HLA compatibility. This fact is especially true when the unrelated persons (or distant relatives) are kidney donors [3, 4]. Living organ transplantation should be considered for each organ recipient. MHC antigens, which have proven to be important in kidney

(2)

_______________________________________________________________________________________________________________________________

transplantation, are HLA-A, HLA-B and HLA-DR markers. As these antigens were determined by the genes of both parents, at least six antigens of the recipient (2A, 2B and 2DR) must correspond with the antigens of the potential donor [5]. If the kidney has been taken from a family member, parents or siblings, it is necessary that there is a correspondence in three loci, and partial coincidence in less important, but other present genes HLA-C, HLA-DP and HLA-DQ [6].

Value of the haplotype synchronizing (0, 1, 2) was determined clinically: siblings with two identical haplotypes can expect survival of 90% of transplants after one year, parents and siblings with the same haplotype after one year achieved graft survival of 75%, and members of the same family without equal haplotypes achieve survival of 50% of the grafts after one year [7]. The main problem in transplantation is the immune response of T and B lymphocytes of the host [8]. The most preferred method for the prevention of transplant rejection is achieving sufficient antigen matching between donor and recipient, as with identical twins. Good acceptance of the transplanted tissue or organ is already achieved if the donor and recipient are matched in MHC-II class of antigens (in particular HLA-DR) since they directly activate T- helper lymphocytes of the recipient [9, 10]. HLA antigens are the main target of immune response which leads to the rejection of transplanted organs.

For the purpose of transplantation, a state of the histocompatibility between donor and recipient exists only when the immune response is absent or controlled to foreign graft survive [11]. The reaction of transplant rejection is an immune response, directed primarily towards the molecules of the main histocompatibility system or MHC antigens and other mismatched graft antigens [12, 13].

The main objective of this research was analysis of distribution and frequency of the allelic groups, genotypes and different haplotypes in the gene loci of HLA class I (HLA-A*, -B, -C*) and HLA class II (HLA-DRB1*, -DQB1*) in patients who have been included in the program of cadaveric renal transplantation in Federation of Bosnia and Herzegovina (FB&H) for period 2007-2012.

Materials and Methods

The sample included 186 patients. All patients were in End Stage Renal Disease (ESRD) who were included in the program of cadaveric kidney transplantation in FB&H, and they are not related by blood kinship relations. Research covered patients from 9 hemodialysis center: Sarajevo (39 male; 16 women), Zenica (29 male; 12 women), Bihać (13 male; 11 women), Tešanj (13 male; 10 women);

Travnik (8 male; 5 women), Mostar +Konjic (4 male; 4

women), Živinice + Gračanica (8 male; 1 women), Odžak (6 male; 2 women). Mean age was 49 ± 7 years. Our research on patients was undertaken in compliance with all applicable guidelines, which aim to ensure the proper implementation of the safety of persons participating in the scientific research, including Fundamentals of Good Clinical Practice, Declaration of Helsinki 1975, as revised in 2008, and in the accordance with the approval of the Ethics Committee of the Institute (Approval No. 01-3-3558 23.6.2016). Genotyping included 59 healthy individuals that are not related to the patients; they represented control group.

HLA genotyping

Samples of venous blood were taken into vacutainer tubes with anticoagulant heparin (serological analysis), and samples of venous blood were taken into vacutainer tubes with anticoagulant EDTA (ethylene diamine tetra acetyl acid – molecular analysis). Isolation of DNA was carried out using the kit for DNA, Ready – DNA Spin Kit (Inno-train, Germany). The HLA genotyping was performed by using three different methods: 1) phenotyping of HLA class I (A, B and C) was done by using micro lymphocyte cytotoxicity test (MLCT); 2) genotyping of HLA class I (HLA-A*, -B* and -C*) and HLA class II (HLA-DRB1* and -DQB1*) was performed by low resolution or by using the Polymerase Chain Reaction (PCR) qualitative detection of sequential specific PCR products by agarose gel electrophoresis (method is based on the PCR-SSP or Sequence Specific Primers); 3) genotyping of HLA class I (HLA-A*, -B*

and -C*) and HLA class II (HLA-DRB1* and -DQB1*) was also performed by low resolution, using asymmetric PCR with different primers for each sample. After amplification of the PCR products labelled with biotin, they were mixed and bind to complementary probes during hybridization process (method based on PCR-SSO or Sequence-Specific Oligonucleotides). This technology is also called Luminex technology or technology of fluoroanalyser with microspheres.

Statistical analysis

The frequency of genotypes, gene variants was estimated according to estimation-maximization (EM) algorithm which has been implemented in a computer software PowerMarker v3.25 (Bioinformatic program, Raleigh, NC, USA) and OpenEpi v3.01. [14].

Software for calculating risk ratios (OR) using 2x2 contingency tables was also used. To calculate the statistical significance of the differences in the frequencies of gene variants, and genotypes of the control group and the patients, the Fisher accuracy test, with P >0.05 was used.

(3)

Fejzić et al. Genotyping of Human Leukocyte Antigens in Bosnia and Herzegovina ______________________________________________________________________________________________________________________________

_______________________________________________________________________________________________________________________________

Open Access Maced J Med Sci. 2017 Mar 15; 5(1):1-5. 3

Results

The frequencies of allelic groups of HLA class I (HLA-A*, -B*, -C*) are shown in Table 1. The highest frequency of the allelic groups of HLA-A* gene locus had HLA-A*02 (0.2930) in patients and control group HLA-A* (0.2797). The lowest frequency allelic groups were HLA-A*30 and A*31, while they weren't recorded in the control group.

