KIR genotype distribution among patients with hepatitis C virus: Higher prevalence of KIR 2DL3 and KIR 2DS4 and a possible role of the B haplotype? Results of a pilot prevalence study from the Mediterranean area

Gene Reports 4 (2016) 33–36

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KIR genotype distribution among patients with hepatitis C virus: Higher prevalence of KIR 2DL3 and KIR 2DS4 and a possible role of the B haplotype? Results of a pilot prevalence study from the Mediterranean area Rouba Hoteit, Georges Ibrahim, Dina Shammaa, Rami Mahfouz ⁎ Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, P.O BOX 11-0236, Riad El Solh, Beirut 1107-2020, Lebanon

a r t i c l e

i n f o

Article history: Received 11 February 2016 Received in revised form 12 February 2016 Accepted 12 February 2016 Available online 27 February 2016 Keywords: Hepatitis C KIR Genotype Lebanon

a b s t r a c t Introduction: Hepatitis C virus (HCV) is a common chronic infection worldwide affecting approximately 170 million people. 80% of acutely infected people fail to eliminate the virus and develop into the chronic phase. Some studies propose that activating KIRs have an important role in the clearance of some viruses including HCV and HIV, however, they might increase the risk of acquiring autoimmune diseases. Aim: The aim of this study is to investigate any possible association between HCV and some KIR genotypes. Methods: KIR genotype was analyzed for 47 HCV patients and 120 healthy Lebanese individuals using the KIR Genotyping SSP kit. Results: Among HCV patients, the AA, AB, and BB genotypes frequencies were, respectively, 34.05%, 42.55% and 23.40% with an A:B ratio of 1.24:1. As for the controls, the AA, AB, and BB genotypes frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.79:1. KIR 2DL3 and KIR 2DS4*001/002 were found to be significantly more prevalent among HCV patients as compared to controls in addition to an increase in the B haplotype. Conclusion: In this first pilot prevalence study in the Mediterranean area studying KIR genotyping in HCV patients, the interesting results warrant further clinical and translational research to assess the protective or predisposing role of KIR genotypes in HCV. © 2016 Elsevier Inc. All rights reserved.

Introduction Natural killer (NK) cells are a category of lymphocyte-like cells that differ from T cells and B cells by being capable of attacking leukemia cells or infected cells without prior sensitization. This property of NK cells allows the cells to protect the human body during the early stages of infection or tumor, yet it is important to regulate the activity of NK cells in order to avoid any auto-reactivity. The function of NK cells is mediated by a family of activating and inhibitory receptors (Cheent and Khakoo, 2008). Killer cells immunoglobulin-like receptors (KIR) belong to the immunoglobulin-like superfamily and play a major role in the regulation of NK cells activity (Middleton and Gonzelez, 2009). KIR genes occupy 150 kb of the leukocyte receptor cluster region (LRC) on chromosome 19q13.4, and they are highly polymorphic. There are two

Abbreviations: KIR, Killer cell immunoglobulin-like receptors; NK, Natural killer; IRB, Institutional Review Board; DNA, Deoxyribonucleic acid; PCR, Polymerase chain reaction; SSP, Sequence specific primers; HCV, Hepatitis C virus; Ig, Immunoglobulin; HLA, Human leukocyte antigens; MHC, Major histocompatibility complex; UV, Ultraviolet; Peg-IFN, Pegylated alpha interferon. ⁎ Corresponding author. Tel.: +961 3 737017; fax: +961 1 370845. E-mail address: [email protected] (R. Mahfouz).

http://dx.doi.org/10.1016/j.genrep.2016.02.007 2452-0144/© 2016 Elsevier Inc. All rights reserved.

