Immunogenetics DOI 10.1007/s00251-015-0829-2
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APOBEC3H polymorphisms associated with the susceptibility to HIV-1 infection and AIDS progression in Japanese Daisuke Sakurai & Yasumasa Iwatani & Hitoshi Ohtani & Taeko K. Naruse & Hiroshi Terunuma & Wataru Sugiura & Akinori Kimura
Received: 23 November 2014 / Accepted: 12 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Human APOBEC3H (A3H) is a member of APOBEC3 cytidine deaminase family that potently restricts HIV-1 replication. Because A3H is genetically divergent with different intracellular stability and anti-HIV-1 activity in vitro, we investigated a possible association of A3H with susceptibility to HIV-1 infection and disease progression in Japanese populations. A total of 191 HIV-1-infected individuals (HIV group), 93 long-term non-progressors to AIDS (LTNP group) and 421 healthy controls were genotyped for two functional APOBEC3H polymorphisms, rs139292 and rs139297. As compared with the controls, minor allele frequency (MAF) for rs139292 was high in the HIV group (MAF in cases vs. controls; 0.322 vs. 0.263, odds ratio (OR)=1.33, 95 % confidence interval (95 % CI)=1.02–1.74, p=0.035) and low in the LTNP group (0.161 vs. 0.263, OR=0.54, 95 % CI=0.36– 0.82, p=0.004, pc=0.007), whereas the MAF for rs139297 was high in the HIV group (0.367 vs. 0.298, OR= 1.36, 95 % CI=1.07–1.76, p=0.017, pc=0.035). In addition, haplotype analyses revealed that the frequencies of A3H-hapC and -hapA were high (0.322 vs. 0.262, OR=1.33, 95% CI= 1.02–1.74, p=0.003) and low (0.634 vs. 0.697, OR=0.75, 95 % CI=0.58–0.97, p=0.002), respectively, in the HIV group, whereas the frequencies of A3H-hapC and -hapB were low (0.161 vs. 0.262, OR=0.54, 95 % CI=0.36–0.82, p= D. Sakurai : H. Ohtani : T. K. Naruse : A. Kimura (*) Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan e-mail: [email protected] Y. Iwatani : W. Sugiura Department of Infection and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan H. Terunuma Biotherapy Institute of Japan, Tokyo, Japan
0.00003) and high (0.097 vs. 0.040, OR=2.55, 95 % CI= 1.40–4.62, p=0.000008), respectively, in the LTNP group, as compared with those in the controls. These observations suggest that the A3H with low anti-HIV-1 activity, A3HhapC, is associated with the susceptibility to HIV-1 infection, whereas the A3H producing a stable protein, A3H-hapB, may confer a low risk of disease progression to AIDS. Keywords APOBEC3H . HIV-1 infection . AIDS progression . Polymorphism . Haplotype . Japanese Introduction Human apolipoprotein B messenger RNA (mRNA)-editing catalytic polypeptide-like 3 (APOBEC3) gene family encodes cellular cytidine deaminases that play a crucial role in antiretroviral activity by inhibiting replication of retroviruses including HIV-1 (Desimmie et al. 2014; Sheehy et al. 2002). Expression of mRNA for a member of APOBEC3 family, APOBEC3H (A3H), can be detected in various tissues and induced by IFN-α in target cells of HIV-1 infection (Refsland et al. 2010; OhAinle et al. 2006; Harari et al. 2009; Tan et al. 2009), although it is difficult to detect cellular protein expression due to low stability of A3H protein (Dang et al. 2008; OhAinle et al. 2008). APOBEC3H is polymorphic with amino acid changes composed of N15del (rs139292), R18L, R105G (rs139297), K121E/D, and E178D. There are at least four major A3H haplotypes, namely haplotype I (hapI; 15N/18R/105G/121K/ 178E), haplotype II (hapII; 15N/18R/105R/121E/D/178D), haplotype III (hapIII; 15del/18R/105R/121E/D/178D), and haplotype IV (hapIV; 15del/18L/105R/121E/D/178D) (OhAinle et al. 2006, 2008; Dang et al. 2008; Harari et al. 2009; Tan et al. 2009; Ooms et al. 2010; Zhen et al. 2012). It has been reported that A3H encoded by hapII has high
Immunogenetics
antiviral activity, because a combination of 15N and 105R increases the protein stability (Zhen et al. 2010). In addition, hapII-A3H has strong antiviral activity against HIV-1 lacking a functional virion infectivity factor (Vif) in vitro (OhAinle et al. 2006; Harari et al. 2009; Tan et al. 2009; Ooms et al. 2010; Zhen et al. 2010). On the other hand, hapIII- and hapIVA3H sharing 15del showed low antiviral activity due to the low protein stability (OhAinle et al. 2006; OhAinle et al. 2008; Dang et al. 2008; Zhen et al. 2012). Interestingly, OhAinle et al. reported that the destabilization of A3H protein had occurred twice during the evolution of primates including humans, by the 15del mutation and the 105G mutation (OhAinle et al. 2008). They also demonstrated the frequency of 15del allele was relatively constant from 0.28 to 0.40 among different human races, while the frequency of 105G allele was largely different from 0.08 in Africans to 0.61 in European Caucasians and 0.69 in Han Chinese, implying natural selection mechanisms shaping the polymorphic nature of APOBEC3H (OhAinle et al. 2008). Susceptibility to HIV-1 infection and progression to AIDS are suggested to be controlled in part by human genome diversity (Le Clerc et al. 2009, 2011; Chinn et al. 2010; Petrovski et al. 2011; Lane et al. 2013). Although the deletion allele of APOBEC3B did not show any association with HIV/ AIDS in Japanese including hemophiliacs (Itaya et al. 2010; Imahashi et al. 2014), we investigated a possible association of APOBEC3H polymorphisms, rs139292 and rs139297, with the susceptibility to HIV-1 infection and AIDS progression.
