The Relationship Between Killer Cell Immunoglobulin-Like Receptors and HLA-C Polymorphisms in Colorectal Cancer in a Saudi Population

Abstract

Aims: We performed an association study to evaluate the contribution of 16 killer cell immunoglobulin-like receptor (KIR) genotype polymorphisms and the HLA-C1 and -C2 ligands in the development of colorectal cancer (CRC) in Saudi Arabian patients. Methods: A total of 52 patients with different stages of malignant CRC as well as 70 healthy Saudi controls were enrolled at the King Khalid University Hospital. Results: Our results showed that the frequency of the activating mutations KIR2DS1, 2DS2, 2DS3, 2DS5, and 3DS1 was significantly higher in CRC patients compared to controls. The 3DS1 gene contributed to the highest risk of CRC (odds ratio [OR] = 16.25, p < 0.0001), followed by 2DS1 (OR = 8.6; p < 0.0001). The distributions of HLA-C1 and -C2 ligands were not significantly different between patients and controls. Analyses of different combinations of KIR genes with their HLA-C1 and -C2 ligands show that the frequency of 2DL3 in the presence of its ligand, the allotype C1, was significantly more prevalent in patients compared to controls. In addition, 2DL2 and 2DL3 that were aggregated in combination with the ligand, HLA-C1, were found to be more highly associated mainly with the homozygote HLA-C1/C1 (p = 0.03; OR = 2.6). The activating mutations 2DS1 and 2DS2 when combined with their respective ligands, HLA-C2 and -C1, showed highly significant associations with CRC development. Conclusion: This study supports a key role for KIR gene mutations in the development of CRC, especially in association with their ligands.

Introduction

Natural killer (NK) cells play a fundamental role in the regulation of immunity against infected and malignantly transformed cells. This function is mainly carried out through their killer cell immunoglobulin-like receptors (KIRs), which interact with human leukocyte antigen (HLA) molecules (Yawata et al., 2006). Sixteen genes coding for receptors that activate (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1), inhibit (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, and KIR3DL3), or both function on the KIR2DL4 gene, and two pseudogenes (KIR2DP1 and KIR3DP1) have been identified (Marsh et al., 2003). Nomenclature of the KIR gene was based on the structure of the number of extracellular immunoglobulin domains (D) of the expressed molecules (double, 2D; or triple, 3D) and the length of the intracytoplasmic tail (long, L; or short, S) (Marsh et al., 2003). Inhibitory KIR molecules are characterized by long cytoplasmic tails and activating molecules are characterized by short tails. These KIR genes are located on chromosome 19 (19q14.3) within the leukocyte receptor complex (Wende et al., 1999; Hsu et al., 2002) and are also characterized by a high genetic, haplotypic, and allelic polymorphism (Martin et al., 2000, 2004). The number and the type of KIR genes may differ substantially between two chromosomes of the same individual due to insertion/deletion events. The number and type of KIR genes also vary between haplotypes and more than 50 different haplotypes have been reported according to the number of KIR loci per chromosome that vary from 4 to 14 (Gonzalez-Galarza et al., 2011). These haplotypes have been classified into two groups, A and B. Group A haplotypes contain a fixed number of inhibitory KIR (iKIR) genes (3DL3-2DL3-2DL1-3DP1-2DL4-3DL1), one activating gene (KIR2DS4), and the two pseudogenes (2DP1 and 3DL2). Group B haplotypes are composed of a variable number of KIR genes and have at least one of the following KIR genes: 2DS1, 2DS2, 2DS3, 2DS5, 2DL5, or 2DL2. A haplotype is identified by the absence of all these genes. Four genes (2DL4, 3DL2, 3DL3, and 3DP1) are frequently shared between all individuals and those that occur in both haplotypes are called framework genes (Robinson et al. 2010).