Table 1: Frequency of allelic groups HLA-A*, HLA-B* and HLA- C* between control and experimental group

a) HLA-A* allelic groups

n Patients 2n Frequency fa

Control 2n Frequency fa

p-values

1. A*01 68 0.1828 A*01 17 0.1441 0.277

2. A*02 109 0.29301 A*02 33 0.2797 0.717

3. A*03 39 0.10484 A*03 10 0.0847 0.503

4. A*11 23 0.06183 A*11 12 0.1017 0.127

5. A*24 36 0.09677 A*24 13 0.1102 0.654

6. A*25 13 0.03495 A*25 4 0.0339 0.956

7. A*26 26 0.06989 A*26 9 0.0763 0.807

8. A*30 9 0.02419 A*30 / - -

9. A*31 9 0.02419 A*31 / - -

10. A*32 12 0.03226 A*32 4 0.0339 0.929

b) HLA-B* allelic groups

p-values

1. B*07 21 0.05645 B*07 4 0.0339 0.318

2. B*08 34 0.0914 B*08 8 0.0678 0.401

3. B*13 15 0.04032 B*13 2 0.0169 0.218

4. B*18 27 0.07258 B*18 11 0.0932 0.445

5. B*27 15 0.04032 B*27 5 0.0424 0.920

6. B*35 45 0.12097 B*35 17 0.1441 0.477

7. B*38 17 0.0457 B*38 4 0.0339 0.572

8. B*44 42 0.1129 B*44 8 0.0678 0.134

9. B*51 54 0.14516 B*51 15 0.1271 0.591

10. B*57 17 0.0457 B*57 5 0.0424 0.820

c) HLA-C* allelic groups

p-values

1. C*01 41 0.11022 C*01 5 0.0424 0.020*

2. C*02 38 0.10215 C*02 8 0.0678 0.239

3. C*03 21 0.05645 C*03 8 0.0678 0.638

4. C*04 66 0.17742 C*04 22 0.1864 0.813

5. C*05 20 0.05376 C*05 4 0.0339 0.371

6. C*06 41 0.11022 C*06 12 0.1017 0.781

7. C*07 120 0.32258 C*07 36 0.3051 0.626

8. C*08 5 0.01344 C*08 1 0.0085 0.667

9. C*12 9 0.02419 C*12 15 0.1271 <0.001*

10. C*14 3 0.00806 C*14 2 0.0169 0.400

*, Statistically different in comparison with control.

Among allelic groups of HLA-B* gene locus the most usual allelic groups were HLA-B*51 (0.1451) in patients group and HLA-B*35 (0.1209) in control group, while allelic groups HLA-B*13 and B*27 in patients group and allelic groups HLA-B*13 in control group had the lowest frequency.

The highest frequency of HLA-C* gene locus had following allelic group HLA-C*04 (0.1774) in patients group and HLA-C*07 (0.3051) at healthy, control group while the lowest frequency value had allelic group C*14 in patients group and C*08 in control group. Significant differences between both groups were recorded for HLA-C*01 and C*12 allelic groups.

Genotyping of HLA class II gene loci determined presence HLA-DRB1*11 allele group with highest frequencies of patients group and HLA- DRB1*13 in control group. Results of analysis of DRB1* gene locus are shown in Table 2.

The highest frequency within the HLA-DQB1* gene locus had allelic group DQB1*03 (0.3333), and HLA- DQB1* 06 is very frequently in both group. It was not noticed an absence of any allelic group in HLA-DQB1*

gene locus. Results of analysis of DQB1* allelic groups are shown in Table 2.

Table 2: Frequency of HLA-DRB1* and HLA-DQB1* gene loci between control and experimental group

HLA-DRB1*

p-values

1. DRB1*01 45 0.12097 DRB1*01 13 0.1102 0.733

2. DRB1*03(17) 43 0.11559 DRB1*03(17) 16 0.1356 0.531

3. DRB1*04 47 0.12634 DRB1*04 9 0.0763 0.110

4. DRB1*07 29 0.07796 DRB1*07 9 0.0763 0.950

5. DRB1*08 14 0.03763 DRB1*08 5 0.0424 0.812

6. DRB1*11 57 0.15323 DRB1*11 15 0.1271 0.443

7. DRB1*13 38 0.10215 DRB1*13 20 0.1695 0.030*

8. DRB1*14 22 0.05914 DRB1*14 5 0.0424 0.751

9. DRB1*15 36 0.09677 DRB1*15 13 0.1102 0.654

10. DRB1*16 29 0.07796 DRB1*16 11 0.0932 0.580

HLA-DQB1*

p-values

1. DQB1*02 68 0.1828 DQB1*02 22 0.1864 0.919

2. DQB1*03 124 0.48334 DQB1*03 36 0.3050 0.427

3. DQB1*04 12 0.03226 DQB1*04 3 0.0254 0.702

4. DQB1*05 106 0.28495 DQB1*05 28 0.2373 0.200

5. DQB1*06 62 0.16667 DQB1*06 29 0.2458 0.028*

*, Statistically different in comparison with control.

HLA-A* gene locus analysis revealed 63 different HLA-A* genotypes for 186 observed patients (Table 3.). The most common genotypes were HLA- A*02/A*02 (0.1022), and HLA-A*01/A*02 (0.0753).

