A Genome-Wide Association Study with DNA Pooling Identifies the Variant rs11866328 in the GRIN2A Gene That Affects Disease Progression of Chronic HBV Infection

Abstract

Host genetics play a vital role in determining clinical outcomes of hepatitis B virus (HBV) infection. To identify novel susceptibility loci to HBV progression, we carried out a genome-wide association study with DNA pooling. This study assessed the relationship between 8 highly-ranked SNPs selected from our DNA pool and disease progression of HBV infection in two independent case-control studies. The first population included 628 asymptomatic HBV carriers (AsC) and 1729 progressed HBV carriers recruited from Hubei Province in south China. The second population was composed of 226 AsC and 215 progressed HBV carriers recruited from Shandong Province in north China. Of the 8 SNPs, variant rs11866328 (G/T), located in the glutamate receptor ionotropic N-methyl D-aspartate 2A (GRIN2A) gene, was replicated and had significant associations with disease progression of HBV infection in the DNA pooling stage both in the Hubei (OR 1.65; 95% CI 1.34,2.02; p=1.96×10⁻⁶; additive model), and in the Shandong (OR 1.73; 95% CI 1.14,2.65; p=1.00×10⁻²; additive model) population. Polymorphism rs11866328 in the GRIN2A gene might be a genetic variant underlying the susceptibility of HBV carriers to disease progression.

Introduction

H epatitis B virus (HBV) infection is a global public health issue because of its worldwide distribution and its potential adverse sequelae, including cirrhosis, hepatocellular carcinoma (HCC), and fulminant liver failure. The prevalence of HBV infection is approximately 5% globally, while it shows regional diversity, with a relatively low incidence in Western countries (0.1–2.0%) (1), but is endemic in China (7.18%) (2,3). Most HBV carriers are thought to be infected through maternal transmission in the neonatal period or infancy in prevalent areas, particularly in China (4 –6). The natural history of chronic HBV infection is a dynamic state involving interaction between virus strain, host hepatocytes, and the immune response, which causes a broad spectrum of liver diseases, ranging from asymptomatic carrier (AsC), fulminant hepatitis, chronic hepatitis, and liver cirrhosis (LC), to HCC (7,8). Persistent HBV infection is multifactorial, with viral, environmental, and host genetic components, including HBV genomic variability, age at infection, sex, chronic alcohol abuse, and co-infection with hepatitis C virus (HCV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) (9 –12). Twin and ethical studies of HBV infection strongly support an important role of genetic susceptibility in HBV persistence and outcomes of HBV infection (13 –17). A significant association between genetic variants in HLA-DPA1 and HLA-DPB1 and chronic hepatitis B persistent infection was identified in Asian populations (18), and 1p36.22 was verified as a susceptibility locus for HCC in HBV carriers by a genome-wide association study (GWAS) (19). To identify susceptibility variants to disease progression of HBV infection, we carried out a GWAS using DNA pooling in Han Chinese HBV carriers. In this study, we evaluate the effects of 8 risk variants selected from our DNA pooling on disease progression of HBV infection in two independent populations with different infection states of HBV (AsC and progressed HBV carriers), one from southern China, and the other from northern China.

Materials and Methods

Study participants

Two different populations were enrolled in this study. The first population was unrelated southern Han Chinese recruited from Hubei Province, and the second was unrelated northern Chinese Han recruited from Shandong Province. They were collected from September 2007 to March 2010. Each population was composed of three subgroups: naïve control, AsC, and progressed HBV carriers (acute liver failure [ALF], liver cirrhosis [LC], and HCC). The Hubei population included 210 naïve controls, 628 AsC, and 1729 progressed HBV carriers (261 with ALF, 631 with LC, and 837 with HCC), recruited from Wuhan Tongji Hospital, Wuhan Union Hospital, and Traditional Chinese Medicine Hospital of Hubei Province. The Shandong population was composed of 210 naïve controls, 226 AsC, and 215 progressed HBV carriers (2 with ALF, 155 with LC, and 58 with HCC), which was recruited from Qingdao Hospital for Infectious Disease and the Affiliated Hospital of Binzhou Medical College. All HBV carriers were positive for both HBsAg and anti-HBcAb for at least 6 mo. The criteria for AsC, ALF, LC, and HCC complied with a previous description (20,21), with minor modifications (Supplementary Table 1; see online supplementary material at http://www.liebertonline.com). Naive controls were community-based healthy Han Chinese (296 male [70.48%], age 48.20±8.63 y), who were HBV serum marker-negative (HBsAg, anti-HBsAb and anti-HBcAb), with no hepatitis B vaccination history. The AsC and naive controls were all over 35 y old to provide adequate time for symptoms to become manifest. All subjects had no serological evidence of co-infection with HCV, HDV, or HIV.

