Mitochondrial DNA haplogroup background affects LHON, but not suspected LHON, in chinese patients

Mitochondrial DNA Haplogroup Background Affects LHON, but Not Suspected LHON, in Chinese Patients A-Mei Zhang 1,5. , Xiaoyun Jia 2. , Rui Bi 1,5 , Antonio Salas 3 , Shiqiang Li 2 , Xueshan Xiao 2 , Panfeng Wang 2 , Xiangming Guo 2 , Qing-Peng Kong 4 , Qingjiong Zhang 2 *, Yong-Gang Yao 1 * 1Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming, China, 2State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, 3Unidade de Xene ´tica, Instituto de Medicina Legal and Departamento de Anatomı ´a Patolo ´xica e Ciencias Forenses, Facultade de Medicina, Universidade de Santiago de Compostela, Galicia, Spain, 4State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China, 5Graduate School of the Chinese Academy of Sciences, Beijing, China Abstract Recent studies have shown that mtDNA background could affect the clinical expression of Leber hereditary optic neuropathy (LHON). We analyzed the mitochondrial DNA (mtDNA) variation of 304 Chinese patients with m.11778G.A (sample #1) and of 843 suspected LHON patients who lack the three primary mutations (sample #2) to discern mtDNA haplogroup effect on disease onset. Haplogroup frequencies in the patient group was compared to frequencies in the general Han Chinese population (n= 1,689; sample #3). The overall matrilineal composition of the suspected LHON population resembles that of the general Han Chinese population, suggesting no association with mtDNA haplogroup. In contrast, analysis of these LHON patients confirms mtDNA haplogroup effect on LHON. Specifically, the LHON sample significantly differs from the general Han Chinese and suspected LHON populations by harboring an extremely lower frequency of haplogroup R9, in particular of its main sub-haplogroup F (#1 vs. #3, P-value = 1.46610 217 , OR = 0.051, 95% CI: 0.016–0.162; #1 vs. #2, P-value = 4.44610 217 , OR = 0.049, 95% CI: 0.015–0.154; in both cases, adjusted P-value ,10 25 ) and higher frequencies of M7b (#1 vs. #3, adjusted P-value = 0.001 and #1 vs. #2, adjusted P-value = 0.004). Our result shows that mtDNA background affects LHON in Chinese patients with m.11778G.A but not suspected LHON. Haplogroup F has a protective effect against LHON, while M7b is a risk factor. Citation: Zhang A-M, Jia X, Bi R, Salas A, Li S, et al. (2011) Mitochondrial DNA Haplogroup Background Affects LHON, but Not Suspected LHON, in Chinese Patients. PLoS ONE 6(11): e27750. doi:10.1371/journal.pone.0027750 Editor: Alfred Lewin, University of Florida, United States of America Received July 5, 2011; Accepted October 24, 2011; Published November 15, 2011 Copyright: ß2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Ministry of Science and Technology of China (2011CB910900), the National Natural Science Foundation of China (30925021), Yunnan Province (2009CI119), and Guangdong Province (2009B091300150). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]om (Y-GY); [email protected] (QZ) .These authors contributed equally to this work. Introduction The three primary mutations (m.3460G.A in the MT-ND1 gene, m.11778G.A in the MT-ND4 gene, and m.14484T.Cin the MT-ND6 gene) on mitochondrial DNA (mtDNA) have been identified to be the essential factors for Leber hereditary optic neuropathy (LHON, OMIM 535000). It is estimated that over 95% of LHON patients have one of the three primary mutations [1–4]. Despite decades of extensive study on LHON, several enigmas remain unresolved. Previous studies suggested that incomplete penetrance and gender bias in the clinical expression of LHON were modulated by many factors, such as mtDNA background, nuclear genes and environmental factors [5–11]. By way of replicating the same findings in different and independent cohorts, it has been well established in European LHON patients that haplogroups J2, J1 and K increase the risk of blindness in patients with m.11778G.A, m.14484T.C and m.3460G.A, respectively, and haplogroup H is suggested to be a