Table 3: Frequency of HLA-A* genotypes between control and experimental group

HLA-A* genotypes

n Patients n Genotype frequency

Control n Genotype frequency

p-values

1. A*01/A*01 7 0.0376 A*01/A*01 / / -

2. A*01/A*02 14 0.0753 A*01/A*02 5 0.0847 0.812

3. A*01/A*03 11 0.0591 A*01/A*03 / / -

4. A*01/A*24 8 0.043 A*01/A*24 3 0.0508 0.800

5. A*01/A*26 6 0.0323 A*01/A*26 1 0.0169 0.460

6. A*02/A*02 19 0.1022 A*02/A*02 4 0.0678 0.424 7. A*02/A*03 14 0.0753 A*02/A*03 4 0.0678 0.848

8. A*02/A*11 8 0.043 A*02/A*11 5 0.0847 0.107

9. A*02/A*24 11 0.0591 A*02/A*24 4 0.0678 0.809 10. A*02/A*26 7 0.0376 A*02/A*26 3 0.0508 0.654

Within the HLA-B* gene locus, in 186 patients, an analysis revealed 96 different genotypes (Table 4). The most common genotypes were HLA- B*35/B*44 (0.0484) and HLA-B*08/51*B (0.0376). In the control group, the most frequent genotypes in B*

locus are HLA-B*08/51 and HLA-B*35/51 (0.0508).

Table 4: Frequency of HLA-B* genotypes between control and experimental group

HLA-B* genotypes

p-values

1. B*07/B*35 5 0.0269 B*07/B*35 1 0.0169 0.667

2. B*07/B*44 4 0.0215 B*07/B*44 / / -

3. B*08/B*44 6 0.0323 B*08/B*44 / / -

4. B*08/B*51 7 0.0376 B*08/B*51 3 0.0508 0.654

5. B*13/B*18 6 0.0323 B*13/B*18 1 0.0169 0.538

6. B*18/B*51 4 0.0215 B*18/B*51 1 0.0169 0.829

7. B*35/B*35 5 0.0269 B*35/B*35 1 0.0169 0.667

8. B*35/B*44 9 0.0484 B*35/B*44 2 0.0339 0.639

9. B*35/B*51 5 0.0269 B*35/B*51 3 0.0508 0.366

10. B*38/B*44 4 0.0215 B*38/B*44 / / -

11. B*38/B*51 4 0.0215 B*38/B*51 1 0.0169 0.829

12. B*39/B*51 4 0.0215 B*39/B*51 1 0.0169 0.829

13. B*51/B*51 6 0.0323 B*51/B*51 / / -

(4)

_______________________________________________________________________________________________________________________________

Within the HLA-C* gene locus it was found 37 different genotypes (Table 5), the most common genotypes were HLA-C*07/07*C (0.1774), HLA- C*04/04*C (0.1022) and HLA-C*04/07*C (0.0699) in patients group. In the control group, the most frequent genotype is HLA-C*07/07*C (0.1356).

Table 5: Frequency of HLA-C* genotypes between control and experimental group

HLA-C* genotypes

p-values

1. C*01/ C*01 10 0.0538 C*01/ C*01 / / -

2. C*02/ C*02 7 0.0376 C*02/ C*02 / / -

3. C*02/ C*07 7 0.0376 C*02/ C*07 1 0.0169 0.436 4. C*03/ C*07 7 0.0376 C*03/ C*07 1 0.0169 0.436 5. C*04/ C*04 19 0.1022 C*04/ C*04 3 0.0508 0.230 6. C*04/ C*07 13 0.0699 C*04/ C*07 5 0.0847 0.703 7. C*07/ C*07 33 0.1774 C*07/ C*07 8 0.1356 0.453

8. C*01/ C*06 7 0.0376 C*01/ C*06 / / -

9. C*06/ C*06 7 0.0376 C*06/ C*06 / / -

10. C*06/ C*07 12 0.0645 C*06/ C*07 5 0.0847 0.086

Within the HLA-DRB1* gene locus it was determined the presence of 58 different genotypes, including the most common HLA-DRB1*01/DRB1*11 (0.0430), HLA-DRB1*04/DRB1*11 and HLA- DRB1*13/DRB1*16 genotype (0.0376) in patients group.

Table 6: Frequency of HLA-DRB1* genotypes between control and experimental group

HLA-DRB1* genotypes

n Patients n Genotype

frequency Control n Genotype Frequency

p- values 1. DRB1*01/ DRB1*11 8 0.043 DRB1*01/ DRB1*11 / / - 2. DRB1*01/ DRB1*04 6 0.0323 DRB1*01/ DRB1*04 1 0.0169 0.538 3. DRB1*03(17)/

DRB1*11 6 0.0323 DRB1*03(17)/

DRB1*11 1 0.0169 0.538 4. DRB1*03(17)/

DRB1*16 6 0.0323 DRB1*03(17)/

DRB1*16 2 0.0339 0.950 5. DRB1*04/DRB1*07 6 0.0323 DRB1*04/DRB1*07 3 0.0508 0.508 6. DRB1*04/ DRB1*11 7 0.0376 DRB1*04/ DRB1*11 2 0.0339 0.894 7. DRB1*11/ DRB1*14 6 0.0323 DRB1*11/ DRB1*14 3 0.0508 0.508 8. DRB1*11/ DRB1*15 6 0.0323 DRB1*11/ DRB1*15 1 0.0169 0.538 9. DRB1*13/ DRB1*16 7 0.0376 DRB1*13/ DRB1*16 / / -

Research results within the HLA-DQB1* gene locus revealed 27 different genotypes. The most usual genotypes were HLA-DQB1*03/DQB1*05 (0.2204) and HLA-DQB1*02/DQB1*03 (0.1667) in patients group. The genotype HLA-DQB1*02/DQB1*03 is the most frequent in the control group.