Materials and methods A total of 191 Japanese HIV-positive patients (HIV group) enrolled at Nagoya Medical Center (Imahashi et al. 2014) and 93 long-term non-progressors (LTNP group) in HIV-1infected hemophiliacs, who were asymptomatic and not progressed to AIDS without antiviral therapy for longer than 10 years (Nakajima et al. 2007), were the subjects. Of note, the median period between diagnosis and starting anti-retrovirus therapy for the HIV group was 68 days and the detailed demographic information on the HIV group was described previously (Imahashi et al. 2014). Japanese control DNA samples from healthy volunteers (n=421) were obtained from the Health Science Research Resources Bank, Japan (http:// www.jhsf.or.jp/index_b.html). The rs139292 insertion/ deletion (N15del) polymorphism was analyzed by electrophoresis of PCR products, amplified with FAM-conjugated sense primer (5′-GGGTTTGAAAAGTGGCTTG-3′) and antisense primer (5′-GCACACATCAGATGGGGTTA-3′), in a 3130xl genetic analyzer (Thermo Fisher Scientific). The rs139297 (G105R) polymorphism was genotyped by digesting PCR products, amplified with sense primer (5′-CTCTCTGTTT GGGACCCTCC-3′) and antisense primer (5′-TGCACTCT
TATAACTGCAAAGCC-3′), by a restriction enzyme HpyAV (New England Biolabs). Genotyping data in about 45 % of samples were confirmed by direct sequencing of the PCR samples. Strength of the association was expressed as odds ratio (OR) with 95 % confidence interval (95 % CI) calculated from two-by-two tables and statistical significance was evaluated by chi-square test. Corrected p (pc) values were obtained by multiplying the p value by the number of tested polymorphism concerning the multiple comparison. Conformity of genotype distribution to Hardy-Weinberg equilibrium law was tested by comparing the observed genotype frequencies and expected genotype frequencies by using two-by-three tables and chi-square test. Estimation of haplotype frequency and haplotype association study to obtain corrected p values by permutation test (n = 10,000) were done by using Haploview software (Barrett et al. 2005). P values less than 0.05 were considered to be significant. Appropriate informed consent was given from each subject and the study protocol was approved by Ethics committees of National Hospital Organization Nagoya Medical Center and Medical Research Institute, Tokyo Medical and Dental University.
Results and discussion To investigate the possible association of APOBEC3H alleles and the susceptibility to HIV-1/AIDS, genomic DNAs from 191 HIV-1-infected patients (HIV group), 93 LTNPs (LTNP group), and 421 controls were analyzed for rs139292 (Asn15del, N15del) and rs139297 (Gly105Arg, G105R). As shown Table 1, the allele frequencies of rs139292 and r139297 in the Japanese controls were similar to those reported in Han Chinese (OhAinle et al. 2008). It was revealed that the minor allele frequency (MAF) for rs139292 (15del) was significantly high in the HIV group (MAF in cases vs. controls: 0.322 vs. 0.263, odds ratio (OR)=1.33, 95 % confidence interval (CI)=1.02–1,74, p=0.035, pc=not significant (ns)) and low in the LTNP group (0.161 vs. 0.263, OR=0.54, 95 % CI=0.36–0.82, p=0.004, pc=0.007). On the other hand, MAF for rs139297 (105R) was high in the HIV group (0.367 vs. 0.298, OR=1.36, 95 % CI=1.06–1.76, p=0.017, pc= 0.035). Genotype distributions in the HIV group, LTNP group, and controls conformed to the Hardy-Weinberg equilibrium for both rs139292 and rs139297 (Table 1). When homozygotes of major allele (15N for rs139292 and 105G for rs139297) was taken as references, the homozygotes of 15del allele showed high OR (0.141 vs. 0.086, OR=1.86, 95 % CI= 1.07–3.24, p=0.026, pc=ns) in the HIV group and low OR in the LTNP group (0.022 vs. 0.086, OR=0.20, 95 % CI= 0.05–0.86, p=0.017, pc=0.034). These observations suggest that the 15del allele might confer the risk for HIV-1 infection and might have been selected out from HIV-1-infected
Immunogenetics Table 1
Association of APOBEC3H polymorphisms with the susceptibility to HIV-1/AIDS HIV
LTNP
Cont
HIV vs. Cont
(n=191)
(n=93)
(n=421)
OR
95 % CI
0.161 0.699 0.280 0.022 ns
0.263 0.561 0.354 0.086 ns
1.33 1.00 (reference) 1.15 1.86
1.02–1.74
0.258 0.548 0.387 0.065 ns
0.298 0.513 0.378 0.109 ns
1.36 1.00 (reference) 1.20 1.84
rs139292 (N15del) MAF (15del) 0.322 N/N 0.497 N/del 0.361 del/del 0.141 HWE-p ns rs139297 (G105R) MAF (105R) 0.367 G/G 0.440 G/R 0.387 R/R 0.173 HWE-p ns
LTNP vs. Cont p
pc
OR
95 % CI
0.035
ns
0.36–0.82
0.004
0.007
0.79–1.67 1.07–3.24
ns 0.026
ns ns
0.54 1.00 (reference) 0.63 0.20