groups of KIR haplotypes, group A and group B. Based on the gene content, two groups of KIR haplotypes, A and B, have been identified in humans. Haplotype A encodes inhibitory receptors and consists of nine genes (3DL3, 2DL3, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, one activating (2DS4), 3DL2, and 2DL5) whereas haplotype B carries a variety of gene combinations and encodes more activating receptors as compared to haplotype A (3DL3, 2DS2, 2DL2, 2DL5B (inhibitory) 2DS3, 2DP1, 2DL1, 3DP1, 2DL4, 3DS1, 2DL5A (inhibitory), 2DS5, 2DS1, and 3DL2) (Bashirova et al., 2011; McQueen et al., 2007; Moretta et al., 2008). Some studies propose that activating KIRs have an important role in the clearance of some viruses including HCV and HIV, however, they might increase the risk of acquiring autoimmune diseases (Khakoo et al., 2004; Gardiner, 2007). Hepatitis C virus (HCV) is a common chronic infection worldwide affecting approximately 170 million people. 80% of acutely infected people fail to eliminate the virus and develop into the chronic phase, and of those, 20% end up with liver cirrhosis and about 2% develop hepatocellular carcinoma (Amadei et al., 2010; De Arias et al., 2009; Kanto and Hayashi, 2007; Vidal-Castiñeira et al., 2010). The treatment of HCV and the responsiveness to therapy depend on various factors including age, gender, fibrosis, presence or absence of cirrhosis, viral load and genotype. According to the listed factors, therapy can vary from

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R. Hoteit et al. / Gene Reports 4 (2016) 33–36

pegylated alpha interferon (Peg-IFN) and ribavirin combined therapy to liver transplantation in severe cases (Vidal-Castiñeira et al., 2010). NK cells are stimulated in HCV infected patients due to the secretion of type I interferon in the liver (Yoon et al., 2009). In order to clear the virus in the primary stage of infection, NK cells secrete interferongamma (Wang et al., 2008). In addition to cytokines secretion, NK cells eliminate HCV by interaction between KIRs and their HLA class I ligands on infected cells (Rauch et al., 2007). The HCV affects NK cells by controlling apoptosis and cytokines secretion, and by interfering with KIRs and their ligands (Jonjić et al., 2008). KIRs, particularly KIR2DL3 and KIR2DL2, control the progression of infection and the reaction to treatment. KIR2DL3 in association with HLA-C1 induces a protective effect against HCV and showed an increase in the response to antiviral treatment; 70.6% of patients having KIR2DL3/KIR2DL3-HLAC1C1 genotype cleared HCV RNA and responded fully to treatment. On the other hand, KIR2DL2 is correlated with a reduced response to treatment or to unresponsiveness to therapy in some cases (Amadei et al., 2010; Mondelli et al., 2010; Vidal-Castiñeira et al., 2010). To our knowledge this is the first study in the Mediterranean region that explores the association between KIR genes and HCV infection. Materials and Methods Study Population This Institutional Review Board (IRB) approved study was conducted at the American University of Beirut Medical Center (AUBMC), a major Lebanese tertiary-care center. KIR genotype was analyzed for 47 HCV cases (age group range 18–60 years with almost equal male to female distribution) using the KIR Genotyping SSP kit. The 120 healthy controls were deducted from the major study by Mahfouz et al. (2006) describing the KIR prevalence in the healthy Lebanese population mainly from donors of a bone marrow transplantation center representing all ethnicities and districts in the country. All consent

forms were signed. The HCV cases were diagnosed in the gastroenterology and/or infectious disease clinics of the American University of Beirut Medical Center based on a panel of serological, liver function analysis, and molecular testing for hepatitis C.

DNA extraction and KIR Genotyping PEL-FREEZ kits (Pel-freez/Dynal, Norway) were used for DNA extraction from 2 to 3 ml of collected peripheral blood. The DNA material was properly labeled and stored at − 80 °C. Based on our Institutional Review Board (IRB) committee rules and study approval protocols, confidentiality was strictly observed for all analyzed samples. Primer mixes were purchased from the PEL-FREEZ/DYNAL company (PEL-FREEZ, Oslo, Norway) as part of the KIR Genotyping SSP kit which is a PCR-based method designed to detect the absence or presence of the following 16 gene loci of KIR (variants also tested): 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. Two variants for KIR2DL5 were typed KIR2DL5A*001 and KIR2DL5B*002/003/004 and two variants for KIR2DS4 were tested and reported as KIR2DS4*001/002 and KIR2DS4*003–006. In addition, two variants for the pseudogene KIR3DP1 were tested: KIR3DP1*001/ 002/004 and KIR3DP1*003. KIR genotyping was performed as recommended by the manufacturer and all steps were strictly followed. Briefly, 25 μl of DNA were added to 150 μl of PCR buffer and 2.4 μl of Taq DNA polymerase and dispensed as aliquots of 8 μl into a supplied 96-well plate for a total reaction volume of 23 μl in each well (reaction + paraffin oil). The thermocycling steps include an initial heating step at 95 °C for 1 min, followed by 30 cycles of 94 °C for 20 s, 63 °C for 20 s, and 72 °C for 90 s. A final holding step was performed at 4 °C. Electrophoresis of the 2% agarose gel was done in ethidium bromide and visualization performed under UV light transillumination.