Table 7: Frequency of HLA-DQB1* genotypes between control and experimental group

HLA-DQB1* genotypes

n Patients n Genotype

frequency

p- values 1. DQB1*02/

DQB1*02 8 0.043 DQB1*02/

DQB1*02 / / -

2. DQB1*02/

DQB1*03 31 0.1667 DQB1*02/

DQB1*03 10 0.1694 0.959 3. DQB1*02/

DQB1*05 21 0.1129 DQB1*02/

DQB1*05 4 0.0678 0.318 4. DQB1*03/

DQB1*03 21 0.1129 DQB1*03/

DQB1*03 3 0.0508 0.162 5. DQB1*03/

DQB1*05 41 0.2204 DQB1*03/

DQB1*05 7 0.1186 0.086 6. DQB1*03/

DQB1*06 22 0.1182 DQB1*03/

DQB1*06 8 0.1356 0.723 7. DQB1*05/

DQB1*05 13 0.0699 DQB1*05/

DQB1*05 6 0.1017 0.426 8. DQB1*05/

DQB1*06 14 0.0753 DQB1*05/

DQB1*06 3 0.0508 0.520

Discussion

HLA class I molecules can be found on the surface of all cells that contain the nucleus, while class II of HLA molecules can be constitutively found on the surface of certain types of cells (dendritic cells, macrophages, B-lymphocytes. HLA-DR (not HLA-DQ, -DP, or -DM) is abundantly expressed on the endothelial cells of peritubular and glomerular capillaries [15].

Table 8: Frequency of allelic group with Odds Ratio (OR) and p- values between control and patients group

Allelic groups 2n

Patients Allele frequency 2n

Control

Allele frequency OR p-values

HLA-C*01 41 0.11022 5 0.0424 2.799 0.020

HLA-C*12 9 0.02419 15 0.1271 0.1702 0.0000063

HLA-DRB1*13 38 0.10215 20 0.1695 0.5575 0.030

HLA-DQB1*06 62 0.16667 29 0.2458 0.6138 0.0280

The explanation for the improved survival of kidney allografts in which HLA have good congruence was: lower occurrence of anti-HLA antibodies [16], lower occurrence of alloreactive CD4+ T-cells or absence of direct CD4+ T-cell response to HLA-DR matched graft [17], fewer peptide epitopes stimulate response of T-helper cells of "indirect way" which includes chronic rejection of allograft [18]. HLA matching in HLA-A, HLA-B and HLA-DR loci increases the likelihood of developing a donor antigen-specific regulatory T-cells [19].

Benefits of HLA matching has an impact on different outcomes in terms of number of days spent in hospital, failure of graft function [20, 21], episodes of rejection, the one-year and three-year levels of serum creatinine [22], on prediction of long-term outcome of the disease, on status of patients and on multivariate analysis [23, 24].

Chronic renal failure (CRF) leads in most cases to ESRD, with final result – kidney transplantation process. There is an interest to assess connection of class I and II of HLA antigens with ESRD or CRF renal diseases [25].

In hemodialysis patients that are the part of cadaveric renal transplantation program in FB&H it was observed that the HLA antigens with the greatest frequency were: HLA-A*02 = 0.29301, HLA-B*51 = 0.14516, HLA-C*07 = 0.32258, HLA-DRB1*11 = 0.15323 and HLA-DQB1*03 = 0.48334. The antigens that showed in control group greatest frequency were:

HLA-A*02= 0.2797, HLA-B*35= 0.1441, HLA-C* = 0.03051, HLA-DRB1*13 = 0.1695 and HLA-DQB1*03

= 0.3050. In the analysis of allelic groups in each locus, with estimation of the p-value, the allelic group HLA-A* showed no statistically significant difference in the aforementioned allelic groups. Such results were also recorded when comparing the frequencies of allelic groups in HLA-B*. Locus C* showed a statistically significant difference in the frequency of the allelic HLA-C*01 with p = 0.020 and OR = 2.601,

(5)

Fejzić et al. Genotyping of Human Leukocyte Antigens in Bosnia and Herzegovina ______________________________________________________________________________________________________________________________

_______________________________________________________________________________________________________________________________

that is considered as an allelic group of high risk, and it showed a difference in the allelic HLA-C*12 with p = 0.0000679. Although the OR value is 0.190, this allelic group can't be considered protective. The HLA-DRB1*

locus also showed statistical significance in frequencies in the allelic HLA-DRB1*13 (p = 0.030) with OR = 0.6027, and it is not at risk of developing ESRD. The allelic group HLA- DQB1*06 showed statistical significance (p = 0.028) and OR = 0.6138, and it is not considered as a group of high risk, as shown in Table 3.

Our research covered hemodialysis patients included in program of cadaveric renal transplantation in FB&H. The HLA class I (HLA-A*, -B*, -C*) genotypes with the highest incidence were: A*02/A*02

= 0.1022, B*35/B*44 = 0.0484, C*07/07 = 0.1774.

HLA class II genotypes with highest incidence were:

DRB1*01/DRB1*11 = 0.043 and DQB1*03/DQB1*05 = 0.2204.

Based on our results and previous research, there were no observed differences between allelic frequencies and genotypes of healthy people and people with ESRD. Differences between allelic groups occurred, but they were not statistically significant, except HLA-C*01, p = 0.020.

References

1. Karamehić J, Dizdarević Z, et al. Komponente imunog sistema (Components of immune system). In: Karamehić J, Dizdarević Z, et al., editors. Klinička imunologija (Clinical Immunology). Sarajevo:

Svjetlost, 2007:pp. 53-62.

2. Hume DM, Merrill JP, Miller BF, Thorne GW. Experiences with renal homotransplantations in humans: report of nine cases. J Clin Invest. 1955;34(2):327. https://doi.org/10.1172/JCI103085 PMid:13233354 PMCid:PMC438633

3. Kramer MR, Sprung CL. Living-related donation in lung transplantation. Ethical con siderations. Arch Int Med.

1995;155(16):1734-8.

https://doi.org/10.1001/archinte.1995.00430160054006 4. Tesi R, Beck R, Lambiase L, Haque S, Flint L, Jaffe B. Living related small bowel transplantation-donor evaluation and outcome.