0.38–1.04 0.05–0.86
ns 0.017
ns 0.034
1.06–1.76
0.017
0.035
0.57–1.17
ns
ns
0.82–1.74 1.10–3.08
ns 0.018
ns 0.037
0.60–1.54 0.22–1.36
ns ns
ns ns
0.82 1.00 (reference) 0.96 0.55
p
pc
Sum of the genotype frequencies may not be 1 due to the rounding at three decimal positions HIV HIV-1-infected subjects, LTNP long-term non-progressors, Cont healthy control subjects, MAF minor allele frequency, OR odds ratio for cases vs. controls, 95 % CI 95 % confidence interval, pc corrected p values, ns not significant, HWE-p p value for deviation from Hardy-Weinberg equilibrium
patients during the disease progression to AIDS, which is consistent with that the 15del variation destabilizes the A3H protein (OhAinle et al. 2008), although a prospective study is required to formally prove the selection of 15del allele under the HIV-1 infection. On the other hand, the homozygotes of 105R allele conferred significantly high OR in the HIV group (0.173 vs. 0.109, OR=1.84, 95 % CI=1.10–3.08, p=0.018, pc=0.037), even though the 105R allele was reported to stabilize the A3H protein and hence could exert higher anti-HIVactivity than the 105G allele (OhAinle et al. 2008). These observations suggested that the 105R allele would contribute the susceptibility to HIV-1 infection or that an allele in strong linkage disequilibrium (LD) with the 105R allele was responsible for the association with the susceptibility. It should be noted here that the heterozygote of 15N and 15del alleles and that of 105G and 105R alleles did not confer significant susceptibility to or
Table 2
protection against HIV-1 infection and/or development of AIDS, implying that the effect of 15del or 105R alleles might not be large enough to give overt effect in the heterozygous states. Although it has recently been reported that A3H could form a homo-multimer to play a role in anti-HIV activity (Li et al. 2014), expression of a mutant allele may not be sufficient to strongly alter the function of A3H hetero-multimers. There is a possibility that the number of subjects was not enough to capture the heterozygous effect or detect such heteromeric interaction, if any, and further study is warranted. From a strong LD between rs139292 and rs139297, A3H haplotype could be categorized into three major groups, hapA (defined by 15N and 105G, representing hapI-A3H), hapB (defined by 15N and 105R, representing hapII-A3H), and hapC (defined by 15del and 105R, representing hapIII- and hapIV-A3H), indicating that the 105R allele could link to either 15N or 15del allele. Alternatively, the 105R allele is
Association between APOBEC3H haplotypes and the susceptibility to HIV-1/AIDS
hapA: hap I (15N-105G) hapB: hap II (15N-105R) hapC: hap III+IV (15del-105R)
HIV
LTNP
Cont
HIV vs. Cont
LTNP vs. Cont
(2n=382)
(2n=186)
(2n=842)
OR
95 % CI
p
OR
95 % CI
p
0.634 0.045 0.322
0.742 0.097 0.161
0.697 0.040 0.262
0.75 1.11 1.33
0.58–0.97 0.61–2.01 1.02–1.74
0.002 ns 0.003
1.25 2.55 0.54
0.87–1.79 1.40–4.62 0.36–0.82
ns 0.000008 0.00003
Sum of the estimated haplotype frequencies may not be 1 due to the rounding at three decimal positions and the presence of a minor haplotype (15del105G) HIV HIV-1-infected subjects, LTNP long-term non-progressors, Cont healthy control subjects, OR odds ratio for cases vs. controls, 95 % CI 95 % confidence interval, ns not significant, p permutation p value calculated from permutation test (n=10,000)
Immunogenetics
mainly linked to the 15del allele, which might explain the association of 105R allele with the susceptibility to HIV-1 infection. To further assess the association of APOBEC3H and HIV/AIDS, especially for evaluating the contribution of 105R in the susceptibility to HIV-1 infection, we performed a haplotype association study by comparing estimated haplotype frequencies. As shown in Table 2, the estimated frequency of hapC (15del-105R) was significantly high in the HIV group as compared with the controls (0.322 vs. 0.262, OR= 1.13, 95 % CI=1.02–1.74, permutation p=0.003), while it was significantly low in the LTNP group (0.161 vs. 0.262, OR=0.54, 95 % CI=0.36–0.82, permutation p=0.00003). On the other hand, the frequency of hapA (15N-105G) was significantly low in the HIV group (0.634 vs. 0.697, OR= 0.75, 95 % CI=0.58–0.97, permutation p=0.002), whereas the frequency of hapB (15N-105R) was significantly high in the LTNP group (0.097 vs. 0.040, OR=2.55, 95 % CI=1.40– 4.62, permutation p=0.000008). Because the hapB represents the hapII-A3H that is known to stabilize A3H protein (OhAinle et al. 2008), significantly higher prevalence of hapB in the LTNP group implied a role