NK cell cytotoxic activity is modulated by their composition of inhibitory and activating KIRs and their interactions with HLA class I ligands; thus, inhibitory KIRs recognize a subset of class I molecules. While specificity toward HLA-A, -B, -C, and -G ligands has been demonstrated for some KIR molecules, for most of these receptors, the ligand is undefined. For the identified ligands, the most studied is the polymorphic HLA-C molecule. Two HLA-C epitopes have been shown to interact with some KIRs, including the C1 epitope carried by HLA-C allotypes with asparagine at position 80 and the C2 epitope characterized by a lysine at the same position. The C1 epitope is also carried by certain HLA-B allotypes that have asparagine at position 80 and valine at position 76 (as reported in some Asian populations). Bw4, another relevant epitope, is carried by HLA-A and HLA-B molecules that have arginine at position 83. The iKIRs 2DL2 and 2DL3 and the aKIR 2DS2 recognize cells that express the group C1 alleles and 2DL2 and 2DS1 interact with C2 alleles (Mandelboim et al., 1996; Du et al., 2007; Moesta et al., 2008). The Bw4 epitope is recognized by KIR3DL1 and 3DS1 (Gumperz et al., 1995). The most important interaction between KIR and HLA has been reported for 2DL2 and 2DL3 with the C1 epitope; such KIR-HLA interactions play an important role in maintaining self-tolerance.

According to the missing self hypothesis, NK cell recognition strategies are based on the level of self-MHC class I molecule expression. The loss or downregulation of self-MHC class I molecules at the surface induces cell destruction by preventing KIR inhibition on NK cells. The reduction of MHC-I molecule expression is one of several strategies used by malignant or infected cells to overcome the host adaptive immune system. Due to the role attributed to KIR and their ligands in tumor immunosurveillance and their polymorphisms, diseases such as cancer, viral infection, autoimmune pregnancy failure, etc. have been found to be associated with KIR gene polymorphisms and HLA-KIR gene combinations (Beelen et al., 2005; Williams et al., 2005; Khakoo and Carrington, 2006; Kulkarni et al., 2008; Knapp et al., 2010; Bashirova et al., 2011; Jamil and Khakoo, 2011).

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. It is considered the third most common cancer worldwide, representing 9.7% of all cancers, and is the fourth most common cause of cancer-related deaths according to 2012 GLOBOCAN estimates (Saika and Yako-Suketomo, 2013; Ferlay et al., 2015). CRC is considered the second most common malignancy in the Saudi Arabian population after breast cancer, ranking first among men and third among women (Ibrahim et al., 2008; Al-Hamdan et al., 2009; Mosli and Al-Ahwal, 2012; Roshandel et al., 2014). In the present study, we evaluated the relationship between the diversity of KIR genotypes and their HLA-C ligands in the development of CRC in a Saudi Arabian population.

Materials and Methods

Participants

A total of 70 Saudi patients (34 females and 36 males; mean age: 56.2 ± 15.2) with CRC at the King Khaled University Hospital in Riyadh were enrolled in this study. The control group was composed of 70 individuals from the general Saudi population with no family history of CRC or any other chronic diseases who were treated at the hospital for minor illnesses. Genomic DNA was obtained by scraping formalin-fixed, paraffin-embedded tissue (FFPE) using a DNeasy Blood & Tissue kit according to the manufacturer's recommendation (Qiagen). For the healthy control group, DNA was extracted from whole blood using the same DNA extraction kit. All participants were asked for their consent according to the permit approved by the Ethics Committee of King Saud University. For quality control, 5 μL of the eluted genomic DNA was run in 1% agarose gel with a 1 kb ladder (Solis Biodyne^®). Only the DNA of 52 FFPE tissue was considered suitable for genetic analysis; the rest of the DNA was degraded. DNA was then quantitated using a NanoDrop 8000 (Thermo Scientific^®).

KIR and HLA-C genotyping

Patients and controls were typed for the 16 common KIR genes by polymerase chain reaction-sequence-specific primer using the commercial KIR Typing Kit (Miltenyi Biotec^®, Inc.) according to the manufacturer's recommendations. For each reaction 50-100 ng of DNA were used in a 15 μL final volume. For HLA-C1 and HLA-C2 group typing, the same primers were used as reported by Tajik et al. (2010). PCR reactions were performed in a final volume of 20 μL containing 4 μL of5 × FIREPol^® Master Mix ready to use (Solis Biodyne), 0.2 pmol of each primer, 50-100 ng of DNA, and ultrapure water. For each reaction, positive, negative, and internal controls were used (human growth hormone [hGH]).

