Association of HLA-DRB1/DQB1 polymorphism with high-risk HPV infection and cervical intraepithelial neoplasia women from Shanghai

Abstract

Persistent human papillomavirus (HPV) infection is the main causative agent for cervical intraepithelial neoplasia (CIN) and cancer. Variability in host immunogenetic factors is important in determining the overall cellular immune response to the HPV infection. This study was carried out to confirm the association of human leukocyte antigen (HLA) class II DRB1 and DQB1 alleles with CIN and HPV persistent infections in women from Shanghai in a case-controlled study. A total of 170 patients, including 105 HPV positive patients and 65 HPV negative women (control) participated in the study. HybriBio’s proprietary flow-through hybridization technique was used to perform HPV genotyping. Low-resolution PCR-sequence specific priming (PCR-SSP) was used to genotype HLA class II for DRB1 and DQB1 loci. Binary and multivariate logistic regression analysis highlighted the association of specific alleles with CIN and HPV persistent infections after adjusting for the confounding factor of age. HLA-DQB1*02 and *06 is significantly associated with increased risk of HPV16 persistent infection (P _c < 0.013). HLA-DRB1*09 is significantly associated with increased risk for CIN, whereas the -DRB1*16 exhibit protective to CIN (P < 0.05). Significant association is found for HLA-DQB1*04 and *06 with increased risk for CIN (P < 0.05). There were possible associations of specific HLA class II alleles either with risk of persistent HPV infection or with developing CIN.

Introduction

Cervical cancer is the second most frequent case of cancer in women with more than 500,000 new cases leading to about 270,000 deaths worldwide annually, among which occur in 40 million CINs and 160 million HPV infections [1,2]. Cervical cancer is one of the 10 most common diseases affecting women with approximately 75,500 new cases (15.1% of all cervical cancer worldwide) and 34,000 deaths (12.6%) per year in China annually [3,4]. Persistent HPV infection plays an important role in the development of CIN and cervical cancer [5]. HPV persistence is defined as the shortest duration of HPV positivity period as 6 to 12 months for most studies [6]. Although the average national estimates of the cervical cancer burden in China are low, the burden may be underestimated because the prevalence of the human papillomavirus (HPV) is high (16.8%) [7,8]. HPV types 16 and 18 are predominant genotypes contributing to cervical cancer worldwide, while other high-risk (HR) genotypes such as HPV 31, 45 and 58 are mainly associated with CIN and cervical cancer [9,10]. In Asian countries, HPV18 is not the most common genotype, and our previous results suggested that HPV16, 52 and 58 are the most common genotype in women from Shanghai [11-13].

Interestingly, the incidence of HR-HPV infection far exceeds the number of individuals who develop HPV-associated CIN and cervical cancer, with 95% of HR-HPV infections of the cervix resolving spontaneously within 1 to 2 years as a result of cell-mediated immunity, which indicates that HPV infection is a necessary but not sufficient risk factor for persistent infection and CIN from the transient infections [14,15]. Host genetic differences in the effective host immune response may influence the risk for cervical cancer among those infected with HPV [16]. Consequently, it’s important to identify biomarkers for distinguishing women with HPV positivity who might develop persistent infection and CIN from the transient infections.

Over the past few decades, researchers have found that the most important Single nucleotide polymorphisms on the genetic susceptibility of cervical cancer were located in 6q12 by candidate gene association and genome-wide association studies, within the human leukocyte antigen (HLA) [17,18]. The human major histocompatibility complex (MHC) is a multi-gene including the highly polymorphic human leucocyte antigen (HLA) class I (HLA-A, HLA-B, HLA-C) and II genes (HLA-DR, HLA-DQ, HLA-DP) that code different types of glycoproteins. These glycoproteins are specialized in the presentation of short peptides derived from infectious agents or self-proteins to T lymphocytes, and play a key role in both the cellular and humoral immune responses [19,20]. The HLA-II (DRB1) gene contains many mutations, and these mutations result in changes of the amino acid sequence of HLA-II. Studies have reported that HLA-II (DRB1) is strongly associated with cervical cancer [21]. Polymorphisms in HLA-II complex variation susceptibility to long-term viral infections such as HIV, EBV, hepatitis B and C have been reported [22-26]. The correlation between HLA-II gene subtype and high risk HPV persistent infection has not been reported in Shanghai, China.