Table 1 KIR genotypic profile among the 47 hepatitis C virus patients. Profile BB AA AA BB BB AA AB AB AB BB AB AB BB BB BB AA AA AA AB AB BB BB AA AB AB AB AB BB BB AB BB AB AB

N

%

1 2.13 3 6.38 6 12.76 1 2.13 1 2.13 1 2.13 2 4.25 2 4.25 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 2 4.25 1 2.13 2 4.25 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 3 6.38 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 1 2.13 2 4.25 1 2.13 47 100

# of genes 2DL1 2DL2 2DL3 2DL4 2DL5A 2DL5B 2DS1 2DS2 2DS3 2DS4*001/002 2DS4*003- 2DS5 3DL1 3DL2 3DL3 3DS1 2DP1 3DP1*001 3DP1*003 007 /002/004 6 7 8 8 8 9 9 9 9 9 10 10 10 10 10 11 11 11 11 11 11 11 12 12 12 13 13 13 13 14 14 15 15

R. Hoteit et al. / Gene Reports 4 (2016) 33–36

35

Table 2 KIR genotypic profile among 120 healthy controls. Profile

No.

%

AA

1

0.83

AA

31

25.83

7

BB

1

0.83

7

No. of Loci

2DL1

2DL2

2DL3

2DL4

2DL5A

2DL5B

3DL1

3DL2

3DL3

2DS1

2DS2

2DS3

2DS4*001/002

2DS4*003

2DS5

3DS1

6

AA

1

0.83

8

AB

17

14.17

9

AA

2

1.67

9

BB

3

2.50

10

BB

1

0.83

10

AB

17

14.17

11

AA

10

8.33

11

AB

8

6.67

11

AA

2

1.67

11

AB

1

0.83

11

BB

1

0.83

11

AB

3

2.50

12

BB

1

0.83

12

BB

1

0.83

12

AB

1

0.83

12

BB

4

3.33

13

AB

1

0.83

13

AB

1

0.83

13

BB

1

0.83

13

AB

6

5.00

14

AB

5

4.17

15

Statistical Analysis We utilized direct counting for the observed phenotype frequencies of KIR and SPSS 15.0 was used to conduct statistical analysis. KIR genetic profiling was expressed as number and frequency. Chi-square analysis was used to test for the association between group (case versus control) and genetic profile. A p-value less than 0.05 was considered statistically significant. Results The KIR genotypic profile distribution among the 47 Lebanese patients with HCV infection is shown in Table 1. The content of KIR genes ranged from 6 to 15 and as per Table 4, the AA, AB, and BB genotypes frequencies were, respectively, 34.05%, 42.55% and 23.4% with an A:B ratio of 1.24:1. Table 2 shows the distribution of the different KIR genotypes among the 120 healthy Lebanese controls. The content of KIR genes ranged from 6 to 15 and, as per Table 4, the AA, AB, and BB genotypes frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.79:1. Table 3 shows the distribution of the different KIR genes among the 47 HCV patients and the 120 healthy controls. KIR 2DL4, KIR 3DL2, KIR 3DL3, and KIR 3DP1*003 were present in all individuals. KIR 2DL3 and KIR 2DS4*001/002 were found to be significantly (with p-values of 0.03 and b 0.001, respectively) more prevalent among HCV patients as compared to controls. Discussion This study results are consistent with the presence of the two major KIR haplotypes, group A and group B with predominance of the group A haplotype among the hepatitis C virus patients similar to its prevalence among the healthy individuals. All our genotypes included between 6 and 15 genes with an average number of 11 KIR loci per patient (excluding