of the product of a particular APOBEC3H allele or haplotype in partly delaying the progression to AIDS. Given that the frequency of hapB was only slightly increased in the HIV group, hapB might not be largely involved in the protection against HIV-1 infection in a short term. On the other hand, the frequency of hapA was significantly low in the HIV group and relatively high in the LTNP group. These data could not be explained merely by the protein stability, because hapA (or hapI) contained the 105G variation that was known to destabilize the A3H protein. Our observations in turn implied that the contributions of 15N to 15del change and 105R to 105G change in altered enzyme activity might be different in vivo. In accordance with our observations, it was reported that an overexpression of hapIA3H could elicit potent antiviral activity and resistant to Vif (Dang et al. 2008). In addition, hapI-A3H could bind viral RNA to less extent to hapII-A3H but with considerably high affinity than A3H carrying the 15del variation (Zhen et al. 2012). Here, we report for the first time that the functional APOBEC3H polymorphisms are associated with the susceptibility to HIV-1 infection and progression to AIDS in vivo. The findings in this study should be investigated in other ethnic groups to confirm the anti-HIV-1 protective role of APOBEC3H in vivo. Acknowledgments We are grateful Drs. Hanabusa H. (Ogikubo Hospital), Matsuda J. (Teikyo University School of Medicine), Sakai M. (University of Occupational and Environmental Health), Ikeda S. (Sasebo Municipal Hospital), and Fujii T. (Hiroshima University School of Medicine) for blood sampling from HIV-1-infected patients. This work was supported in part by research grants from the Ministry of Health, Labor and Welfare, Japan, and a Joint Research Program of National Hospital Organization Nagoya Medical Center, Japan.
References Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21:263– 265 Chinn LW, Tang M, Kessing BD, Lautenberger JA, Troyer JL, Malasky MJ, McIntosh C, Kirk GD, Wolinsky SM, Buchbinder SP, Gomperts ED, Goedert JJ, O'Brien SJ (2010) Genetic associations of variants in genes encoding HIV-dependency factors required for HIV-1 infection. J Infect Dis 202:1836–1845 Dang Y, Siew LM, Wang X, Han Y, Lampen R, Zheng YH (2008) Human cytidine deaminase APOBEC3H restricts HIV-1 replication. J Biol Chem 283:11606–11614 Desimmie BA, Delviks-Frankenberrry KA, Burdick RC, Qi D, Izumi T, Pathak VK (2014) MultipleAPOBEC3 restriction factors for HIV-1 and one Vif to rule them all. J Mol Biol 426:1220–1245 Harari A, Ooms M, Mulder LC, Simon V (2009) Polymorphisms and splice variants influence the antiretroviral activity of human APOBEC3H. J Virol 83:295–303 Imahashi M, Izumi T, Watanabe D, Imamura J, Matsuoka K, Ode H, Masaoka T, Sato K, Kaneko N, Ichikawa S, Koyanagi Y, TakaoriKondo A, Utsumi M, Yokomaku Y, Shirasaka T, Sugiura W, Iwatani Y, Naoe T (2014) Lack of association between intact/deletion polymorphisms of the APOBEC3B gene and HIV-1 risk. PLoS One 9: e92861 Itaya S, Nakajima T, Kaur G, Terunuma H, Ohtani H, Mehra N, Kimura A (2010) No evidence of an association between the APOBEC3B deletion polymorphism and susceptibility to HIV infection and AIDS in Japanese and Indian populations. J Infect Dis 202:815–816 Lane J, McLaren PJ, Dorrell L, Shianna KV, Stemke A, Pelak K et al (2013) A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A. Hum Mol Genet 22:1903–1910 Le Clerc S, Limou S, Coulonges C, Carpentier W, Dina C, Taing L et al (2009) Genomewide association study of a rapid progression cohort identifies new susceptibility alleles for AIDS (ANRS Genomewide Association Study 03). J Infect Dis 200:1194–1201 Le Clerc S, Coulonges C, Delaneau O, Van Manen D, Herbeck JT, Limou S et al (2011) Screening low-frequency SNPS from genome-wide association study reveals a new risk allele for progression to AIDS. J Acquir Immune Defic Syndr 56:279–284 Li J, Chen Y, Li M, Carpenter MA, McDougle RM, Luengas EM, Macdonald PJ, Harris RS, Mueller JD (2014) APOBEC3 multimerization correlates with HIV-1 packaging and restriction activity in living cells. J Mol Biol 426:1296–1307 Nakajima T, Ohtani H, Naruse T, Shibata H, Mimaya J, Terunuma H, Kimura A (2007) Copy number variations of CCL3L1 and longterm prognosis of HIV-1 infection in asymptomatic HIV-infected Japanese with hemophilia. Immunogenetics 59:793–798 OhAinle M, Kerns JA, Malik HS, Emerman M (2006) Adaptive evolution and antiviral activity of the conserved mammalian cytidine deaminase APOBEC3H. J Virol 80:3853–3862 OhAinle M, Kerns JA, Li MM, Malik HS, Emerman M (2008) Antiretroelement activity of APOBEC3H was lost twice in recent human evolution. Cell Host Microbe 4:249–259 Ooms M, Majdak S, Seibert CW, Harari A, Simon V (2010) The localization of APOBEC3H variants in HIV-1 virions determines their antiviral activity. J Virol 84:7961–7969 Petrovski S, Fellay J, Shianna KV, Carpenetti N, Kumwenda J, Kamanga G et al (2011) Common human genetic variants and HIV-1 susceptibility: a genome-wide survey in a homogeneous African population. AIDS 25:513–518 Refsland EW, Stenglein MD, Shindo K, Albin JS, Brown WL, Harris RS (2010) Quantitative profiling of the full APOBEC3 mRNA