The internal positive control was amplified with the primers hGH forward (5′-GCCTTCCCAACCATTCCCTTA-3′) and hGH reverse (5′-GTCCATGTCCTTCCTGAAGCA-3′) (Tajik et al., 2009, 2010). The PCR protocol used for this reaction consisted of an initial denaturation step at 94°C followed by 5 cycles of 20 s at 94°C, 30 s at 64°C, and 60 s at 72°C; 25 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s; 5 cycles of 94°C for 30 s, 55°C for 30 min, and 72°C for 90 s, followed by a final extension step for 10 min at 72°C. All PCR reactions were performed with the thermocycler apparatus (Techne TC-Plus Satellites^®). PCR products were electrophoresed in 2% agarose gel stained with ethidium bromide and visualized on an UV transilluminator using a gel documentation system (Biometra^®).

Statistical analyses

The frequencies of KIR genes and genotypes and HLA-C1 and -C2 for patients and controls were determined by direct counting. Differences in the distribution of KIR genes and genotypes, HLA-C, -C2, and KIR-HLA-C combinations were estimated using a two-tailed Fisher's exact probability (p) test. A p < 0.05 was used as the criterion for statistical significance. Odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated to assess the relative risk conferred by specific KIR genes, HLA-C1, -C2 groups, and KIR-HLA-C combinations. All statistical analyses were performed using the SigmaPlot software version 11.

Results

We evaluated the role of 16 genes coding for KIR and one of the most relevant ligands, HLA-C, in the susceptibility to CRC in a Saudi Arabian population. Seventy patients were enrolled in this study that was composed of 34 females (48%) and 36 males (51.4%). The average age was 56.2 ± 15.2. The control group consisted of 70 individuals with an average age of 52.4 ± 13.6 (25 males and 45 females). Table 1 shows the clinical data of all patients. The TNM classification shows that there were 2 patients in stage 0, 2 patients in stage I, 20% of patients were in stage II, 60% of patients were in stage III, and 14.3% of patients were in stage IV. In the CRC patient group, 88.6% of tumors were localized in different parts of the colon and 11.42% were located in the rectum. Due to the degradation of genomic DNA for 18 FFPE tissues, KIR and HLA-C1 and -C2 polymorphisms were typed for only 52 patients.

Table 1.

Demographic and Main Clinical Data of CRC Patients and Controls Used for Genotyping

Characteristic	Cancer (70)	Controls (70)
Gender
Male	34 (48%)	25 (35.7%)
Female	36 (51.4%)	45 (64.2%)
Age	56.2 ± 15.2	52.4 ± 13.6
Localization
Colon	62 (88.6%)	—
Rectum	8 (11.42%)	—
Cell type
Adenocarcimona
Mucinous carcinoma
Therapy
Chemotherapy
Yes	0	—
No	70	—
Radiotherapy
Yes	3	—
No	67	—
Metastasis
Yes	8	—
No	62	—
Stage
II	14	—
III	42	—
IV	10	—

CRC, colorectal cancer.

Associations between KIR gene polymorphisms and CRC

Logistic regression analysis of the 16 KIR genes is represented in Table 2. The framework genes appear at the highest frequencies. 3DL3 was absent from only two patients and the rest of these framework genes appeared in all subjects. For all of the inhibitory genes, no significant differences were found in the distribution between patients and controls; however, 2DL3 and 2DL2 are more frequent in patients than in controls, but these differences were not significant (p = 0.072, p = 0.09, respectively). In contrast, all the activating genes appeared at significantly higher frequencies in patients than in controls. The highest difference was observed for 3DS1 (OR = 16.25, 95% CI = 6.49-40.04, and p < 0.00001). The genes 2DS1 and 2DS5 were also more prevalent in patients than in controls (OR = 8.6 and OR = 4.5, respectively; p < 0.0001). For 2DS3 and 2DS2, while significant, they are at the limit of significance and their lower limit of CI is slightly above 1 (Table 2).

Table 2.