protective factor for LHON patients with m.11778G.A [5,12– 14]. Our previous study of Chinese families with m.11778G.A showed that haplogroup M7b192 could increase the risk of vision loss but haplogroup M8a might produce a protective effect [15]. However, frequencies of haplogroups M7b192 and M8a are not significantly altered in LHON pedigrees compared to the normal controls despite their apparent background effect on the penetrance [15]. This pattern is quite different from that of European patients, in which there is an internal consistency of the haplogroup association, namely, haplogroup J is present at an increased frequency in LHON families with m.11778G.A and m.14484T.C, and subdivisions of this haplogroup show increased penetrance [5]. Dissecting haplogroups in European LHON families have shown that the increased frequency of J was indeed due to an increased frequency of two specific nested sub-clades of J (one deriving from J1 and another deriving from J2) [5,14]. Insufficient sampling (limited statistical power), and/or potential population stratification may account for the lack of internal consistency in our observation [15]. Besides the LHON patients with the primary mutations, there are many suspected LHON patients expressing LHON clinical features but carrying none of the three primary mutations [16]. PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27750 The exact pathogenic mechanism of these heterogeneous patients with suspected LHON has not been well examined. Rare pathogenic mutations, e.g. m.3635G.A, might account for the disease expression in a very small portion of these patients (Ref. [17,18] and references therein). The MT-ND1 and MT-ND6 genes were found to be mutational hotspots for LHON patients without the three primary mutations in European populations [19,20]. Similarly, we found that the MT-ND1 and MT-ND5 genes are mutational hotspots in Chinese suspected LHON patients with a family history but lacking the three primary mutations [21]. Whether there is an effect of mtDNA background on suspected LHON has not been tested so far. In order to further verify the mtDNA haplogroup effect on LHON and to discern the potential effect of mtDNA background in cases of suspected LHON, we dissected the matrilineal pool of 304 LHON samples with m.11778G.A and 843 suspected LHON samples lacking the three primary mutations. We further sequenced four complete M7b mtDNA genomes and evaluated haplogroup-specific genetic variants that may be relevant for the haplogroup association with LHON. Our study provided an updated profile for the effect of mtDNA background on LHON in Chinese populations. Materials and Methods Ethics statement Written informed consents conforming to the tenets of the Declaration of Helsinki were obtained from each participant prior to the study. The institutional review boards of the Zhongshan Ophthalmic Center and the Kunming Institute of Zoology approved this study. Patients The patients received ophthalmological examinations at the Pediatric and Genetic Clinic of the Eye Hospital, Zhongshan Ophthalmic Center and/or local medical centers, and were categorized according to the expression of LHON clinical features (e.g. painless, acute or sub-acute vision deterioration without apparent reasons). LHON patients with primary mutation m.14484T.C or m.3460G.A were reported elsewhere [22,23] and were not discussed in this study. 175 LHON patients with m.11778G.A had a family history and were described in our previous study [15]; these patients were reanalyzed with the new samples in this study. LHON patients with both m.11778G.A and m.593T.C (n = 14) and suspected LHON patients with m.593T.C (n = 12) were discussed in a separate paper [24]. Patients without the three primary mutations of LHON were simply regarded as patients with suspected LHON, despite that this grouping included a very small proportion of LHON patients that were caused by other rare primary LHON mutations. Blood samples were collected from patients in vacuum