the pseudogenes KIR2DP1 and KIR3DP1) which is comparable to the 11 KIR loci in the general healthy population (Mahfouz et al., 2006). The distribution of the KIR genotypes (34.05%, 42.55%, and 23.40% respectively for AA, AB, and BB) shows a significant increase in the BB genotype among the HCV patients as compared to the healthy controls which is also applicable to the haplotype distribution (an increase in the B haplotype) (Table 4). Our current study does not include clinical data related to the HCV patients involved as it is a genetic prevalence study, thus, no major phenotypic–genotypic correlations can be deduced at this level. However, the interesting observation of a significant increase of KIR2DS4 and KIR2DL3 genes among the HCV patients as compared to

Table 3 Comparison and statistically significant difference between HCV patients and healthy controls.

2DL1 2DL2 2DL3 2DL4 2DL5A 2DL5B 2DS1 2DS2 2DS3 2DS4*001/002 2DS4*003/007 2DS5 3DL1 3DL2 3DL3 3DS1 2DP1 3DP*001/002/004 3DP1*003

Negative N = 120

HCV N = 47

p-value

119 (99.2%) 73 (60.8%) 106 (88.3%) 120 (100%) 38 (31.7%) 45 (37.5%) 49 (40.8%) 73 (60.8%) 45 (37.5%) 20 (16.7%) 99 (82.5%) 34 (28.3%) 115 (95.8%) 120 (100%) 120 (100%) 45 (37.5%) 116 (96.7%) 33 (27.5%) 120 (100%)

45 (95.7%) 31 (66.0%) 35 (74.5%) 47 (100%) 13 (27.7%) 19 (40.4%) 17 (36.2%) 31 (66.0%) 22 (46.8%) 21 (44.7%) 40 (85.1%) 11 (23.4%) 46 (97.9%) 47 (100%) 47 (100%) 15 (31.9%) 46 (97.9%) 11 (23.4%) 47 (100%)

0.19 0.33 0.03 – 0.38 0.43 0.35 0.33 0.18 b0.001 0.44 0.33 1 – – 0.31 1 0.7 –

A p-value b 0.05 was considered significant.

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Table 4 Frequency distribution of AA, AB, and BB genotypes among controls and HCV patients. Genotype

AA AB BB Total

Controls

HCV patients

No.

%

No.

%

47 60 13 120

39.17 50.00 10.83 100

16 20 11 47

34.05 42.55 23.40 100

Haplotype

Controls

HCV patients

A B A:B ratio

154 86 1.79:1

52 42 1.24:1

healthy individuals is worth noting as it has not been previously elucidated or described in the pathophysiology of hepatitis C. As a typical example, KIR2DS4 and KIR2DL3 can be assessed in correlation with virus clearance and outcome in these patients or maybe a direct association with the disease development and prognostication itself. The B haplotype is known to harbor mostly activating KIR genes, thus, this may be of a major significance in the predisposition for HCV infection or activation of defense mechanisms against the virus. KIRs, particularly KIR2DL3 and KIR2DL2, are reported to control the progression of HCV infection and the reaction to treatment. KIR2DL3 is known to induce a protective effect against HCV as reported in the international literature but may not seem to be the case in the Lebanese population. This study does not reflect on any clinical follow-up for the treatment response and prognosis of the included patients and is only a prevalence report for the KIR genes in this common condition in our region. Our study, however, is the first to report on the high frequency of two KIR genes among HCV patients as compared to a healthy control population. Further clinical and translational studies will definitely be informative in this regard and may use the results of this report as a template to build on further biological processes for a better understanding of the disease pathogenesis and to address specifically the question whether the higher prevalence of KIR 2DL3 and KIR 2DS4 and the B haplotype have any role in predisposing some populations to hepatitis C infection like may be preliminarily the case in our region.