Informed written consent was obtained from all participants, and the study was performed with the approval of the ethical committee of Huazhong University of Science and Technology.

DNA isolation, DNA pooling, and genotyping

Genomic DNA was isolated from EDTA-anticoagulated blood using a QuickGene DNA whole blood kit S (DB-S) with QuickGene-Mini80 equipment (Fujifilm, Tokyo, Japan). Affymetrix Genome-Wide Human Mapping SNP6.0 Arrays were performed for DNA pooling, which were constructed by pooling 120 ng of DNA from each participant. Four independent pools were created: case A was the ALF group (n=86), case B was the LC group (n=88), case C was the HCC group (n=90), and case D was the AsC group (n=66), that was considered as control. Twelve chips (each pool was replicated in triplicate) were finished according to the manufacturer's instructions. The detailed process of DNA pooling has been submitted to GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jfobnoyaukyemry&acc=GSE26034). Raw probe intensity data (CEL files) were used to calculate relative allele signal (RAS) scores and to estimate allele frequencies (22). The silhouette method was used to analyze the results of DNA pooling. The criteria for selection of SNPs for further investigation were Silhouette width exceeding 0.7 (23,24), and minor allele frequency (MAF) >0.05. Eight SNPs, all high-ranked and located in a known gene, were included in the panel. They were all tagSNPs using data from the International HapMap project (www.hapmap.org; CHB + JPT population), and their characteristics are shown in Supplementary Table 2 (see online supplementary material at http://www.liebertonline.com). The TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) was used for genotyping according to the manufacturer's instructions. Each assay was carried out using 10 ng DNA in a 5-μL reaction system consisting of TaqMan universal polymerase chain reaction master mix (Applied Biosystems), forward and reverse primers, and 6-carboxyfluorescein (FAM)- and 4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein (VIC)-labeled probes designed by Applied Biosystems (ABI Assay on Demand). To validate the TaqMan assay, we analyzed 118 (5%) randomly selected samples recruited from Hubei Province by both direct sequencing (Invitrogen, Shanghai, China) and TaqMan assay. The concordance rate of these two methods was 99.9%, indicating that the TaqMan assay was reliable. A total of 10% of all genotypes were repeated in independent PCR reactions using the same TaqMan assay to check for consistency and to ensure intra- and inter-plate genotype quality control. No genotyping discrepancies were detected between the repeated samples. Patients detected by TaqMan genotyping were independent samples, with no overlap with those in DNA pooling.

Statistical analysis

The Hardy-Weinberg equilibrium of genotype frequencies was evaluated online using the website of the Technical University Munich (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Statistical analysis was performed using SPSS software (version 17.0; SPSS Inc., Chicago, IL). Chi-square (χ²) tests were used to examine the differences in clinical characteristics of the participants. The adjusted odds ratio (OR) and 95% confidence interval (95% CI) were tested by multivariable unconditional logistic regression under genetic models with adjustment for gender, age, and alcohol consumption. All tests were two-sided with a significance level of p<0.05.

Results

Characteristics of HBV carriers are shown in Table 1. Although an effort was made to obtain a good match for gender and age between AsC and progressed HBV carriers, there were more men in the progressed HBV carrier group than the AsC group (p<0.01) in our hospital-based case-control study. The subjects in the progressed HBV group were younger in Hubei Province (p=0.01), and older in Shandong Province (p<0.01). More individuals had first-degree relatives with HBV infection in the progressed HBV carrier group than the AsC group (p<0.01). A large difference in alcohol consumption between AsC and progressed HBV carriers was not evident. Genotype distributions of the participants are shown in Table 2. All genotypic distributions of variants in naive controls, AsC, and progressed HBV carriers were consistent with Hardy-Weinberg equilibrium (p≥0.05).