In this study, we performed a case-controlled study to explore the association between the immunogenotype of HLA genes and the risk of HR-HPV persistent infection and CIN in women from Shanghai.

Materials and methods

Clinical samples

We investigated the association of HLA class II (DRB1 and DQB1) alleles with susceptibility to HPV persistent infection and HPV-associated CIN in a hospital-based case-control study. A total of 105 patients, comprising of 78 CINs and 27 HPV positive without lesion cases, participated in the study. The patients were recruited from Cervical Disease Centre of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine (Shanghai, China), with histopathology confirmed pre-cancer of the uterine cervix. The patients had a mean age of 41.2±12.3 years, whereas the controls without HPV infection were 42.8±11.2 years old. The age and ethnicity matched the control group consisting of 65 healthy women with no self or familial history of any neoplastic diseases were selected from patients who came into the Cervical Disease Centre of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine (Shanghai, China) for routine check-ups.

Ethics approval and consent to participate

This study was approved by the ethics committee of the University of Shanghai First Maternity and Infant Health Hospital (No: KS 1533), Tongji University School of Medicine. Written, informed consent had been obtained from the reported patients.

Genomic DNA isolation

DNA from cervical cell brush samples was purified by silica gel columns (TIANamp Genomic DNA Kit No: 3304-9) according to the manufacturer’s procedure. Final elution of DNA was performed in 50 μL of distilled water. And the concentration (C) ≥ 80 ng/μL was used to the latter experiment.

HPV detection and typing

The HPV GenoArray test kit (HybriBio Ltd) was used to perform Human papillomavirus genotyping [13]. It was used in both DNA amplification and HybriBio’s proprietary flow-through hybridization technique. HPV Blot contains 21 types of genotypes, including 5 low-risk types (6, 11, 42, 43, and 44), 14 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), and 2 intermediate-risk types (CP8304 and 53) common in Chinese people.

HLA typing

Low-resolution PCR-sequence-specific priming (SSP) was used for the amplification of HLA DR-DQ alleles in both the cases and the controls (Morgan^TM HLA SSP DRB Typing Kit, DQB Low SSP Morgan^TM Kit) according to the manufacturer’s instructions.

Statistical analysis

Binary and multivariate logistic regression analysis was applied to compare the number of cases and controls who were positive for HLA DR-DQ alleles. Allelic frequencies; odds ratios (OR) with respective 95 per cent confidence intervals were estimated using the SPSS software version 20.0 (SPSS, Chicago, Il, USA). Multivariate analysis highlighted the association of specific alleles with CIN and HPV persistent infection after adjusting for the confounding factor of age. P _c < 0.013 is considered statistically significant.

Results

HPV typing

Of the 105 women with identified persistent HPV infection, HPV type 16 was the most common type (44.8%, 47/105). HPV co-infection with two or three different types were 40, 38.1%. Women with HPV 16/52/58 co-infection were 34, 32.4%, and 65 HPV negative women participated in the study. (Tables 1 and 2) Figure 3 depicts the representative gel picture for low-resolution PCR-SSP analysis for the HLA DRB1-DQB1 region.

Table 1.