Immunogenetics repertoire in lymphocytes and tissues: implications for HIV-1 restriction. Nucleic Acids Res 38:4274–4284 Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002) Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646–650 Tan L, Sarkis PT, Wang T, Tian C, Yu XF (2009) Sole copy of Z2-type human cytidine deaminase APOBEC3H has inhibitory activity against retrotransposons and HIV-1. FASEB J 23:279–287
Zhen A, Wang T, Zhao K, Xiong Y, Yu XF (2010) A single amino acid difference in human APOBEC3H variants determines HIV-1 Vif sensitivity. J Virol 84:1902–1911 Zhen A, Du J, Zhou X, Xiong Y, Yu XF (2012) Reduced APOBEC3H variant anti-viral activities are associated with altered RNA binding activities. PLoS One 7:e38771
BRIEF COMMUNICATION
APOBEC3H polymorphisms associated with the susceptibility to HIV-1 infection and AIDS progression in Japanese Daisuke Sakurai & Yasumasa Iwatani & Hitoshi Ohtani & Taeko K. Naruse & Hiroshi Terunuma & Wataru Sugiura & Akinori Kimura
Received: 23 November 2014 / Accepted: 12 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Human APOBEC3H (A3H) is a member of APOBEC3 cytidine deaminase family that potently restricts HIV-1 replication. Because A3H is genetically divergent with different intracellular stability and anti-HIV-1 activity in vitro, we investigated a possible association of A3H with susceptibility to HIV-1 infection and disease progression in Japanese populations. A total of 191 HIV-1-infected individuals (HIV group), 93 long-term non-progressors to AIDS (LTNP group) and 421 healthy controls were genotyped for two functional APOBEC3H polymorphisms, rs139292 and rs139297. As compared with the controls, minor allele frequency (MAF) for rs139292 was high in the HIV group (MAF in cases vs. controls; 0.322 vs. 0.263, odds ratio (OR)=1.33, 95 % confidence interval (95 % CI)=1.02–1.74, p=0.035) and low in the LTNP group (0.161 vs. 0.263, OR=0.54, 95 % CI=0.36– 0.82, p=0.004, pc=0.007), whereas the MAF for rs139297 was high in the HIV group (0.367 vs. 0.298, OR= 1.36, 95 % CI=1.07–1.76, p=0.017, pc=0.035). In addition, haplotype analyses revealed that the frequencies of A3H-hapC and -hapA were high (0.322 vs. 0.262, OR=1.33, 95% CI= 1.02–1.74, p=0.003) and low (0.634 vs. 0.697, OR=0.75, 95 % CI=0.58–0.97, p=0.002), respectively, in the HIV group, whereas the frequencies of A3H-hapC and -hapB were low (0.161 vs. 0.262, OR=0.54, 95 % CI=0.36–0.82, p= D. Sakurai : H. Ohtani : T. K. Naruse : A. Kimura (*) Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan e-mail: [email protected] Y. Iwatani : W. Sugiura Department of Infection and Immunology, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan H. Terunuma Biotherapy Institute of Japan, Tokyo, Japan
0.00003) and high (0.097 vs. 0.040, OR=2.55, 95 % CI= 1.40–4.62, p=0.000008), respectively, in the LTNP group, as compared with those in the controls. These observations suggest that the A3H with low anti-HIV-1 activity, A3HhapC, is associated with the susceptibility to HIV-1 infection, whereas the A3H producing a stable protein, A3H-hapB, may confer a low risk of disease progression to AIDS. Keywords APOBEC3H . HIV-1 infection . AIDS progression . Polymorphism . Haplotype . Japanese Introduction Human apolipoprotein B messenger RNA (mRNA)-editing catalytic polypeptide-like 3 (APOBEC3) gene family encodes cellular cytidine deaminases that play a crucial role in antiretroviral activity by inhibiting replication of retroviruses including HIV-1 (Desimmie et al. 2014; Sheehy et al. 2002). Expression of mRNA for a member of APOBEC3 family, APOBEC3H (A3H), can be detected in various tissues and induced by IFN-α in target cells of HIV-1 infection (Refsland et al. 2010; OhAinle et al. 2006; Harari et al. 2009; Tan et al. 2009), although it is difficult to detect cellular protein expression due to low stability of A3H protein (Dang et al. 2008; OhAinle et al. 2008). APOBEC3H is polymorphic with amino acid changes composed of N15del (rs139292), R18L, R105G (rs139297), K121E/D, and E178D. There are at least four major A3H haplotypes, namely haplotype I (hapI; 15N/18R/105G/121K/ 178E), haplotype II (hapII; 15N/18R/105R/121E/D/178D), haplotype III (hapIII; 15del/18R/105R/121E/D/178D), and haplotype IV (hapIV; 15del/18L/105R/121E/D/178D) (OhAinle et al. 2006, 2008; Dang et al. 2008; Harari et al. 2009; Tan et al. 2009; Ooms et al. 2010; Zhen et al. 2012). It has been reported that A3H encoded by hapII has high