tubes containing EDTA. For some cases, blood samples were collected on filtered papers. Screening of primary LHON mutations, sequencing the mtDNA control region and complete genome The PCR-single strand conformation polymorphism (PCRSSCP) and allele specific PCR (AS-PCR) methods were jointly used to detect the three primary mutations as previously described [25]. The presence of the three primary mutations in the patients was independently screened in Kunming using method described by Bi et al. [26]. The mtDNA control region sequence of each patient was amplified by using primer pair (L15594: 59-CGCCTACACAATTCTCCGATC-39and H901: 59-ACTTGGGTTAATCGTGTGACC-39) and the following PCR condition: one pre-denaturation cycle at 94uC for 5 min; 30 cycles at 94uC for 30 sec, 52uC for 30 sec, 72uC for 2 min; and one extension cycle at 72uC for 10 min. The PCR products were purified and sequenced by sequencing primers, which were used in our previous study [27], on a 3730 DNA analyzer (Applied Biosystems). Each sample was sequenced for an approximate 1.4 kb fragment (mtDNA regions 16033–16569 and 1–850; numbering according to the revised Cambridge reference sequence (rCRS) [28]) which cover the mtDNA control region, the MT-TF gene and part of the MT-RNR1 gene. We determined the entire mtDNA sequences for four LHON patients (Fig. S1) belonging to haplogroup M7b192 using the same procedure and condition described in our previous study [27]. Novelty and potential pathogenicity for those non-synonymous private variants in these lineages were analyzed using the same method reported by Bandelt et al. [29]. mtDNA haplogroup classification and statistical analysis The sequences were compared with the rCRS [28] and the variants in each mtDNA sequence were recorded and were further checked by using the MitoTool (www.mitotool.org) [30]. We classified each sample based on the haplogroup motifs that were identified according to the PhyloTree (http://www.phylotree.org; mtDNA tree Build 9, 20 Jun 2010) [31]. For samples which could not be unambiguously determined by the mtDNA control region sequence variants, PCRrestriction fragment length polymorphism (RFLP) analysis for specific coding region variant(s) was performed, e.g. a check of 25176AluI was performed to confirm haplogroup D status. We first compared the matrilineal composition of suspected LHON patients (n= 843) to 1,689 pooled Han Chinese mtDNAs from the general populations (Table S1), to discern the potential effect of mtDNA background on the onset of suspected LHON. Under the null hypothesis that the suspected LHON patients had a similar matrilineal composition as the pooled Han Chinese, the suspected LHON patients could be used as a second control group for comparison to the LHON patients. We then compared the newly obtained 304 LHON patients with m.11778G.A to the suspected LHON samples and the pooled Han Chinese samples, respectively, to discern the mtDNA haplogroup background effect on LHON and to verify the pattern as observed in our previous studies based on limited sample sizes [15]. We next pooled the newly obtained LHON patients (n= 304) with the reported LHON patients (n= 175; [15]). Combining these two different datasets provide more statistical power than the strategy of analyzing both data sets separately (Methods S1) [32]. Pearson’s chi-square test with a one degree of freedom was used to assess the significance of the differences observed in haplogroup frequencies between cases and controls. The Fisher Exact test (two tailed) was applied to those cases with cell counts below five. A permutation test was used to address the issue of multiple testing in haplogroup association test; 100,000 permutations were used in all comparisons. The statistical package SPSS (version 15.0) and Stata v.8 (http://www.stata.com) were used for all the computations; adjusted P-values below 0.05 were considered to be statistically significant. Results Patient features A total of 1,322 unrelated subjects collected from different provinces