Comparison of KIR Gene Frequencies Between CRC Patients and Controls

Genes	Patients (52)	Controls (70)	OR	95% CI	p	χ ²
3DL2	52 (100)	70 (100)	Na	Na	1	Inf
3DL3	50 (96.1)	70 (100)	Na	Na	1	Inf
2DL4	52 (100)	70 (100)	Na	Na	1	Inf
3DP1	52 (100)	70 (100)	Na	Na	1	Inf
2DL1	51 (98.0)	70 (100)	0	0	0.42	0
2DL3	48 (92.3)	56 (80)	3	0.92-9.72	0.072	2.68
3DL1	51 (98.1)	66 (94.3)	3.09	0.33-28.5	0.39	0
2DS4	51 (98.1)	70 (100)	0	0	0.42	0
2DL2	47 (90.4)	55 (78.6)	2.56	0.86-7.58	0.09	2.24
2DL5	45 (86.5)	53 (75.7)	2.06	0.78-5.41	0.169	1.58
2DS1	43 (82.7)	25 (35.7)	8.6	3.6-205	Inf 0.0001	24.8
2DS2	46 (88.5)	50 (71.4)	3	1.13-8.3	0.02	4.2
2DS3	40 (76.9)	40 (57.1)	2.5	1.12-5.56	0.033	4.33
2DS5	35 (67.3)	22 (31.4)	4.5	2.08-9.68	0.0001	14.02
3DS1	43 (82.7)	16 (22.8)	16.25	6.49-40.04	Inf 0.0001	40.41
2DP1	52 (100)	70 (100)	Na	Na	1	Inf

Significant associations are shown in bold.

CI, confidence interval; Inf, infinity; KIR, killer cell immunoglobulin-like receptor; Na, not applicable; OR, odds ratio.

Associations between HLA-C groups 1 and 2 and CRC

Comparative analysis data on the basis of the presence or absence of HLA-C group 1 or group 2 alleles are shown in Table 3. While the frequencies of the C1 group and the genotype C1C2 are slightly higher in patients (conversely for C2 and its homozygotes), these differences are not significant.

Table 3.

Frequencies of the KIR Ligand C1/C2 in Patient and Healthy Control Groups

	Patients	Controls	OR	IC	p	χ ²
C1C1	20 (38.5)	19 (27.1)	1.67	0.77-3.61	0.24	1.28
C2C2	11 (21.2)	22 (31.4)	0.58	0.25-1.34	0.22	1.12
C1C2	21 (40.4)	29 (41.4)	0.95	0.46-1.98	1	0
C1	41 (78.8)	48 (68.6)	1.70	0.74-3.93	0.22	0.28
C2	32 (61.5)	51 (72.8)	0.59	0.27-1.28	0.23	1.28

Associations between KIR genes in the presence and absence of their HLA-C ligands C1 and C2 and CRC

Inhibitory KIR molecules recognize distinct HLA class I molecules. KIR2DL2 and 2DL3 bind HLA-C1 allotypes and KIR2DL1 binds HLA-C2. Table 4 shows the results of the distribution of activating and inhibitory KIR genes in combination with their HLA-C1 and -C2 allotypes. We found that the entire inhibitory genes combined with their ligands were more frequent in CRC patients than in controls and these differences were significant for the 2DL3/C1 (p = 0.039, OR = 2.28). Moreover, the cumulative signal allowed by the association of 2DL2/3 and their C1 ligand were significantly associated with the development of CRC (p = 0.01, OR = 2.67). This association with 2DL2/3 persists with the homozygote C1C1 (p = 0.03, OR = 2.6).

Table 4.