HLA-DQB1* alleles associated with HPV Persistent Infection

HLA-DQB1	HPV16 (N=47)		no-HPV16 (N=58)		Total Cases (N=105)		Controls (N=65)		P values	OR	95% CI	p c value
	n	Af (%)	n	Af (%)	n	Af (%)	n	Af (%)
*02	23	24.47	11	9.48	34	16.19	12	9.23	0.003 a			0.013
									0.633b
									0.067c
									0.031 d
*03	37	39.36	52	44.83	89	42.38	66	50.77	0.231a
									0.46b
									0.716c
									0.043 d	0.393	0.159-0.972
*04	5	5.32	7	6.03	12	5.71	8	6.15	0.067a
									0.125b
									0.054c
									0.685d
*05	6	6.38	18	15.52	24	11.43	23	17.69	0.384a
									0.477b
									0.359c
									0.83d
*06	23	24.47	28	24.14	51	24.29	21	16.15	0.009 a	4.198	1.432-12.31	0.013
									0.212b
									0.03 c	2.327	1.083-5.002
									0.1d

n, n=2N, number of alleles; Af, allelic frequency; OR, odds ratio; CI, confidence interval; OR, odds ratio; CI, confidence interval; P _c, corrected P value after applying Bonferroni’s correction; P value, probability from logistic regression analysis after adjusting for the confounding factor of age comparing the haplotype frequency distribution among the following:

^aHPV16 infection cases versus controls;

^bno-HPV16 infection versus controls;

^ctotal cases versus controls;

^dHPV16 infection versus no-HPV16 infection.

Bold type refers to statistically significant results.

Table 2.

HLA-DRB1* alleles associated with HPV Persistent Infection

HLA-DRB1	HPV16 (N=47)		no-HPV16 (N=58)		Total Cases (N=105)		Controls (N=65)		P values	OR	95% CI	p c value
	n	Af (%)	n	Af (%)	n	Af (%)	n	Af (%)
*01	1	1.06	4	3.45	5	2.38	1	0.77	0.526
									0.27
									0.188
									0.736
*02	1	1.06	0	0	1	0.48	0	0	1.0
									0.107
									1.0
*03	9	9.57	4	3.45	13	6.19	5	3.85	1.0
									0.155
									1.0
									1.0
*04	10	10.64	12	10.34	22	10.48	18	13.85	1.0
									0.772
									0.103
									1.0
*07	12	12.77	8	6.9	20	9.52	11	8.46	1.0
									0.898
									0.372
									1.0
*08	8	8.51	12	10.34	20	9.52	7	5.38	1.0
									0.824
									0.177
									1.0
*09	16	17.02	24	20.69	40	19.05	22	16.92	1.0
									0.228
									0.067
									1.0
*10	1	1.06	3	2.59	4	1.9	3	2.31	1.0
									0.448
									0.154
									1.0
*11	5	5.32	10	8.62	15	7.14	13	10	1.0
									0.641
									0.45
									1.0
*12	9	9.57	6	5.17	15	7.14	15	11.54	1.0
									0.768
									0.427
									1.0
*13	2	2.13	2	1.72	4	1.9	9	6.92	1.0
									0.643
									0.532
									1.0
*14	6	6.38	10	8.62	16	7.62	11	8.46	1.0
									0.353
									0.751
									1.0
*15	14	14.89	18	15.52	32	15.24	10	7.69	1.0
									0.625
									0.285
									1.0
*16	0	0	3	2.59	3	1.43	5	3.85	1.0
									0.186
									0.04	0.187	0.038-0.927	0.013
									1.0

n, n=2N, number of alleles; Af, allelic frequency; OR, odds ratio; CI, confidence interval; OR, odds ratio; CI, confidence interval; ud, undetermined. P _c, corrected P value after applying Bonferroni’s correction; P value, probability from logistic regression analysis after adjusting for the confounding factor of age comparing the haplotype frequency distribution among the following: ^aHPV16 infection cases versus controls; ^bno-HPV16 infection versus controls; ^ctotal cases versus controls; and ^dHPV16 infection versus no-HPV16 infection. Bold type refers to statistically significant results.

Figure 3.