in China were enrolled in the current analysis based on the availability of DNA after the initial primary mutation mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27750 screening. These patients included 479 LHON patients with mutation m.11778G.A (including 175 samples reported in our previous study [15] and 304 samples newly sequenced) and 843 suspected LHON patients who expressed clinical features but did not harbor any of the three primary mutations. Only one out of 479 patients had the heteroplasmic mutation m.11778G.A. The overall frequency of patients with heteroplasmic mutation m.11778G.A was thus very low (0.21%; 1/479) in these Chinese patients based on the limitation of detection sensitivity of our approach. The mtDNA control region sequence variations for each patient are listed in the online Tables S2 and S3, and the sequences can be retrieved from GenBank under the Accession Numbers HM632037–HM632340 (for 304 LHON patients with m.11778G.A) and HM632341–HM633183 (for 843 suspected LHON patients). mtDNA Haplogroup distribution in patients with suspected LHON We first dissected the matrilineal genetic components in the suspected LHON (sample #2) and compared to the pooled Han Chinese from the general populations (sample #3, n= 1,689). As shown in Table 1 and Fig. 1, the overall matrilineal composition of the 843 suspected LHON patients resembled that of the pooled Han Chinese. There was no statistically significant difference for any of the haplogroup classes between the two populations. Therefore, the suspected LHON sample, despite of its miscellaneous nature of the disease, could be used as the second independent control sample to compare with LHON patients with m.11778G.A in the subsequent analyses. mtDNA Haplogroup distribution in LHON patients with m.11778G.A To compare with the pattern that was reported in our previous studies based on limited number of patients [15], we treated the newly obtained 304 LHON patients as an independent sample (sample #1) and compared to the two control population groups. We found that haplogroups R9 (including its subhaplogroups F, F1, F1a) and M7b presented remarkable differences between the LHON patients and the controls after adjusting for multiple test (Tables 2 and 3). The LHON sample significantly differs from the Table 1. Haplogroup frequencies and Pearson’s chi-square test in 843 patients with suspected LHON and 1,689 Han Chinese from the general populations. Haplogroup Suspected LHON Pooled Han Chinese a P -value b Adjusted P -value c OR 95% CI A 31 92 0.064 0.686 0.663 0.437–1.004 B4 126 196 0.021 0.314 1.339 1.052–1.703 B5 54 94 0.448 1.000 1.161 0.822–1.640 C 35 51 0.172 0.969 1.391 0.897–2.157 D4 115 252 0.423 1.000 0.901 0.710–1.143 D5 32 88 0.139 0.934 0.718 0.475–1.085 R9 d 164 305 0.394 0.999 1.413 0.804–2.483 F 143 274 0.677 1.000 1.055 0.845–1.317 F1 99 193 0.229 0.991 1.175 0.903–1.530 F1a 71 132 0.281 0.997 1.165 0.882–1.541 F2 17 48 0.216 0.988 1.421 0.812–2.487 F2a 16 22 0.246 0.994 1.466 0.766–2.806 F3 12 15 0.216 0.988 1.612 0.751–3.458 F3a 12 13 0.117 0.887 1.862 0.846–4.098 F4 7 8 0.270 0.996 1.759 0.636–4.868 G 31 71 0.598 1.000 0.870 0.566–1.338 M10 14 35 0.579 1.000 0.798 0.427–1.492 M12 8 4 0.031 0.436 4.036 1.212–13.441 M7b 59 119 1.000 1.000 0.993 0.718–1.372 M7c 32 54 0.504 1.000 1.195 0.765–1.865 M8a 27 63 0.575 1.000 0.854 0.540–1.351 M9a 11 29 0.539 1.000 0.757 0.376–1.522 N9a 33 62 0.847 1.000 1.069 0.695–1.645 R11 5 10 1.000 1.000 1.002 0.341–2.940 Y 7 16 0.944 1.000 0.876 0.359–2.136 Z 21 50 0.585 1.000 0.837 0.500–1.404 a Pooled Han Chinese were from reported populations (see Table S1 for more information). b Two tailed Fisher exact test was applied instead of a Pearson chi-square test in cases containing cell counts below five. c Adjusted P-value: adjustment of P-values was carried out with a permutation-based approach; number of permutations =100,000; OR (95% CI): Odds Ratio (95% Confidence Interval). d Note that haplogroup F is a sub-haplogroup of haplogroup