Table 1.

Clinical and Demographic Characteristics of Hepatitis B virus (HBV) carriers

	Hubei Province		Shandong Province
	AsC	Progressed HBV carriers	AsC	Progressed HBV carriers
Age, y (SD)	47.78 (9.28)	46.93 (10.34)^*	45.71 (12.63)	53.42 (9.59)^**
^bAlcoholics, no. (%)	223 (35.51)	684 (39.56)	69 (30.53)	77 (35.81)
HBeAg-positive no. (%)	43 (6.85)	195 (11.28)	39 (17.26)	44 (20.47)
^aHBV family history, no. (%)	25 (3.98)	218 (12.61)^**	1 (0.44)	65 (30.23)^**

Compared with AsC (p=0.01).

Compared with AsC p<0.01).

HBV family history: individuals who have known parents or siblings who are HBV carriers.

Defined as alcohol consumption of >40 g/wk for men and >20 g/wk for women.

The differences of clinical characteristics were calculated using chi-square test.

M, male; F, female; SD, standard deviation; no., number; AsC, asymptomatic HBV carriers; HBeAg, hepatitis B early antigen.

Table 2.

Association Results for SNPs in Two Case-Control Studies

		Hubei Province				Shandong Province
SNP	Gene	Control ^a n=628	Case ^a n=1729	P	OR (95% CI)	Control ^a n=226	Case ^a n=215	p	OR (95% CI)
rs11866328	GRIN2A	93/173/362	70/527/1126	1.96×10⁻⁶	1.65 (1.34,2.02)^c	22/96/108	10/70/135	1.00×10⁻²	1.73 (1.14,2.65)^c
rs2013562	UGT2B4	122/310/196	346/872/507	0.59	0.93 (0.71,1.22)^c
rs7861010	BNC2	36/268/318	111/670/948	1.20×10⁻²	1.31 (1.06,1.61) ^c	25/97/104	12/93/110	0.19	0.73 (0.45,1.17)^c
rs10485138	ASCC3	23/172/429	60/553/1113	0.1	0.83 (0.66,1.04)^c
rs6909880	ASCC3	55/245/328	113/662/949	0.47	1.08 (0.88,1.34)^c
rs12206945	ASCC3	63/255/310	95/676/958	3.00×10⁻³	1.74 (1.21,2.51)^b	16/83/127	12/71/132	0.32	1.68 (0.60,4.68)^b
rs10845858	GRIN2B	114/323/191	322/874/525	0.84	0.97 (0.74-1.28)^c
rs1041236	GPA33	67/240/321	150/683/891	0.88	0.98 (0.79-1.22)^c

Number of minor homozygote/number of heterozygote/number of major homozygote.

Dominant model.

Additive model.

Total numbers for each polymorphism vary because not all samples were successfully genotyped.

SNP, single nucleotide polymorphism; Control, asymptomatic HBV carriers; Case, progressed HBV carriers.

p Values, odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression compared with asymptomatic HBV carriers, adjusting for gender, age and alcohol consumption.