In: Abstract book of XVI International Congress of the Transplantation Society; Barcelona, Spain,1996.

5. Hirata M, Terasaki PI. Renal re-transplantation. Clin Transplant.

1994;8:419-33.

6. Đurinović-Bello I, Pasini J, Barašić D, Thune S, Puretić Z, Gerenčer M, et al. Early detection of allograft rejection by donor- specific lymphocyte –mediated cytolysis: cloning of cytotoxic lymphocytes from positive patients. Transplantant Proc.

1987;19(5):4283-5. PMid:3314029

7. Busson M, Raffoux C, Bouteiller AM, Betuel H, Cambom- Thomsen A, Fizet D, et al. Influence of HLA-A, -B, and –DR matching on the outcome of kidney transplant survival in pre- immunized patens. Transplantation. 1984;38(3):227-9.

https://doi.org/10.1097/00007890-198409000-00006 PMid:6382711

8. Salvatierra Jr O, Melzer J, Potter D, Garovoy M, Vincent F, Amend WJ, et al. Seven-year experience with donor-specific blood transfusions (DST): Results and considerations for maximum efficacy. Transplantation. 1986;40:654-8.

https://doi.org/10.1097/00007890-198512000-00016

9. Abbas AK, Lichtman AH. Basic Immunology: Functions an Disorders of the Immune System. New York: Elsevier Inc., 2006- 2007: pp. 161-77.

10. Abbas KA, Lichtman HA. Basic Immunology. 2nd ed. Beograd:

Data status, 2007: pp. 48-51.

11. Lakkis FG. Transplantation immunobiology. In: Karamehić J.

and contributing authors, editors. Transplantation of kidney.

University of Sarajevo-Yale University, 2004: pp. 57-68.

PMCid:PMC311434

12. Janeway CA, Travers P, Walport M, Shlomchik M, editors. The Immune system in health and disease. In: Immunobiology. New York: Garland Publishing, 2005: pp. 557-96.

13. Janeway CA, Travers P, Walport M, Shlomchik M, editors.

Extrinsic regulation of unwanted immune responses. In:

Immunobiology. New York: Garland Science Publishing, 2005: pp.

613-30.

14. Liu K, Spencer VM. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics.

2005;21(9):2128-29. https://doi.org/10.1093/bioinformatics/bti282 PMid:15705655

15. Muczynski KA, Cotner T, Anderson SK. Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium. Kidney Int. 2001;59(2):488-97.

https://doi.org/10.1046/j.1523-1755.2001.059002488.x PMid:11168931

16. Terasaki PI. Humoral theory of transplantation. Am J Transplant. 2003;3(6):665-73. https://doi.org/10.1034/j.1600- 6143.2003.00135.x PMid:12780557

17. Vandekerckhove BA, Datema G, Zantvoort F, Claas FH. An increase of donor-specific T helper precursors resulting from blood transfusions. Transplantation. 1990; 49(5): 987-90.

https://doi.org/10.1097/00007890-199005000-00029 PMid:2139988

18. Ciubotariu R, Liu Z, Colovai AI, Ho E, Itescu S, Ravalli S, et al.

Persistent allopeptide reactivity and epitope spreading in chronic rejection of organ allografts. J Clin Invest. 1998;101(2):398.

https://doi.org/10.1172/JCI1117 PMid:9435312 PMCid:PMC508579 19. Rodriguez DS, Jankowska‐Gan E, Haynes LD, Leverson G, Munoz A, Heisey D, et al. Immune regulation and graft survival in kidney transplant recipients are both enhanced by human leukocyte antigen matching. Am J Transplant. 2004;4(4):537-543.

https://doi.org/10.1111/j.1600-6143.2004.00385.x PMid:15023145 20. Gjertson DW. Impact of delayed graft function and acute rejection on kidney graft survival. In: Terasaki P, editor. Clinical Transplants. Los Angeles: UCLA Immunogenetics Center, 2001:

pp. 467.

21. Shoskes DA, Cecka JM. Effect of delayed graft function on short- and longterm kidney graft survival. In: Terasaki P, editor.

Clinical Transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1997: pp. 297. PMid:9919413

22. Gjertson DW. Two-factor reference tables for renal transplantation. In: Terasaki P, editor. Clinical Transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1996: pp. 433.

23. Takemoto SK, Cho YW, Gjertson DW. Transplant risks. In:

Terasaki PI, editor. Clinical Transplants. Los Angeles: UCLA Immunogenetics Center, 2000: pp. 325.

24. Cho YW, Terasaki PI. Impact of new variables reported to the UNOS registry. In: Terasaki P, editor. Clininical Transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1998: pp. 305.

25. Crispim JC, Mendes-Junior CT, Wastowski IJ, Palomino GM, Saber LT, Rassi DM, Donadi EA. HLA polymorphisms as incidence factor in the progression to end-stage renal disease in Brazilian patients awaiting kidney transplant. Transplantation proceedings.

2008;40(5):1333-1336.

https://doi.org/10.1016/j.transproceed.2008.02.086 PMid:18589099

(6)

ID Design 2012/DOOEL Skopje, Republic of Macedonia

Basic Science

Sensitivity to Antimicrobial Drugs of Pseudomonas Aeruginosa Extreme-Resistant Strains Isolated in the Major Hospitals of Central Kazakhstan

Ilya S. Azizov¹, Alyona V. Lavrinenko²*, Ilya A. Belyaev², Dmitry B. Babenko², Natalya A. Shambilova¹, Nelya M. Bissenova²

1Institute of Antimicrobial Hemotherapy, Smolensk, Russia; ²Karaganda State Medical University, National Scientific Medical Center, Karaganda, Kazakhstan

Citation: Azizov IS, Lavrinenko AV, Belyaev IA, Babenko DB, Shambilova NA, Bissenova NM. Sensitivity to Antimicrobial Drugs of Pseudomonas Aeruginosa Extreme-Resistant Strains Isolated in the Major Hospitals of Central Kazakhstan. Open Access Maced J Med Sci.