R9 and therefore the number of F mtDNAs are also included here. doi:10.1371/journal.pone.0027750.t001 mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27750 general Han Chinese and suspected LHON populations by harboring an extremely lower frequency of haplogroup R9 (#1 vs. #3, P-value = 7.62610 219 , OR = 0.061, 95% CI: 0.022–0.164; #1 vs. #2, P-value = 5.19610 219 , OR = 0.055, 95% CI: 0.020– 0.150; in both cases, adjusted P-value ,10 25 ), in particular of its main sub-haplogroup F (#1 vs. #3, P-value = 1.46610 217 , OR = 0.051, 95% CI: 0.016–0.162; #1 vs. #2, P-value = 4.32610 217 , OR = 0.049, 95% CI: 0.015–0.154; in both cases, adjusted P-value ,10 25 ) and higher frequencies of M7b (#1 vs. #3, P-value = 7.00610 25 , adjusted P-value = 0.001 and #1 vs. #2, P-value = 3.23610 24 , adjusted P-value = 0.004). Distribution of the other haplogroups had no statistical difference between the two comparisons after adjustment of P-values. We further pooled the newly analyzed LHON patients with the previously reported LHON patients [15] as one population for comparison, to increase the power of the statistical test [32]. The dissection of matrilineal genetic components in the enlarged LHON population [479 LHON patients with m.11778G.A (sample #4)] and comparison to the two control groups [suspected LHON (sample #2) and pooled Han Chinese from the general populations (sample #3)] revealed several interesting yet important features (Fig. 1 and Tables S4 and S5). First, haplogroups M7b (#4 vs. #3, adjusted P-value = 0.018; #4 vs. #2, adjusted Pvalue = 0.047) and possibly D4 (#4 vs. #3, adjusted Pvalue = 0.085; #4 vs. #2, adjusted P-value = 0.031) are both more prevalent in patients with LHON than in the general Han Chinese group. Most of LHON patients with m.11778G.A belonging to haplogroup M7b could be further classified into M7b192 (77.19%, 44/57). Second, haplogroup R9, and more specifically, its sub-haplogroup F has a strikingly lower frequency in LHON patients with m.11778G.A than in the other two control populations (#4 vs. #3, nominal P-value = 1.999610 224 , adjusted P-value ,10 25 ;#4 vs. #2, nominal P-value = 9.133610 223 , adjusted P-value ,10 25 ). This significance is still strongly maintained for several F sub-haplogroups (F, F1, F1a), as also indicated when analyzing the newly sequenced LHON patients (Tables 2 and 3) and the reported LHON patients with m.11778G.A in our previous study (Ref. [15] and references therein) (Tables S6 and S7), separately. Complete mtDNA tree for LHON patients belonging to haplogroup M7b192 In order to further evaluate the effect of the haplogroupspecific variants that account for the haplogroup association with LHON, we determined four M7b complete mtDNAs (GenBank accession numbers JN645818–JN645821), and analyzed these sequences with those from published sources [24]. Five private variants (including four non-synonymous and one rRNA variant) were found in the four patients, but none of these variants (with the exception of m.8999T.C [p.V158A]) were conserved according to the evolutionary analysis performed by the MitoTool web server (Table 4). Two of these private variants (m.13105A.G and m.14978A.G) were reported as haplogroupspecific variants for other haplogroups. Interestingly, m.6228C.T (p.L109F) in patient Le777 and m.13105A.G (p.I257V) in patient Le666 were previously reported in LHON patients belonging to haplogroup M7b192 [15]. We presented sequence variants in each mtDNA in a classification tree following our previous strategy [15] (Fig. 2). The newly determined M7b sequences indicated two novel clades within M7b1, which were defined by m.13105A.G and m.6228C.T, respectively. We counted the number of private variants that were located in the terminal branches of the phylogenetic tree in each of the 13 mitochondrial DNA encoding genes in 12 LHON patients sequenced in this study and our previous study [15], and compared to that of 69 reported complete mtDNAs from the general populations (refer to [21] and