Immunogenetics
antiviral activity, because a combination of 15N and 105R increases the protein stability (Zhen et al. 2010). In addition, hapII-A3H has strong antiviral activity against HIV-1 lacking a functional virion infectivity factor (Vif) in vitro (OhAinle et al. 2006; Harari et al. 2009; Tan et al. 2009; Ooms et al. 2010; Zhen et al. 2010). On the other hand, hapIII- and hapIVA3H sharing 15del showed low antiviral activity due to the low protein stability (OhAinle et al. 2006; OhAinle et al. 2008; Dang et al. 2008; Zhen et al. 2012). Interestingly, OhAinle et al. reported that the destabilization of A3H protein had occurred twice during the evolution of primates including humans, by the 15del mutation and the 105G mutation (OhAinle et al. 2008). They also demonstrated the frequency of 15del allele was relatively constant from 0.28 to 0.40 among different human races, while the frequency of 105G allele was largely different from 0.08 in Africans to 0.61 in European Caucasians and 0.69 in Han Chinese, implying natural selection mechanisms shaping the polymorphic nature of APOBEC3H (OhAinle et al. 2008). Susceptibility to HIV-1 infection and progression to AIDS are suggested to be controlled in part by human genome diversity (Le Clerc et al. 2009, 2011; Chinn et al. 2010; Petrovski et al. 2011; Lane et al. 2013). Although the deletion allele of APOBEC3B did not show any association with HIV/ AIDS in Japanese including hemophiliacs (Itaya et al. 2010; Imahashi et al. 2014), we investigated a possible association of APOBEC3H polymorphisms, rs139292 and rs139297, with the susceptibility to HIV-1 infection and AIDS progression.
Materials and methods A total of 191 Japanese HIV-positive patients (HIV group) enrolled at Nagoya Medical Center (Imahashi et al. 2014) and 93 long-term non-progressors (LTNP group) in HIV-1infected hemophiliacs, who were asymptomatic and not progressed to AIDS without antiviral therapy for longer than 10 years (Nakajima et al. 2007), were the subjects. Of note, the median period between diagnosis and starting anti-retrovirus therapy for the HIV group was 68 days and the detailed demographic information on the HIV group was described previously (Imahashi et al. 2014). Japanese control DNA samples from healthy volunteers (n=421) were obtained from the Health Science Research Resources Bank, Japan (http:// www.jhsf.or.jp/index_b.html). The rs139292 insertion/ deletion (N15del) polymorphism was analyzed by electrophoresis of PCR products, amplified with FAM-conjugated sense primer (5′-GGGTTTGAAAAGTGGCTTG-3′) and antisense primer (5′-GCACACATCAGATGGGGTTA-3′), in a 3130xl genetic analyzer (Thermo Fisher Scientific). The rs139297 (G105R) polymorphism was genotyped by digesting PCR products, amplified with sense primer (5′-CTCTCTGTTT GGGACCCTCC-3′) and antisense primer (5′-TGCACTCT
TATAACTGCAAAGCC-3′), by a restriction enzyme HpyAV (New England Biolabs). Genotyping data in about 45 % of samples were confirmed by direct sequencing of the PCR samples. Strength of the association was expressed as odds ratio (OR) with 95 % confidence interval (95 % CI) calculated from two-by-two tables and statistical significance was evaluated by chi-square test. Corrected p (pc) values were obtained by multiplying the p value by the number of tested polymorphism concerning the multiple comparison. Conformity of genotype distribution to Hardy-Weinberg equilibrium law was tested by comparing the observed genotype frequencies and expected genotype frequencies by using two-by-three tables and chi-square test. Estimation of haplotype frequency and haplotype association study to obtain corrected p values by permutation test (n = 10,000) were done by using Haploview software (Barrett et al. 2005). P values less than 0.05 were considered to be significant. Appropriate informed consent was given from each subject and the study protocol was approved by Ethics committees of National Hospital Organization Nagoya Medical Center and Medical Research Institute, Tokyo Medical and Dental University.
