Mitochondrial DNA Haplogroup A may confer a genetic susceptibility to AIDS group from Southwest China

Abstract The acquired immunodeficiency syndrome (AIDS) in humans was one of the chronic infections caused by human immunodeficiency virus (HIV), and the interactions between viral infection and mitochondrial energetic implicated that mitochondrial DNA (mtDNA) variation(s) may effect genetic susceptibility to AIDS. Thus, to illustrate the maternal genetic structure and further identify whether mtDNA variation(s) can effect HIV infection among southwest Chinese AIDS group, the whole mtDNA control region sequences of 70 AIDS patients and 480 health individuals from southwest China were analyzed here. Our results indicated the plausible recent genetic admixture results of AIDS group; comparison of matrilineal components between AIDS and matched Han groups showed that mtDNA haplogroup A (p = 0.048, OR = 3.006, 95% CI = 1.109–8.145) has a significant higher difference between the two groups; further comparison illustrated that mtDNA mutations 16,209 (p = 0.046, OR = 2.607, 95% CI = 0.988–6.876) and 16,319 (p = 0.009, OR = 2.965, 95% CI = 1.278–6.876) have significant differences between AIDS and matched control groups, and both of which were the defining variations of mtDNA haplogroup A, they further confirmed that mtDNA haplogroup A may confer genetic susceptibility to AIDS. Our results suggested that haplogroup A may confer a genetic susceptibility to AIDS group from Southwest China.


Introduction
The acquired immunodeficiency syndrome (AIDS) in humans was one of the chronic infections caused by human immunodeficiency virus (HIV) (Shioda & Nakayama, 2006). The mortality of people with the infection of HIV without treated was more than 90% (O'Brien & Nelson, 2004). Highly active antiretroviral treatment (HAART) has significantly reduced the morbidity associated with HIV infection and AIDS (Mocroft et al., 2003), however, the HAART treatment can also inhibit human mitochondrial DNA (mtDNA) polymerase-g which may lead to mtDNA depletion and mitochondrial dysfunction (Martin et al., 2003) and the interactions between viral infection and mitochondrial energetics implied that mtDNA variations may play crucial role in viral disease progression (Hendrickson et al., 2008).
Mitochondria played crucial role in cellular energy metabolism, free radical production, and the apoptotic pathways. Nevertheless, lacking of protective histones, an extremely inefficient DNA mismatch repair mechanism, as well as high level of reactive oxygen species (ROS) caused its higher mutation rate (Beal, 1996;DiMauro & Schon, 2001;Lightowlers et al., 1997;Wallace, 2005;Wallace et al., 1999). The specific combination of the mutations formed mtDNA haplogroup, which was widely used to trace the origin and prehistory of modern human (Cann et al., 1984(Cann et al., , 1987 for having the regional specific mtDNA haplogroups, such as mtDNA haplogroup L, was mainly distributed to African groups (Soares et al., 2012), haplogroups H, UV, WXI, JT, and K were prevalent among European groups (Richards et al., 2000), M7, M8, M9, N9, and R9 were the major mtDNA haplogroups among East Asian groups (Hill et al., 2006;Kivisild et al., 2002), and M2, M3, M4, M5, M6, N5, R5, R6, R30, R31were prevalent in South Asian (Palanichamy et al., 2004;Sun et al., 2006). For the crucial role of mitochondrion on regulating cellular energy metabolism, producing free radicals, and initiating and executing the apoptotic pathways, thus the mtDNA variation(s) or combination of different mtDNA mutation (mtDNA haplogroup) were deduced to be associated with different diseases. MtDNA haplogroup F1 can significantly increase the risk of nasopharyngeal carcinoma (Hu et al., 2014); haplogroup JT was associated with the higher survival rates for severe septic patients (Lorente et al., 2013); haplogroup R was a strong independent predictor of sperm motility decreased chance of asthenozoospermia (Feng et al., 2013). One of the plausible explanations for the association between mtDNA haplogroup was the subtle functional effects on affecting ATP production, ROS, heat generation, and apoptosis for mtDNA variation represented by different haplogroups (Gomez-Duran et al., 2010). However, the regional distributing pattern of different mtDNA haplogroups among different continents implied that there should be different maternal haplogroup on affecting HIV/AIDS susceptible from different regions.
In current study, to illustrate the maternal genetic structure and further identify whether any mtDNA variation(s) can effect HIV infection among southern of East Asia AIDS groups, the whole mtDNA control region of 70 AIDS patients were sequenced and further analyzed by incorporating with 168 matched control individuals of 3 Han Chinese groups retrieved from 480 individuals belonging to 11 ethnic/Han Chinese populations.

Samples
Blood samples of 70 unrelated HIV seropositives patients were collected from southwest of China, which received informed consent before sample collection. All procedures were supervised and approved by the human tissue research committee of our hospital.

DNA amplification, sequencing
Genome DNA was isolated using the genomic DNA extraction kit (Axygen, Union City, CA). DNA was stored at À80 C. The whole mtDNA control region was amplified with one primer pair and purified as described in previous work (Wang et al., 2012). The purified PCR product was sequenced with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on a 3730 DNA sequencer according to the manufacturer's manual.