Distribution of the Frequencies of Different KIR Genes in the Presence and Absence of Their C1C2 Ligand Between CRC Patients and Controls

	Controls		Patients
	n	%	n	%	p	OR	95% CI	χ ²
2DL2+C1+	38	54.3	37	71.2	0.063	2.07	0.96-4.45	2.91
2DL2+C1C1+	15	21.4	19	36.5	0.07	2.11	0.94-4.71	2.68
2DL2+C1C2+	23	32.9	18	34.6	0.8	1.08	0.5-2.3	0
2DL2+C1−	17	24.3	11	21.2	0.82	0.83	0.35-1.97	0.04
2DL3+C1+	38	54.3	38	73.1	0.039	2.28	1.05-4.94	3.72
2DL3+C1C1+	14	20.0	18	34.6	0.09	0.47	0.20-1.07	2.58
2DL3+C1C2+	24	34.3	20	38.5	0.7	1.19	0.56-2.52	0.08
2DL3+C1−	18	25.0	10	19.2	0.51	0.71	0.29-1.7	0.29
2DL2/3+C1+	29	41.4	34	65.4	0.01	2.67	1.26-5.61	5.93
2DL2/3+C1C1+	11	15.7	17	32.7	0.03	2.6	1.09-6.19	3.95
2DL2/3+C1C2+	18	25.7	17	32.7	0.42	1.4	0.63-3.08	0.41
2DL2/3+C1−	13	18.6	9	17.3	0.98	0.91	0.35-2.34	0
2DL1+C2+	51	72.9	32	61.5	0.23	0.59	0.27-1.28	1.28
2DL1+C2C2+	22	31.4	11	21.2	0.22	0.58	0.25-1.34	0.14
2DL1+C1C2+	29	41.4	19	36.5	0.7	0.81	0.38-1.7	0.13
2DL1+C2−	19	27.1	19	36.5	0.32	1.24	0.71-3.34	0.83
2DS2+C1+	35	50.0	36	69.2	0.41	2.25	1.06-4.77	3.78
2DS2+C1C1+	12	17.1	19	36.5	0.02	2.78	1.2-6.44	4.94
2DS2+C1C2+	23	32.9	17	32.7	1	0.9	0.46-2.13	0.03
2DS2+C1−	15	30.0	10	21.7	0.48	0.64	0.25-1.63	0.47
2DS1+C2+	18	34.6	27	38.57	0.004	3.12	1.45-6.69	7.71
2DS1+C2C2	8	15.4	9	12.85	0.43	1.61	0.57-4.53	0.44
2DS1+C1C2	10	14.3	18	34.6	0.0003	4.76	2.00-11.34	12.2
2DS1+C2−	7	13.5	16	22.85	0.004	4	1.5-10.63	7.11
2DS1+2DS2
+C1C2	9	12.86	16	30.77	0.022	3.01	1.2-7.5	4.83
+C1C1	6	8.57	8	15.38	0.26	1.93	0.62-5.97	0.78
+C2C2	7	10.00	16	30.77	0.004	4.0	1.5-10.63	7.11
+C1	16	22.86	32	61.54	Inf 0.0001	5.4	2.45-11.89	17.12
+C2	15	21.43	24	46.15	0.005	3.14	1.4-6.9	7.92

Significant associations are shown in bold.

For the activating receptors, significant associations were found between 2DS2 in the presence of the homozygote C1 ligand only (p = 0.02, OR = 2.78). The 2DS1 gene is significantly associated with CRC for all combinations, except when combined with its homozygous C2. To understand the effect of the C2 on the association of KIR 2DS1/2DS2 with CRC, we assessed the distribution of both 2DS1 and 2DS2 with different combinations of their ligand genotypes. We found that all combinations are positively associated with CRC only with the homozygote C2C2 (Table 3), demonstrating that the presence of the C1 allotype with its KIR and/or the absence of C2 may influence the activity of the NK cells against CRC.

Discussion

CRC is one of the most important contributors of mortality in the world, with over 1 million new cases diagnosed every year (Harrison and Benziger, 2011). Environmental, genetic, and epigenetic factors underlie the main cause of this malignant disease (Berg and Soreide, 2011; Choong and Tsafnat, 2012; Wong et al., 2013; Coppede et al., 2014), as the risk attributed to heritable factors could explain 35% of CRC cases (Lichtenstein et al., 2000). Hundreds of genetic markers have been tested, but few of them have been oriented to molecules that play important roles in the immune defense against malignant cells, specifically innate immunity. NK cells have been considered important factors in protecting patients from the early development of cancer and its progression (Xu et al., 2011). The cytotoxic activity of NK cells is mainly modulated by a multitude of activating and inhibitory receptors at the cell surface that interact with ligands in target cells. The interactions of KIR receptors with HLA ligands are considered crucial for the regulation of NK cell activity.