Results and discussion To investigate the possible association of APOBEC3H alleles and the susceptibility to HIV-1/AIDS, genomic DNAs from 191 HIV-1-infected patients (HIV group), 93 LTNPs (LTNP group), and 421 controls were analyzed for rs139292 (Asn15del, N15del) and rs139297 (Gly105Arg, G105R). As shown Table 1, the allele frequencies of rs139292 and r139297 in the Japanese controls were similar to those reported in Han Chinese (OhAinle et al. 2008). It was revealed that the minor allele frequency (MAF) for rs139292 (15del) was significantly high in the HIV group (MAF in cases vs. controls: 0.322 vs. 0.263, odds ratio (OR)=1.33, 95 % confidence interval (CI)=1.02–1,74, p=0.035, pc=not significant (ns)) and low in the LTNP group (0.161 vs. 0.263, OR=0.54, 95 % CI=0.36–0.82, p=0.004, pc=0.007). On the other hand, MAF for rs139297 (105R) was high in the HIV group (0.367 vs. 0.298, OR=1.36, 95 % CI=1.06–1.76, p=0.017, pc= 0.035). Genotype distributions in the HIV group, LTNP group, and controls conformed to the Hardy-Weinberg equilibrium for both rs139292 and rs139297 (Table 1). When homozygotes of major allele (15N for rs139292 and 105G for rs139297) was taken as references, the homozygotes of 15del allele showed high OR (0.141 vs. 0.086, OR=1.86, 95 % CI= 1.07–3.24, p=0.026, pc=ns) in the HIV group and low OR in the LTNP group (0.022 vs. 0.086, OR=0.20, 95 % CI= 0.05–0.86, p=0.017, pc=0.034). These observations suggest that the 15del allele might confer the risk for HIV-1 infection and might have been selected out from HIV-1-infected
Immunogenetics Table 1
Association of APOBEC3H polymorphisms with the susceptibility to HIV-1/AIDS HIV
LTNP
Cont
HIV vs. Cont
(n=191)
(n=93)
(n=421)
OR
95 % CI
0.161 0.699 0.280 0.022 ns
0.263 0.561 0.354 0.086 ns
1.33 1.00 (reference) 1.15 1.86
1.02–1.74
0.258 0.548 0.387 0.065 ns
0.298 0.513 0.378 0.109 ns
1.36 1.00 (reference) 1.20 1.84
rs139292 (N15del) MAF (15del) 0.322 N/N 0.497 N/del 0.361 del/del 0.141 HWE-p ns rs139297 (G105R) MAF (105R) 0.367 G/G 0.440 G/R 0.387 R/R 0.173 HWE-p ns
LTNP vs. Cont p
pc
OR
95 % CI
0.035
ns
0.36–0.82
0.004
0.007
0.79–1.67 1.07–3.24
ns 0.026
ns ns
0.54 1.00 (reference) 0.63 0.20
0.38–1.04 0.05–0.86
ns 0.017
ns 0.034
1.06–1.76
0.017
0.035
0.57–1.17
ns
ns
0.82–1.74 1.10–3.08
ns 0.018
ns 0.037
0.60–1.54 0.22–1.36
ns ns
ns ns
0.82 1.00 (reference) 0.96 0.55
p
pc
Sum of the genotype frequencies may not be 1 due to the rounding at three decimal positions HIV HIV-1-infected subjects, LTNP long-term non-progressors, Cont healthy control subjects, MAF minor allele frequency, OR odds ratio for cases vs. controls, 95 % CI 95 % confidence interval, pc corrected p values, ns not significant, HWE-p p value for deviation from Hardy-Weinberg equilibrium
patients during the disease progression to AIDS, which is consistent with that the 15del variation destabilizes the A3H protein (OhAinle et al. 2008), although a prospective study is required to formally prove the selection of 15del allele under the HIV-1 infection. On the other hand, the homozygotes of 105R allele conferred significantly high OR in the HIV group (0.173 vs. 0.109, OR=1.84, 95 % CI=1.10–3.08, p=0.018, pc=0.037), even though the 105R allele was reported to stabilize the A3H protein and hence could exert higher anti-HIVactivity than the 105G allele (OhAinle et al. 2008). These observations suggested that the 105R allele would contribute the susceptibility to HIV-1 infection or that an allele in strong linkage disequilibrium (LD) with the 105R allele was responsible for the association with the susceptibility. It should be noted here that the heterozygote of 15N and 15del alleles and that of 105G and 105R alleles did not confer significant susceptibility to or
Table 2
protection against HIV-1 infection and/or development of AIDS, implying that the effect of 15del or 105R alleles might not be large enough to give overt effect in the heterozygous states. Although it has recently been reported that A3H could form a homo-multimer to play a role in anti-HIV activity (Li et al. 2014), expression of a mutant allele may not be sufficient to strongly alter the function of A3H hetero-multimers. There is a possibility that the number of subjects was not enough to capture the heterozygous effect or detect such heteromeric interaction, if any, and further study is warranted. From a strong LD between rs139292 and rs139297, A3H haplotype could be categorized into three major groups, hapA (defined by 15N and 105G, representing hapI-A3H), hapB (defined by 15N and 105R, representing hapII-A3H), and hapC (defined by 15del and 105R, representing hapIII- and hapIV-A3H), indicating that the 105R allele could link to either 15N or 15del allele. Alternatively, the 105R allele is