Of the 8 SNPs, 3 SNPs: rs11866328 (OR 1.65; 95% CI 1.34,2.02; p=1.96×10⁻⁶; additive model), rs7861010 (OR 1.31; 95% CI 1.06,1.61; p=1.20×10⁻²; additive model), and rs12206945 (OR 1.74; 95% CI 1.21,2.51; p=3.00×10⁻³; dominant model) were replicated and had strong correlations with disease progression of HBV infection, as in the DNA pooling stage in the Hubei population, based on multivariable logistic regression analysis with adjustment for age, sex, and alcohol use (Table 2). The three strongly correlated SNPs were further genotyped in an additional independent case-control population recruited from Shandong Province in northern China using the same TaqMan assay. On the basis of logistic regression adjusting for sex, age, and alcohol consumption, only SNP rs11866328 was found to affect susceptibility to HBV progression (OR 1.73; 95% CI 1.14,2.65; p=1.00×10⁻²; additive model) (Table 2). Combining the two case-control studies, the association of rs11866328 with HBV progression reached a p value of 1.60×10⁻⁸ (OR 1.68; 95% CI 1.40,2.02; additive model) (Table 3). In stratification analysis of SNP rs11866328 in all studied HBV carriers, the results remained significant in males (OR 1.61; 95% CI 1.30,1.99; p=1.10×10⁻⁵; additive model), females (OR 1.78; 95% CI 1.25,2.53; p=1.00×10⁻³; additive model), and patients 35 y or older (OR 1.63; 95% CI 1.35,1.97; p=3.10×10⁻⁷; additive model) (Table 3). As shown in Table 4, GG genotype was a risk factor for ALF (p=1.70×10⁻²), LC (p=6.60×10⁻⁷), and HCC (p=4.90×10⁻⁴), in the Hubei population. This variant might perform a crucial role in determining the outcomes of HBV infection.

Table 3.

Association Tests for SNP rs11866328 in All HBV Carriers and Stratification Analysis by Sex and Age

				Additive model
Population	Genotype	Asymptomatic HBV carriers	Progressed HBV carriers	P	OR (95% CI)
All HBV carriers		n=854	n=1938	1.60×10⁻⁸	1.68 (1.40,2.02)
	TT	84 (9.84)	80 (4.13)
	TG	342 (40.05)	597 (30.80)
	GG	428 (50.11)	1261 (65.07)
Male		n=507	n=1670	1.10×10⁻⁵	1.61 (1.30,1.99)
	TT	47 (9.27)	74 (4.43)
	TG	200 (39.45)	515 (30.84)
	GG	260 (51.28)	1081 (64.73)
Female		n=347	n=268	1.00×10⁻³	1.78 (1.25,2.53)
	TT	37 (10.66)	6 (2.24)
	TG	143 (41.21)	82 (30.60)
	GG	167 (48.13)	180 (67.16)
Age ≥35 y		n=854	n=1467	3.10×10⁻⁷	1.63 (1.35,1.97)
	TT	84 (9.84)	58 (3.95)
	TG	342 (40.05)	463 (31.56)
	GG	428 (50.11)	946 (64.49)

The number of progressed HBV carriers for SNP rs11866328 was 1723 because not all samples were successfully genotyped.

p Values, odds ratios (OR), and 95% confidence intervals (CI) were calculated by logistic regression compared with asymptomatic HBV carriers, adjusting for gender, age, and alcohol consumption.

Table 4.

Logistic Regression of SNP rs11866328 to ALF, LC, and HCC in the Hubei Population

				Additive model
	TT	TG	GG	p	OR (95% CI)
AsC no. (%)	93 (14.81)	173 (27.55)	362 (57.64)	Reference	Reference
ALF no. (%)	10 (3.86)	83 (32.05)	166 (64.09)	1.70×10⁻²	1.50 (1.08,2.09)^a
LC no. (%)	29 (4.63)	178 (28.39)	420 (66.98)	6.60×10⁻⁷	1.90 (1.48,2.44)
HCC no. (%)	31 (3.70)	266 (31.78)	540 (64.52)	4.90×10⁻⁴	1.52 (1.20,1.93)
ALF + LC + HCC no. (%)	70 (4.06)	527 (30.59)	1126 (65.35)	1.96×10⁻⁶	1.65 (1.34,2.02)

Recessive model (GG/TG+TT); the additive model was p=0.11.

The number of progressed HBV carriers for SNP rs11866328 was 1723 because not all samples were successfully genotyped.

p Values, odds ratios (OR), and 95% confidence intervals (CI) were calculated by logistic regression compared with asymptomatic HBV carriers, adjusting for gender, age, and alcohol consumption.

AsC, asymptomatic HBV carrier; ALF, acute liver failure; LC, liver cirrhosis; HCC, hepatocellular carcinoma; HBV, hepatitis B virus.