references therein). There was no difference regarding the occurrence of private nonsynonymous (NS) and synonymous (S) substitutions between the LHON patients and controls from the general populations; note that the sample size was very limited and this result should be treated with caution (Table S8). Figure 1. mtDNA haplogroup distribution frequency of different cohorts of LHON patients with m.11778G . A and control samples. Detailed information for the newly sequenced LHON patients with m.11778G.A (sample #1, n= 304), reported LHON patients with m.11778G.Ain our previous study (sample #5, n = 175; Ref. [15]), pooled LHON patients (sample #4, n = 479), suspected LHON patients (sample #2, n= 843) and the reported Han Chinese from the general populations (sample 3, n= 1,689; Table S1) are listed in Tables 2 and 3 and Tables S4, S5, S6, S7. Haplogroup R9 contains samples belonging to haplogroup F and its subhaplogroups. We lumped together all these haplogroups that occurred in less than five individuals per haplogroup as others. doi:10.1371/journal.pone.0027750.g001 mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27750 Discussion In recent years, many studies reported that mtDNA background affects the expression of human disorders [33–37], although some positive findings could respond to false positives [38]. Among these studies, LHON is probably the most extensively studied disease that has been reported to be modulated by mtDNA haplogroups [5,12–15]. In this study, we analyzed 479 unrelated LHON samples with m.11778G.A (including 175 LHON patients reported in our previous study [15] and 304 patients newly sequenced) and 843 suspected LHON patients without the three primary mutations, with the aim of primarily addressing the following questions: (1) does mtDNA haplogroup background contribute to the suspected LHON? (2) will the enlarged LHON patients with m.11778G.A discern the internal consistency of the haplogroup association of LHON penetrance? Dissecting the matrilineal components of the enlarged sample size of LHON patients with m.11778G.A and the suspected LHON patients did provide new insightful information to both questions. In contrast to the pattern that was observed in the LHON patients with m.11778G.A [15], the overall matrilineal composition of the suspected LHON patients resembles that of the pooled Han Chinese, showing no evident effect from mtDNA background (Fig. 1 and Tables 1, 2, 3). This result was not surprising, as the suspected LHON cohort was quite heterogeneous and might be caused by nuclear gene defects, as well as, some rare mtDNA pathogenic mutations. Indeed, we analyzed some of these patients and identified mutations in the OPA1 gene, suggesting that these patients should be classified as autosomal dominant optic atrophy (ADOA; authors’ unpublished data). Nonetheless, the suspected LHON population could be used as a suitable control population for the LHON patients carrying m.11778G.A. In addition, both LHON carriers and none carriers of m.11778G.A were collected in the same geographic origin, a fact that helps to prevent the undesirable effects of population stratification in inflating type I error. A direct comparison of the frequencies of the matrilineal components between the LHON population and the suspected LHON population or between the LHON population and the pooled Han Chinese from the general populations yields meaningful information on the mtDNA background effect on LHON. Haplogroup M7b192, which was found to increase vision loss risk in the presence of m.11778G.A [15], had a significantly higher occurrence in LHON patients with this mutation than in patients with suspected LHON or in Han Chinese from the general populations (Tables 2 and 3 and Tables S4 and S5). This Table 2. Haplogroup frequencies and Pearson’s chi-square test in 304 LHON patients with m.11778G.A and 843 patients with suspected LHON. Haplogroup LHON Suspected LHON P -value a Adjusted P -value b OR 95% CI A 6 31 0.150 0.931 0.527 0.218–1.277 B4 43 126 