Association between APOBEC3H haplotypes and the susceptibility to HIV-1/AIDS
hapA: hap I (15N-105G) hapB: hap II (15N-105R) hapC: hap III+IV (15del-105R)
HIV
LTNP
Cont
HIV vs. Cont
LTNP vs. Cont
(2n=382)
(2n=186)
(2n=842)
OR
95 % CI
p
OR
95 % CI
p
0.634 0.045 0.322
0.742 0.097 0.161
0.697 0.040 0.262
0.75 1.11 1.33
0.58–0.97 0.61–2.01 1.02–1.74
0.002 ns 0.003
1.25 2.55 0.54
0.87–1.79 1.40–4.62 0.36–0.82
ns 0.000008 0.00003
Sum of the estimated haplotype frequencies may not be 1 due to the rounding at three decimal positions and the presence of a minor haplotype (15del105G) HIV HIV-1-infected subjects, LTNP long-term non-progressors, Cont healthy control subjects, OR odds ratio for cases vs. controls, 95 % CI 95 % confidence interval, ns not significant, p permutation p value calculated from permutation test (n=10,000)
Immunogenetics
mainly linked to the 15del allele, which might explain the association of 105R allele with the susceptibility to HIV-1 infection. To further assess the association of APOBEC3H and HIV/AIDS, especially for evaluating the contribution of 105R in the susceptibility to HIV-1 infection, we performed a haplotype association study by comparing estimated haplotype frequencies. As shown in Table 2, the estimated frequency of hapC (15del-105R) was significantly high in the HIV group as compared with the controls (0.322 vs. 0.262, OR= 1.13, 95 % CI=1.02–1.74, permutation p=0.003), while it was significantly low in the LTNP group (0.161 vs. 0.262, OR=0.54, 95 % CI=0.36–0.82, permutation p=0.00003). On the other hand, the frequency of hapA (15N-105G) was significantly low in the HIV group (0.634 vs. 0.697, OR= 0.75, 95 % CI=0.58–0.97, permutation p=0.002), whereas the frequency of hapB (15N-105R) was significantly high in the LTNP group (0.097 vs. 0.040, OR=2.55, 95 % CI=1.40– 4.62, permutation p=0.000008). Because the hapB represents the hapII-A3H that is known to stabilize A3H protein (OhAinle et al. 2008), significantly higher prevalence of hapB in the LTNP group implied a role of the product of a particular APOBEC3H allele or haplotype in partly delaying the progression to AIDS. Given that the frequency of hapB was only slightly increased in the HIV group, hapB might not be largely involved in the protection against HIV-1 infection in a short term. On the other hand, the frequency of hapA was significantly low in the HIV group and relatively high in the LTNP group. These data could not be explained merely by the protein stability, because hapA (or hapI) contained the 105G variation that was known to destabilize the A3H protein. Our observations in turn implied that the contributions of 15N to 15del change and 105R to 105G change in altered enzyme activity might be different in vivo. In accordance with our observations, it was reported that an overexpression of hapIA3H could elicit potent antiviral activity and resistant to Vif (Dang et al. 2008). In addition, hapI-A3H could bind viral RNA to less extent to hapII-A3H but with considerably high affinity than A3H carrying the 15del variation (Zhen et al. 2012). Here, we report for the first time that the functional APOBEC3H polymorphisms are associated with the susceptibility to HIV-1 infection and progression to AIDS in vivo. The findings in this study should be investigated in other ethnic groups to confirm the anti-HIV-1 protective role of APOBEC3H in vivo. Acknowledgments We are grateful Drs. Hanabusa H. (Ogikubo Hospital), Matsuda J. (Teikyo University School of Medicine), Sakai M. (University of Occupational and Environmental Health), Ikeda S. (Sasebo Municipal Hospital), and Fujii T. (Hiroshima University School of Medicine) for blood sampling from HIV-1-infected patients. This work was supported in part by research grants from the Ministry of Health, Labor and Welfare, Japan, and a Joint Research Program of National Hospital Organization Nagoya Medical Center, Japan.
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