2017 Mar 15; 5(1):6-8.

https://doi.org/10.3889/oamjms.2017.023

Keywords: Pseudomonas aeruginosa; carbapenemases gene; Antibiotic resistance; MALDI-TOF; VIM.

*Correspondence: Alyona V. Lavrinenko. Karaganda State Medical University, National Scientific Medical Center, Karaganda, Kazakhstan. E-mail:

[email protected]

Received: 26-Dec-2016; Revised: 25-Jan-2017;

Shambilova, Nelya M. Bissenova. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Abstract

AIM: The article presents the current data on the sensitivity of the main 37 strains of eXtremaly Drugs Resistance (XDR) category to anti-pseudomonas drugs.

MATERIAL AND METHODS: The strains were collected during the prospective multicenter study in large multidisciplinary hospitals of Central Kazakhstan. Susceptibility to antimicrobial drugs was carried out by disk method and the serial dilution method with the interpretation of the results according to EUCAST criteria. Detection of carbapenemases gene of VIM, IMP, NDM and GES classes was carried out by PCR method using the commercial kits.

RESULTS: All identified carbapenemases were sorted to VIM class and accounted for 63.64%.

Resistance to aminoglycoside drugs exceeded 80%. All the strains were susceptible to polymyxin.

CONCLUSION: Thus, at the present stage the circulation of P. aeruginosa strains of XDR category continues in major hospitals in Kazakhstan. The strains remain sensitiveness only to polymyxin.

Introduction

Antibiotic resistance as a phenomenon of microorganisms' insensibility to achievable concentrations of antibiotics in clinical conditions has become in the last 20 years the pattern of the global problem, migrated from the level of individual departments and hospitals to the level of a global epidemic process, threatening the future of humanity.

Every year more than 20 thousand patients die in the USA in the result of infectious processes caused by multidrug-resistant microorganisms. The USA government spends more than 20 billion dollars a year for the control of antibiotic-resistant strains spreading [1].

The EU countries annually spend more than 9 billion euros for the solution of the problem. At the same time in the European Union more than 25 thousand patients a year die because of ineffective antimicrobial chemotherapy, and more than half of the cases caused by Gram-negative microorganisms [2].

The last in most cases have multidrug resistance mechanisms, leading to a significant narrowing of the list of choice drugs and in the cases of pan-resistance – to almost no alternative solutions [3].

Till recently carbapenems were regarded as the drugs of extreme selection. However wide spreading of carbapenemases genes significantly expanded the list of problematic strains in which of Pseudomonas aeruginosa is regarded as a classic representative [4]. Modern strains of P. aeruginosa in addition to many natural mechanisms and

(7)

Azizov et al. Pseudomonas Aeruginosa Extreme-Resistant Strains Isolated in Central Kazakhstan ______________________________________________________________________________________________________________________________

_______________________________________________________________________________________________________________________________

antimicrobial resistance mechanisms due to the high genetic lability constantly replenish its arsenal of acquired resistance mechanisms [5] and have the predilection to the clonal global spreading [6]. From this perspective, the continuous supervision for the local spread of multidrug-resistant strains is important for practical health care as well as for fundamental science. The strains of XDR (eXtremaly Drugs Resistance) category [7] are the particular problem for medicine because of extreme multi-resistance to a wide range of antimicrobial agents.

Our study focuses on the description of the sensitivity of antimicrobial agents and detection of genes that determine resistance to carbapenems in of P. aeruginosa strains of XDR category, isolated in large hospitals of Central Kazakhstan.

Materials and Methods

The study included strains collected in the period from 2015 to 2016 during the prospective multicenter microbiological research covering large multidisciplinary hospitals of Central Kazakhstan (Karaganda and Astana).

Isolation of strains was conducted in local bacteriological laboratories of participating centres, and after the strains were forwarded to the microbiology laboratory of the Scientific-Research Center of Karaganda State Medical University, where it was conducted the re-identification methods of time- of-flight mass-spectrometry (MALDI-TOF) using MALDI-Biotyper software (Bruker). Determination of sensitivity to antimicrobial agents was conducted by disk- diffusion method and by the method of serial microdilution in a liquid medium according to EUCAST recommendations [8].

The primary test for detection of Metallo-beta- lactamase activity was carried out with 100 mM EDTA by the recommendations [9]. Additional screening CIM test for detection of carbapenemases activity was carried out by the recommendations [10].

The presence of carbapenemases genes of VIM and IMP classes were performed by Real-Time PCR methods using the commercial kit «AmpliSens MDR MBL-FL» produced by Interlab Service (Russia).

Statistical processing was performed by determining the average values, the definition of rank correlation coefficient by Spearman and determining the 95% confidence interval for the mean values by Klopper-Pearson with the use of MS Excel and Whonet 6.5 [11].

Results

As a result of screening, it was selected 37 strains with XDR phenotypic profile among 270 strains collected in large multidisciplinary hospitals of Central Kazakhstan.

Data on antimicrobial resistance is shown in Figure 1.

Figure 1: The share of non-sensitive (%R+%I) hospital P.

aeruginosa strains to antimicrobial drugs. The dotted squares represent 95% confidence intervals

The studied strains were characterised by resistance to the absolute majority of drugs with anti- pseudomonas activity. The exception was polymyxin, to which we did not reveal any resistant strain. Taking into account the trend towards the emergence of resistant strains of P. aeruginosa to colistin [12] we carried out a quantitative evaluation of the sensitivity of the studied strains to polymyxin.