Discussion

We performed this study using DNA pooling in the screening stage. DNA pooling has been confirmed to be an effective and efficient method to select candidate susceptibility loci for follow-up by individual genotyping (23,25 –27). The results of DNA pooling were analyzed using silhouettes, which were introduced in 1987 as a general graphical aid for interpretation and validation of cluster analysis (24,28).

There was a strong difference in the distribution of sex and age between our AsC and progressed HBV carriers (Table 1), hence we controlled for the effect of sex and age in all analyses by logistic regression. To decrease the bias of gender on the effect estimates, we conducted a stratification analysis for sex. Interestingly, the association remained significant for male and female patients (Table 3). Long-term cohort studies show that advanced age is a risk factor for HBV persistence and disease progression (29 –31), so we limited our AsC subjects to those 35 y or older to minimize the deviation of age. In stratification analysis for age (≥35 y), subjects bearing the GG genotype showed a strong correlation with adverse outcomes of HBV infection (Table 3). This is in accord with our observation that the variant rs11866328 is associated with disease progression in HBV carriers. There were more individuals with parents or siblings who were HBV carriers in the progressed HBV carrier group, which is consistent with the fact that genetic factors play an important role in HBV progression.

The genotype frequencies of rs11866328 in our naive subjects (TT: 3.1%, TG: 35.0%, GG: 61.9%) were similar to the frequency distributions seen in chronic hepatitis B patients reported in dbSNP (TT: 2.2%, TG: 31.1%, GG: 66.7%; www.ncbi.nlm.nih.gov). The correlation between variant rs11866328 and HBV progression in south China (p=2.0×10⁻⁶) was stronger than that seen in north China (p=1.00×10⁻²) (Table 2). One explanation for this distinction is that the number of subjects recruited from north China was relatively small. More samples need to be recruited in north China to verify this association. The differences in genotype distribution seen between Hubei and Shandong Province may be due to the population substructure of the Han Chinese (32). Studies based on analyses of archeology, anatomy, linguistics, and genetic data consistently suggest that there is a significant difference between the North Chinese and South Chinese (33). Compared with the T allele (GT + TT), those with the GG genotype displayed an increased susceptibility to ALF (p=1.70×10⁻²; recessive model), while in comparison with G/T heterozygotes, the significance was not great (p=0.11; additive model) (Table 4). This deviation may be due to the small ALF sample, as there were only 261 ALF patients in the Hubei population. Because of the relatively small number of progressed HBV carriers in the Shandong population, genotype frequencies of SNP rs11866328 in the ALF, LC, and HCC subgroups were not large.

The GRIN2A gene is located on chromosome 16p13.2, which encodes the N-methyl-D-aspartate (NMDA) glutamate receptor NR2A subunit. NMDA receptors (NMDARs) are a major class of excitatory neurotransmitter receptors in the central nervous system. They form glutamate-gated ion channels that are highly permeable to calcium, and mediate activity-dependent synaptic plasticity (34). SNP rs11866328 is located in the tenth intron of the GRIN2A gene, and does not alter the amino acid sequence of the protein. Further studies are needed to more precisely map and identify the causative polymorphism, and to clarify which genes drive the genetic association. Further functional studies are required to establish the role of this gene in the pathogenesis of disease progression of HBV infection. We realize that the ideal model for studying disease progression in HBV carriers is a community-based, long-term cohort study. From this initial population an HBV carrier cohort can be established, and time-to-progression analyses may then be conducted.

Conclusion

We revealed the important role of SNP rs11866328 on the gene GRIN2A in disease progression of HBV infection in two independent case-control studies in Han Chinese HBV carriers. To our knowledge, this is the first report showing that rs11866328 may affect an individual's susceptibility to HBV progression. Our research indicates that SNP rs11866328 may be a genetic variant underlying chronic HBV carriers' susceptibility to disease progression.

Footnotes

Acknowledgments

This study was supported by the National Basic Research Program of China (973 Program) grant 2007CB512900, and the Chinese National Natural Science Foundation grant 30872237.

Author Disclosure Statement

No competing financial interests exist.

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