0.735 1.000 0.938 0.645–1.363 B5 21 54 0.761 1.000 1.084 0.643–1.827 C 14 35 0.738 1.000 1.114 0.591–2.101 D4 58 115 0.023 0.304 1.493 1.055–2.112 D5 13 32 0.712 1.000 1.132 0.586–2.187 R9 c 4 164 5.190610 219 ,10 25 0.055 0.020–0.150 F 3 143 4.319610 217 ,10 25 0.049 0.015–0.154 F1 2 99 1.003610 211 ,10 25 0.050 0.012–0.203 F1a 1 71 6.614610 29 ,10 25 0.036 0.005–0.259 F2 0 17 9.725610 23 0.141 0.731 0.706–0.757 F3 1 12 0.203 0.979 0.229 0.030–1.765 F3a 1 12 0.203 0.979 0.229 0.030–1.765 F4 0 7 0.200 0.977 0.733 0.708–0.759 G 14 31 0.475 1.000 1.265 0.663–2.411 M10 13 14 0.010 0.144 2.645 1.229–5.694 M12 0 8 0.119 0.872 0.733 0.708–0.759 M7b 42 59 3.233610 24 0.004 2.130 1.400–3.241 M7c 15 32 0.391 0.999 1.315 0.702–2.465 M8a 11 27 0.729 1.000 1.135 0.556–2.316 M9a 9 11 0.059 0.631 2.308 0.947–5.624 N9a 12 33 0.980 1.000 1.009 0.514–1.979 R11 0 5 0.333 0.999 0.734 0.709–0.760 Y 5 7 0.232 0.989 1.997 0.629–6.340 Z 8 21 0.894 1.000 1.058 0.464–2.414 a Two tailed Fisher exact test was applied instead a Pearson chi-square test in cases containing cell counts below five. b Adjusted P-value: adjustment of P-values was carried out with a permutation-based approach; number of permutations = 100,000; OR (95% CI): Odds Ratio (95% Confidence Interval). c Note that haplogroup F is a sub-haplogroup of haplogroup R9 and the number of F mtDNAs are also included here. doi:10.1371/journal.pone.0027750.t002 mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27750 Table 3. Haplogroup frequencies and Pearson’s chi-square test in 304 LHON patients with m.11778G.A and 1,689 Han Chinese from general populations. Haplogroup LHON Pooled Han Chinese a Pvalue b Adjusted P -value c OR 95% CI A 6 92 0.010 0.144 0.350 0.152–0.806 B4 43 196 0.210 0.981 1.255 0.880–1.790 B5 21 94 0.355 1.000 1.259 0.772–2.055 C 14 51 0.152 0.948 1.551 0.847–2.838 D4 58 252 0.065 0.673 1.344 0.980–1.844 D5 13 88 0.494 1.000 0.813 0.448–1.474 R9 d 4 304 7.621610 219 ,10 25 0.061 0.022–0.164 F 3 274 1.461610 217 ,10 25 0.051 0.016–0.162 F1 2 193 2.822610 212 ,10 25 0.051 0.013–0.208 F1a 1 132 5.356610 29 ,10 25 0.039 0.005–0.280 F2 0 48 7.125610 24 0.012 0.844 0.828–0.860 F3 1 15 0.492 1.000 0.368 0.049–2.799 F3a 1 13 0.708 1.000 0.425 0.055–3.265 F4 0 8 0.616 1.000 0.847 0.831–0.863 G 14 71 0.750 1.000 1.100 0.612–1.978 M10 13 35 0.021 0.288 2.111 1.104–4.039 M12 0 4 1.000 1.000 0.847 0.831–0.863 M7b 42 119 6.669610 25 0.001 2.115 1.453–3.078 M7c 15 54 0.127 0.908 1.572 0.875–2.822 M8a 11 63 0.925 1.000 0.969 0.505–1.861 M9a 9 29 0.144 0.936 1.746 0.818–3.727 N9a 12 62 0.814 1.000 1.078 0.574–2.026 R11 0 10 0.376 1.000 0.847 0.831–0.863 Y 5 16 0.273 0.995 1.749 0.636–4.809 Z 8 50 0.754 1.000 0.886 0.416–1.888 a Pooled Han Chinese were from reported populations (see Table S1 for more information). b Two tailed Fisher exact test was applied instead a Pearson chi-square test in cases containing cell counts below five. c Adjusted P-value: adjustment of P-values was carried out with a permutation-based approach; number of permutations =100,000; OR (95% CI): Odds Ratio (95% Confidence Interval). d Note that haplogroup F is a sub-haplogroup of haplogroup R9 and the number of F mtDNAs are also included here. doi:10.1371/journal.pone.0027750.t003 Table 4. Private non-synonymous and mt-rRNA variants in four Chinese LHON patients with m.11778G.A and a haplogroup status of M7b192. Sample Haplogroup Nucleotide variant (Amino acid change) Gene Conservation Index a Reported b (population context) Reported b (disease context) Haplogroup specific variant c Le95 M7b2 m.8999T.C (p.V158A) MT-ATP6 1.000 Yes No No m.866A.GMT-RNR1 0.256 Yes No No Le666 M7b1 m.13105A.G (p.I257V) MT-ND5 0.256 Yes Yes L5c, L3b, L3e2a, etc. Le777 M7b1 m.6228C.T (p.L109F) MT-CO1 0.140 Yes Yes No Le978 M7b19294 m.A14978G (p.I78V) MT-CYB 0.651 Yes Yes C7a1 a The evolutionary conservation analysis was performed by comparing human mtDNA (GenBank accession no. J01415) to 43 different primate species by using the MitoTool (http://www.mitotool.org) [30]. A