MIC50 for studied strains was 0.5 µg/ml but MIC range was 0.5-2 µg/ml. This pattern suggests polymyxin as the only available anti-pseudomonas drug with high activity, and it actualizes the questions on the development of technologies increasing the bioavailability of the drug [13]. Sensitivity to aztreonam was observed in more than half of the cases (51.43%; 95% CI, 31.25-71.15). A similar pattern is due to the high frequency of occurrence of strains producing Metallo-beta-lactamase (B class) hydrolyzing all beta-lactams except aztreonam [14], which proportion was 68.75%.

Genetic typing of the mechanisms of resistance to carbapenems identified the carbapenemases genes of VIM class, the proportion of strains producing carbapenemases of VIM class was 63.64% (95% CI 39.63-81.17). Meanwhile, the test with chelating agent EDTA showed inhibitory activity in 31 strains that in combination with MIC values corresponding to the expected moderate stability permit to expect the low-affinity carbapenemases of GES class. However, conducted research has not revealed GES carbapenemases genes. Ecoff analysis of distributions on imipenem (Fig. 2) allows surmising sampling heterogeneity.

According to that 75% isolates have MIC higher 32 µg/ml we expected an equal number of

(8)

_______________________________________________________________________________________________________________________________

producers of carbapenemases. At the same time, PCR detection of VIM producers was positive in 63.64% which is clearly linked to other mechanisms of resistance. Strains with moderate resistance imipenem were moderately resistant to meropenem and also to aminoglycosides that can be connected with other on- enzymatic resistance mechanisms [15].

Figure 2: Ecoff distribution of MIC imipenem for studied P.

aeruginosa strains (boundary values 4-16 µg/ml), S – sensitive population, I – moderately resistant, R – resistant population

Resistance to fluorinated quinolones was detected in more than 90%, the average MIC values were extremely high too (>32 µg/ml). Resistance to ciprofloxacin had a strong positive correlation with resistance to levofloxacin, which is obviously connected with the general resistance mechanisms and can approximate these results to the whole group of fluorinated quinolones.

Discussion

At present the only drug with a high-clinically significant activity against studied pan-resistant P.

aeruginosa strains is polymyxin. We did not reveal any cases of resistance; all the studied strains had MIC less than 1 µg/ml. The average values of the MIC of polymyxin totalled 0.49 µg/ml.

The resulting picture clearly shows that there is no alternative situation on the choice of drugs for the causal treatment of infections caused by extremely resistant P. aeruginosa strains.

Thus, at the present stage, the circulation of P. aeruginosa strains of XDR category continues in major hospitals in Kazakhstan, which have been shown previously [16]. The strains remain sensitiveness only to polymyxin.

References

1. Michael CA, Dominey-Howes D, Labbate M. The antimicrobial resistance crisis: causes, consequences, and management. Front

Public Health. 2014; 2: 145.

https://doi.org/10.3389/fpubh.2014.00145 PMid:25279369 PMCid:PMC4165128

2. The Center for Diseases Dynamic, E.a.P. The State of the World's Antibiotics, 2015. CDDEP: Washington, D.C., 2015.

3. Centers for Disease Control and Prevention, O.o.I.D. Antibiotic resistance threats in the United States, 2013. 2013; Available from:

http://www.cdc.gov/drugresistance/threat -report-2013.

4. Zavascki AP, et al. Multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii: resistance mechanisms and implications for therapy. Expert Rev Anti Infect Ther. 2010; 8(1):71- 93. https://doi.org/10.1586/eri.09.108 PMid:20014903

5. El Zowalaty ME, et al. Pseudomonas aeruginosa: arsenal of resistance mechanisms, decades of changing resistance profiles, and future antimicrobial therapies. Future Microbiol. 2015;10(10):

1683-706. https://doi.org/10.2217/fmb.15.48 PMid:26439366 6. Woodford N, Turton JF, Livermore DM. Multiresistant Gram- negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev. 2011;35(5): 736-55.

https://doi.org/10.1111/j.1574-6976.2011.00268.x PMid:21303394 7. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G,

Olsson‐Liljequist B, Paterson DL. Multidrug‐resistant, extensively drug‐resistant and pandrug‐resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical microbiology and infection. 2012;18(3):268-81.

https://doi.org/10.1111/j.1469-0691.2011.03570.x PMid:21793988 8. EUCAST, European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 6.0, valid from 2016-01-01, 2016.

9. Arakawa Y, et al., Convenient test for screening metallo-beta- lactamase-producing gram-negative bacteria by using thiol compounds. J Clin Microbiol. 2000;38(1): 40-3. PMid:10618060 PMCid:PMC86013

10. van der Zwaluw K, et al. The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

PLoS One. 2015;10(3): e0123690.

https://doi.org/10.1371/journal.pone.0123690 PMid:25798828 PMCid:PMC4370852

11. Stelling JM, O'Brien TF. Surveillance of antimicrobial

resistance: the WHONET program. Clin Infect Dis. 1997;24 (Suppl 1): S157-68. https://doi.org/10.1093/clinids/24.Supplement_1.S157 PMid:8994799

12. Fernandez-Barat L, et al. Phenotypic shift in Pseudomonas aeruginosa populations from cystic fibrosis lungs after 2-week antipseudomonal treatment. J Cyst Fibros. 2016.

https://doi.org/10.1016/j.jcf.2016.08.005 PMid:27651273 13. Lin B, Zhang C. Xiao X. Toxicity, bioavailability and

pharmacokinetics of a newly formulated colistin sulfate solution. J Vet Pharmacol Ther. 2005; 28(4): 349-54.

https://doi.org/10.1111/j.1365-2885.2005.00666.x PMid:16050814 14. Poirel L, Pitout JD, Nordmann P. Carbapenemases: molecular diversity and clinical consequences. Future Microbiol.