Data analysis
The sequence variations were recorded by comparing with the revised Cambridge Reference Sequence (rCRS) (Andrews et al., 1999). The 70 AIDS patients and 480 health individuals were assigned to specific haplogroup, respectively according to the most recently updated mtDNA phylogeny (van Oven & Kayser, 2009) based on the specific mtDNA variations of each individual. The principle component analysis (PCA) was conducted by taking the haplogroup frequency as input factor to show the overall clustering pattern of AIDS and ethnic/Han Chinese populations from the similar geographic region, and the populations showing closer genetic affinity with AIDS group were selected as matched control groups. To assess the significance of differences observed for haplogroup distribution frequencies and mtDNA mutations frequencies (ranging from 16,024 bp to 16,365 bp) between AIDS and controls, the Pearson's chi-square test with a one degree of freedom was used, and the Fisher's exact test (two tailed) was applied to those cases with cell counts below five with the SPSS software package (version 16.0, SPSS company, Chicago, IL).

Results and discussion
As shown in Table S1 (supporting material online), all the 70 sequences of AIDS patients were allocated to known mtDNA haplogroup of East Asian (Kivisild et al., 2002;Kong et al., 2006), Southeast Asian (Hill et al., 2006(Hill et al., , 2007Peng et al., 2010) with a few lineages of unassigned M* and N* status. The southern of East Asian prevalent haplogroups, such as haplogroups B, M7, M9 and R9 (including F) (Hill et al., 2006(Hill et al., , 2007 accounted 55.71% (39/70) of all the invested AIDS patients, which was much higher than that of northern of East Asian prevalent haplogroups A, C, D, G and N9 (Kong et al., 2003). In detail, haplogroups B, F and M7 accounted more than 51.43% of the maternal components, and pattern for haplogroups prevalent in northern East Asia group (including A, C, D, and G) was similar with that of Tibetan groups, which may reflect rather recent genetic contributions from northern of East Asia. Thus, our results indicated that the AIDS group was admixed with southern and northern of eastern Asian groups.
To get more insights into the genetic affinities between AIDS group and 11ethnic/Han Chinese groups from the adjacent regions, the principal component map was constructed ( Figure S1, supporting material online) based on the first two principal components (accounting for 65.99% of the total variations), which indicated that AIDS group has the similarity of matrilineal genetic structures with 3 Han Chinese groups, however, which was separated with other ethnic groups from the similar geographic regions ( Figure S1). Our PC map further supported that the AIDS group was the recent admixture history with components from southern and northern of East Asian populations, and the former accounted much higher ratios, this pattern was different from that of ethnic groups, and the current mtDNA variation may reveal their ethnohistory to some extent.
Previous works have revealed that many human diseases were associated with mtDNA haplogroup (Achilli et al., 2011;Fernandez-Caggiano et al., 2013;Hendrickson et al., 2008). More evidences have suggested that the essential role of mitochondria in the innate immunity response to infection (Arnoult et al., 2011;McWhirter et al., 2005), and AIDS in humans was an infection caused by HIV, thus, we hypothesized that mtDNA haplogroup may confer a genetic susceptibility to AIDS. Three Han Chinese were pooled as control group, because these populations have the similarity of matrilineal genetic structures with AIDS group. The 2 test was performed for mtDNA haplogroups with frequency higher than 5% in the AIDS and control groups. As shown in Table 1, haplogroup A (p ¼ 0.048, OR ¼ 3.006, 95% CI ¼ 1.109-8.145) showed significant difference between AIDS and Han Chinese groups (p50.05), which indicated that individuals of haplogroup A may has an increasing risk of AIDS. To test whether any mtDNA variation(s) may contribute to the increasing risk of AIDS, the 2 test were performed for 120 mtDNA mutations of hypervariable region HVSI (HVSI, spanning nucleotide position 16,024-16,365, was covered by both AIDS and control groups) for AIDS patients and control samples with frequency higher than 5%. As shown in  (Dement et al., 2007), affect mtDNA replication and lead to electron transport chain alteration, and which would result in the release of highly ROS (Gille & Joenje, 1992) and the increasing of the ROS may cause the susceptible of HIV. In summary, by studying the mtDNA of 70 AIDS patients from southwest China and that of 480 individuals of 11 ethnic/Han Chinese groups, our results indicated that AIDS group may represent the recent genetic admixture results of southern and northern of East Asian maternal components, which were different from that of ethnic groups from the surrounding regions. Further statistic analyses indicated that mtDNA mutations 16,209 (p ¼ 0.046, OR ¼ 2.607, 95% CI ¼ 0.988-6.876) and 16,319 (p ¼ 0.009, OR ¼ 2.965, 95% CI ¼ 1.278-6.876) pointed that mtDNA haplogroup A (p ¼ 0.048, OR ¼ 3.006, 95% CI ¼ 1.109-8.145) may confer a susceptible genetic factor to AIDS from southwest China.  -Shou Li (No: LJS-201302). Authors declare that they do not have any financial, consulting, and personal relationships with other people or organizations that could influence the author's work. Haplogroup occurred in less than four individuals in the entire case or control population and those unassigned M*, N* and R* mtDNAs were pooled together. We presented these nested haplogroups according to their phylogenetic status, e.g. B contains B4, B5, B6 and B*. *p value was calculated by Pearson's chi-square test; Fisher's exact test was used when haplogroup occurred in less than five individuals.