A representative 2% agarose gel picture showing PCR products of low resolution PCR-SSP analysis for the HLA DR-DQ region. (A-C): Correspond to HLA DRB1* alleles; (D): Corresponds to HLA DQB1* alleles, a corresponds to control band, b corresponds to control band Primer dimer band. 1 (A), contamination control. The internal control fragment is 600 bp in size. The DNA fragments amplified by allele- or group-specific primer pairs are designed to have a size in the range between 70-275 bp (shown in white rectangle). Refer to the worksheet for the exact size of the specifically amplified DNA fragment in each reaction tube. The positive wells of (A-C) are 6, 12, 19, 22, 23, 24; the positive wells of (D) are 3, 7. Document the positive reactions for the test and determine the HLA types by using the worksheet provided.

Distribution of HLA-DRB1* and HLA-DQB1* alleles in case-controlled study population

HLA DRB1 and DQB1 typing was completed in all 105 cases (HPV positive) and 65 controls (HPV negative). There were 14 HLA-DRB1 and 5 HLA-DQB1 alleles identified among 170 women, including 105 cervical patients and 65 control subjects (Figures 1, 2). The allelic distribution for the HLA DQB1 locus revealed DQB1*03 was the most frequent allele both in cases (42.4%; 89/210) and in controls (50.8%; 66/130). The allelic distribution for the HLA DRB1 locus revealed DRB1*09 was the most frequent allele in HPV-positive cases (19.1%; 40/210) and controls (16.9%; 22/130).

Figure 1.

Distribution of HLA-DRB1* alleles in case-control study population. 14 HLA-DRB1 alleles were identified among 170 women, including 105 HPV-positive patients and 65 HPV-negative subjects from Shanghai. The HLA-DRB1*09 was the most frequent allele in HPV-positive cases (19.1%; 40/210) and controls (16.9%; 22/130).

Figure 2.

Distribution of HLA-DQB1* alleles in case-control study population. 5 HLA-DQB1 alleles were identified among 170 women, including 105 HPV-positive patients and 65 HPV-negative subjects from Shanghai. HLA-DQB1*03 allele was the most frequent allele both in cases (42.4%; 89/210) and in controls (50.8%; 66/130).

HLA-DRB1/DQB1 polymorphisms associated with HPV persistent infection

HLA-DQB1*02 and *06 were significantly associated with increased risk for HPV16 persistent infection (P _c < 0.013). A positive association was also established for the DQB1*02 and *03 allele with respect to HPV infection, for the DQB1*06 allele in HPV16 infection compared with no-HPV16 infection cases. After application of Bonferroni’s correction, statistical significance were not retained (P _c=0.013) (Table 1). A positive association was also established for the DRB1*16 allele in HPV16 infection compared with no-HPV16 infection cases. After application of Bonferroni’s correction, statistical significance were not retained (P _c=0.013) (Table 2).

HLA-DRB1/DQB1 polymorphisms associated with CIN

HLA-DQB1*04 and *06 were significantly associated with increas-ed risk for CIN (P < 0.05) (Table 3). Significant association was found for HLA-DRB1*09 with increased risk for CIN (P < 0.05). Conversely, the allele frequencies for DRB1*16 among cases was significantly lower than that among control subjects, thereby indicating their protective effect (P < 0.05) (Table 4).

Table 3.

HLA-DQB1* alleles associated with Cervical Intraepithelial Neoplasia

HLA-DQB1	Cases (N=78)		Controls (N=65)		P values	OR	95% CI
	n	Af (%)	n	Af (%)
*02	21	13.46	12	9.23	0.06
*03	63	40.38	66	50.77	0.688
*04	10	6.41	8	6.15	0.014	2.195	1.176-4.097
*05	18	11.54	23	17.69	0.342
*06	44	28.21	21	16.15	0.017	2.677	1.195-5.998

n, n=2N, number of alleles; Af, allelic frequency; OR, odds ratio; CI, confidence interval; P value, probability from logistic regression analysis after adjusting for the confounding factor of age comparing the haplotype frequency distribution among CIN and controls.

Table 4.