2007;2(5):501-12. https://doi.org/10.2217/17460913.2.5.501 PMid:17927473

15. Tangden T, Giske CG. Global dissemination of extensively drug-resistant carbapenemase- producing Enterobacteriaceae:

clinical perspectives on detection, treatment and infection control. J Intern Med. 2015;277(5): 501-12.

https://doi.org/10.1111/joim.12342 PMid:25556628

16. Edelstein MV, et al. Spread of extensively resistant VIM-2- positive ST235 Pseudomonas aeruginosa in Belarus, Kazakhstan, and Russia: a longitudinal epidemiological and clinical study.

Lancet Infect Dis. 2013;13(10): 867-76.

https://doi.org/10.1016/S1473-3099(13)70168-3

(9)

_______________________________________________________________________________________________________________________________

Open Access Maced J Med Sci. 2017 Mar 15; 5(1):9-13. 9 ID Design 2012/DOOEL Skopje, Republic of Macedonia

Basic Science

Mismatch Repair Proteins and Microsatellite Instability in Colorectal Carcinoma (MLH1, MSH2, MSH6 and PMS2):

Histopathological and Immunohistochemical Study

Nour El Hoda S. Ismael¹, Samar A. El Sheikh¹, Suzan M. Talaat^2*, Eman M. Salem²

1Faculty of Medicine, Cairo University, Cairo, Egypt; ²Ahmed Maher Teaching Hospital, Cairo, Egypt

Citation: Ismael NEHS, El Sheikh SA, Talaat SM, Salem EM. Mismatch Repair Proteins and Microsatellite Instability in Colorectal Carcinoma (MLH1, MSH2, MSH6 and PMS2): Histopathological and Immunohistochemical Study. Open Access Maced J Med Sci. 2017 Mar 15;

5(1):9-13. https://doi.org/10.3889/oamjms.2017.003 Keywords: Colorectal cancer; Immunohistochemistry;

Lynch Syndrome; Microsatellite instability; Mismatch repair proteins.

*Correspondence: Suzan M. Talaat. Ahmed Maher Teaching Hospital, Cairo, Egypt. E-mail:

[email protected]

Received: 29-Dec-2016; Revised: 01-Feb-2017;

Accepted: 03-Feb-2017; Online first: 12-Feb-2017 Copyright: © 2017 Nour El Hoda S. Ismael, Samar A. El Sheikh, Suzan M. Talaat, Eman M. Salem. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Abstract

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide.

Microsatellite instability (MSI) is detected in about 15% of all colorectal cancers. CRC with MSI has particular characteristics such as improved survival rates and better prognosis. They also have a distinct sensitivity to the action of chemotherapy.

AIM: The aim of the study was to detect microsatellite instability in a cohort of colorectal cancer Egyptian patients using the immunohistochemical expression of mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2).

MATERIAL AND METHODS: Cases were divided into Microsatellite stable (MSS), Microsatellite unstable low (MSI-L) and Microsatellite unstable high (MSI-H). This Microsatellite stability status was correlated with different clinicopathological parameters.

RESULTS: There was a statistically significant correlation between the age of cases, tumor site &

grade and the microsatellite stability status. There was no statistically significant correlation between the gender of patients, tumor subtype, stage, mucoid change, necrosis, tumor borders, lymphocytic response, lymphovascular emboli and the microsatellite stability status.

CONCLUSION: Testing for MSI should be done for all colorectal cancer patients, especially those younger than 50 years old, right sided and high-grade CRCs.

Introduction

Globally, more than one million people get colorectal cancer annually [1]. It is the second most common cause of cancer in women (after breast cancer) and the third most common in men (after lung and prostate cancers) [2].

In Egypt, there was a rapid increase in colorectal cancer incidence, where the occurrence was formerly low. Egypt reveals an unusually high rate of colorectal carcinoma under age 40, low prevalence of colorectal polyps in cancer patients and a predominant cancer site in the rectum [3].

Colorectal cancer is a heterogeneous disease, and so far four main molecular pathways have been identified. These four pathways are the Chromosomal Instability (CIN) Pathway, CpG Island Methylator Phenotype (CIMP) pathway, Microsatellite

Instability (MSI) pathway and the Serrated pathway [4].

MSI is a kind of genomic instability that arises when mutations occur in nucleotide repeat sequences throughout the genome. These repeat sequences are known as microsatellites, and the discrepancy that arises between these sequences in tumor and germline cells is known as microsatellite instability [5]. MSI arises from defects in the DNA mismatch repair (MMR) system which corrects any errors made by DNA polymerases during the replication of DNA [6].

Lynch Syndrome (LS), previously termed hereditary non-polyposis colorectal cancer, is an inherited condition of defective DNA MMR and predisposes people to a variety of cancers [7].

Colorectal cancer is the most common type of cancer associated with Lynch syndrome [8].

(10)

_______________________________________________________________________________________________________________________________

LS is caused by autosomal dominant heterozygous germline mutations in one of the four key MMR genes — the mutL homologue 1 (MLH1)(chromosome 3p21.3),mutS homologue 2(MSH2)(chromosome 2p22–21), mutS homologue 6 (MSH6)(chromosome 2p16) or postmeiotic segregation increased 2 (PMS2)(chromosome 7p22.2) genes [9].

The loss of function occurs due to a germline mutation in one allele of one of the DNA- MMR genes; however, inactivation of the second allele, which is mostl