In the present study, we investigated the role of individual KIR genes and their HLA ligands C1 and C2 groups, individually or combined, in the development of CRC in a Saudi Arabian population. Analysis of the distribution of 16 KIR genes between CRC patients and healthy control groups demonstrated that five activating KIR genes (2DS1, 2DS2, 2DS3, 2DS5, and 3DS1) are significantly more prevalent in the CRC patient group. The highest risk was found to be associated with the 3DS1 gene (OR = 16.25), followed by the 2DS1 gene (OR = 8.6). Significant differences were not observed between the distributions of inhibitory and pseudogenes between patients and controls. A positive association was found between KIR2DS5 and both CRC and rectal cancer subgroups in a case/control study in a Korean population (Kim et al., 2014); similar results were also reported for the 2DS5 gene in thyroid cancer patients in Iran (Ashouri et al., 2012). In other diseases, such as ankylosing spondylitis, endometriosis, and in the acute rejection of kidney grafts, KIR2DS5 was also shown to confer protection, but these results were not replicated in studies of nonsmall-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (Nowak et al., 2010). It should also be noted that the ligand of the KIR2DS5 receptor has not yet been identified.

Kim et al. (2014) reported that higher frequencies of 3DL1, 2DS2, and 2DS4 exhibited protective effects against CRC. In our study, there were no associations found between 3DL1 and 2DS4 and CRC, and 2DS2 was found to be inversely associated with CRC development. These differences in the susceptibility/protection effect of some genes could be related to other factors, such as ethnicity or other undetermined genetic or epigenetic factors. In other diseases, such as Vogt-Koyanagi-Harada disease in Japanese patients, the activating 3DS1, 2DS1, and 2DS5 genes, were observed to be associated with the development of this pathology (Levinson et al., 2010); the same 3D1, 2DS1, and 2DS5 genes were associated with increased risk of developing cervical neoplasms (Carrington et al., 2005; Stringaris et al., 2010). Remarkably, these three genes exhibited strong linkage disequilibrium, which could explain in part the trend in the association with certain diseases (Norman et al., 2002; Gourraud et al., 2010; Stringaris et al., 2010; Ashouri et al., 2012; Vierra-Green et al., 2012).

It is worth noting that the function of KIR genes in the immune response depends highly on HLA ligand expression on the surface of target cells (Purdy and Campbell, 2009). KIR-HLA interaction can therefore result in either activation or inhibition of NK cells. The specificity toward HLA ligands has been well established for some KIRs with an hierarchy of inhibition (Yu et al., 2007), as 2DL1-C2/C2 has the greatest inhibitory potential, followed by 2DL2-C1 and 2DL3-C1 (Yu et al., 2007). In this study, we found an association with an elevation of the frequency of 2DL3-C1 and CRC. The development of CRC and the strength of this association increases when 2DL3 and 2DL2 are accumulated together with the C1 this association disappears in the presence of the C2. The opposite effect of KIR2DL2/3 and its C1 on 2DL3/C1C1 was observed in the development of kidney cancer as well as in nonsmall-cell lung cancer (Al Omar et al., 2010); however, the authors found that KIR2DL3 and its ligand HLA-C1 homozygote have a protective effect in nonsmall-cell lung cancer.

Combinations of the activating receptors 2DS2 and 2DS1 with their respective C1 and C2 ligands demonstrate opposite effects, as 2DS2 is positively associated with CRC in combination with C1 and 2DS1 has a protective effect when associated with its C2 in the presence of C1 or in the absence of C2.

Finally, the present study highlights the existence of positive associations between KIR genes and the development of CRC in the Saudi population. Furthermore, different KIR-HLA ligand associations may be relevant in the development of the disease. This supports a major role of the innate immune response, mainly the cytotoxic cells in the development of CRC.

Footnotes

Acknowledgment

This project was supported by NSTIP Strategic Technologies Program number (11MED1900-02) in the Kingdom of Saudi Arabia.

Author Disclosure Statement

No competing financial interests exist.

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