HLA-DRB1* alleles associated with Cervical Intraepithelial Neoplasia

HLA-DRB1	Cases (N=78)		Controls (N=65)		P values	OR	95% CI
	n	Af (%)	n	Af (%)
*01	2	1.28	1	0.77	0.0620
*02	0	0	0	0
*03	8	5.13	5	3.85	0.161
*04	16	10.26	18	13.85	0.0920
*07	14	8.97	11	8.46	0.1940
*08	18	11.54	7	5.38	0.113
*09	33	21	22	17	0.031	0.078	0.008-0.79
*10	4	2.56	3	2.31	0.074
*11	11	7.05	13	10	0.155
*12	8	5.13	15	11.54	0.217
*13	1	0.64	9	6.92	0.406
*14	16	10.26	11	8.46	0.699
*15	24	15.38	10	7.69	0.0880
*16	1	0.64	5	3.85	0.032	0.1	0.009-0.807

n, n=2N, number of alleles; Af, allelic frequency; OR, odds ratio; CI, confidence interval; P value, probability from logistic regression analysis after adjusting for the confounding factor of age comparing the haplotype frequency distribution among CIN and controls.

Discussion

In this study, we identified the high risk HLAII allelic associated with HR HPV persistent infection. This is the first comprehensive study in women from Shanghai. Approximately 80% of the female population is exposed to HPV sometime in their life, but the infection is usually transient, with 70-90% of infected individuals ‘clearing’ the virus (HPV DNA undetectable by assays) within 6-12 months [27]. Fewer than 4% of individuals infected with HPV develop persistent infections and CIN and even fewer proportion (1-2%) will progress to cervical cancer [28,29]. Therefore in addition to HPV infection, other viral risk factors and host susceptibilities are required to initiate the transformation process [30,31]. Significant associations were observed in the major histocompatibility complex region on chromosome 6 for influenza A virus, Epstein-Barr virus, JC polyomavirus, and Merkel cell polyomavirus. And common HLA-DRB1 haplotypes showed virus-specific patterns of humoral-response regulation [32].

In our study, of the 105 women with identified persistent HPV infection, HPV type 16 was the most common type (44.8%, 47/105). The frequency of HPV52 and HPV58 was 24.8% and 20%. HPV co-infection with two or three different types were 40, 38.1%. Women with HPV 16/52/58 coinfection were 34, 32.4%. Our previous results suggested that HPV16 and 52, instead of HPV18, are the top two prevalent types in China [13,33,34].

There were 14 HLA-DRB1 and 5 HLA-DQB1 alleles identified among 170 women, including 105 cervical patients and 65 control subjects. The allelic distribution for the HLA-II locus revealed DRB1*09, and DQB1*03 was the most frequent allele in Shanghai population. HLA-DQB1*02 and *06 were significantly associated with increased risk for HPV16 persistent infection (P _c < 0.013). These results indicate that the HLAII allele is closely related to the persistent HPV infection.

HPV persistent infection is an essential factor in the development of cervical cancer, but not all of the persistent infections develop into cervical cancer. Some patients have persistent infection for many years but malignant transformations have not occurred. So we speculate that this may be related to the different HLA gene subtype. Sankhadeep Dutta1’s data suggest persistent HPV16/18 infection in the cervix due to the presence of the HLA-DQB1*03 [35]. Hu JM’s study suggests that HLA-DRB1*1501 and DQB1*0602 alleles may influence the immune response to HPV16 infection and decrease the risk of ICC among Uighurs and Hans in Xinjiang, China [36].

Our results show that HLA-DRB1*09, DQB1*04 and *06 were significantly associated with increased risk for CIN (P < 0.05). Conversely, the allele frequencies for DRB1*16 among cases was significantly lower than that among control subjects, thereby indicating their protective effect (P < 0.05). This is consistent with the results of previous studies [37].

Our study indicates that different polymorphic human leukocyte antigen (HLA) genes are involved in the clearance and maintenance of HPV infection. To fully evaluate the effects of the HLA class II alleles on the natural course of HPV persistent and cervical lesions, prospective studies with larger sample sizes and longitudinal design may be needed in various ethnic populations [38].

Disclosure of conflict of interest

None.