References Amadei, B., Urbani, S., Cazaly, A., Fisicaro, P., Zerbini, A., Ahmed, P., Missale, G., Ferrari, C., Khakoo, S.I., 2010. Activation of natural killer cells during acute infection with hepatitis C virus. Gastroenterology 138 (4), 1536–1545. Bashirova, A.A., Thomas, R., Carrington, M., 2011. HLA/KIR restraint of HIV: surviving the fittest. Annu. Rev. Immunol. 29, 295–317. Cheent, K., Khakoo, S.I., 2008. Natural killer cells: integrating diversity with function. Immunology 126, 449–457. De Arias, A.E., Haworth, S.E., Belli, L.S., Burra, P., Pinzello, G., Vangeli, M., Minola, E., Guido, M., Boccagni, P., De Feo, T.M., Torelli, R., Cardillo, M., Scalamogna, M., Poli, F., 2009. Killer cell immunoglobulin-like receptor genotype and killer cell immunoglobulinlike receptor-human leukocyte antigen C ligand compatibility affect the severity of hepatitis C virus recurrence after liver transplantation. Liver Transpl. 15 (4), 390–399. Gardiner, C.M., 2007. Killer cell immunoglobulin-like receptors on NK cells: the how, where and why. Int. J. Immunogenet. 35, 1–8. Jonjić, S., Babić, M., Polić, B., Krmpotić, A., 2008. Immune evasion of natural killer cells by viruses. Curr. Opin. Immunol. 20 (1), 30–38. Kanto, T., Hayashi, N., 2007. Innate immunity in hepatitis C virus infection: interplay among dendritic cells, natural killer cells and natural killer T cells. Hepatol. Res. (Suppl. 3), S319–S326. Khakoo, S.I., Thio, C.L., Martin, M.P., Brooks, C.R., Gao, X., Astemborski, J., Cheng, J., Goedert, J.J., Vlahov, D., Hilgartner, M., Cox, S., Little, A.M., Alexander, G.J., Cramp, M.E., O'Brien, S.J., Rosenberg, W.M.C., Thomas, D.L., Carrington, M., 2004. HLA and NK Cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 305 (5685), 872–874. Mahfouz, R., Rayes, R., Mahfoud, Z., Bazarbachi, A., Zaatari, G., 2006. Distribution of killer cell immunoglobulin-like receptors genotypes in the Lebanese population. Tissue Antigens 68 (1), 66–71 Jul. McQueen, K.L., Dorighi, K.M., Guethlein, L.A., Wong, R., Sanjanwala, B., Parham, P., 2007. Donor–recipient combinations of group A and B KIR haplotypes and HLA class I ligand affect the outcome of HLA-matched, sibling donor hematopoietic cell transplantation. Hum. Immunol. 68 (5), 309–323. Middleton, D., Gonzelez, F., 2009. The extensive polymorphism of KIR genes. Immunology 129, 8–19. Mondelli, M.U., Varchetta, S., Oliviero, B., 2010. Natural killer cells in viral hepatitis: facts and controversies. Eur. J. Clin. Investig. 40 (9), 851–863. Moretta, A., Locatelli, F., Moretta, L., 2008. Human NK cells: from HLA class I-specific killer Ig-like receptors to the therapy of acute leukemias. Immunol. Rev. 224, 58–69. Rauch, A., Laird, R., McKinnon, E., Telenti, A., Furrer, H., Weber, R., Smillie, D., Gaudieri, S., Swiss HIV Cohort Study, 2007. Influence of inhibitory killer immunoglobulin-like receptors and their HLA-C ligands on resolving hepatitis C virus infection. Tissue Antigens (Suppl. 1), 237–240. Vidal-Castiñeira, J.R., López-Vázquez, A., Díaz-Peña, R., Alonso-Arias, R., Martínez-Borra, J., Pérez, R., Fernández-Suárez, J., Melón, S., Prieto, J., Rodrigo, L., López-Larrea, C., 2010. Effect of killer immunoglobulin-like receptors in the response to combined treatment in patients with chronic hepatitis C virus infection. J. Virol. 84 (1), 475–481. Wang, S.H., Huang, C.X., Ye, L., Wang, X., Song, L., Wang, Y.J., Liang, H., Huang, X.Y., Ho, W.Z., 2008. Natural killer cells suppress full cycle HCV infection of human hepatocytes. J. Viral Hepat. 15 (12), 855–864. Yoon, J.C., Shiina, M., Ahlenstiel, G., Rehermann, B., 2009. Natural killer cell function is intact after direct exposure to infectious hepatitis C virions. Hepatology 49 (1), 12–21.