conservation index of 1 for certain variant means that this position is conserved in all species considered for comparison. b Web and dataset based searches were performed on September 5, 2011, following the same strategy described in our previous study [29] (e.g. both ‘G6249A mtDNA’ and ‘m.6249G.A mtDNA’ were queried). c The column ‘‘Haplogroup specific variant’’ refers to the presence of the corresponding variant in the world mtDNA phylogenetic tree displayed at http://www. phylotree.org/tree/main.htm (mtDNA tree Build 12; 20 Jul 2011) [31]. We listed the haplogroup name if it was characterized by the private variants identified in our newly generated M7b sequences. doi:10.1371/journal.pone.0027750.t004 mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27750 observation provides direct evidence for the internal consistency of association of haplogroup M7b192 with clinical expressions of LHON, as predicted by our previous study [15]. Note that in our previous study [15], the sample size for the control Han Chinese population was relatively small (n = 408) and this would bias the comparison for certain haplogroup. This defect had been corrected by including more Han Chinese samples from the general populations (n = 1,689) in the current analysis (Tables S6 and S7). As shown in Tables S2 and S3, most of the mtDNAs belonging to M7 and its subhaplogroups carry different control region profiles, indicating no close maternal relationship between these carriers; the different frequencies observed between cohorts are unlikely to be caused by close kinship. Analysis of the complete mtDNA sequence variation in those LHON patients belonging to M7b further supported our previous result that variant m.12811T.C would account for the haplogroup association with Figure 2. Classification tree of M7b complete mtDNA sequences, plus the revised Cambridge reference sequence (rCRS) [28].Five Chinese LHON mtDNAs (including Le53 (GenBank accession number JF896798) which was reported in our recent study [24]) belonged to haplogroup M7b192 were analyzed. The length polymorphisms of the C-tracts in region 303–309 were not considered. Mutations on each uninterrupted branch segment are listed in an arbitrary order. Recurrent mutations are underlined. The synonymous and non-synonymous coding-region variants in each mtDNA are denoted by ‘‘/s’’ and ‘‘/ns’’, respectively. Variants in the rRNA genes and tRNA genes are denoted by ‘‘/r’’ and ‘‘/t’’, respectively. doi:10.1371/journal.pone.0027750.g002 mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27750 LHON [15]. It seems that no other private variants would account for the overall increased risk for visual loss for pedigrees belonging to haplogroup M7b192. On the other hand, haplogroup M8a was found to reduce the clinical penetrance of mutation m.11778G.A in Chinese families in our previous study [15]. However, even with increased sample size of LHON patients, there was no indication, as it was expected, of a reduced frequency of M8a in LHON patients relative to control populations. Consistent with the previous observations [15], haplogroup D4 has a substantially higher frequency in the LHON patients (for 304 new patients, 58/304 = 19.08%; for pooled 479 patients, 98/ 479 = 20.50%) than in patients with suspected LHON (13.64%, 115/843) or in the pooled Han Chinese population (14.92%, 252/ 1689), though the adjusted P-value is not statistically significant (but marginal for pooled samples) between LHON patients and pooled Han Chinese. Because more than half of the LHON patients analyzed in this study were sporadic cases and we lacked the detailed clinical information for some families with selfreported disease history, we were unable to perform a correlation analysis to discern the effect of haplogroup D4 on the clinical expression of LHON. We failed to discern such an effect in our previous analysis for LHON families with m.11778G.A (Ref. [15] and references therein). Haplogroup D4 is defined by a string of non-synonymous variants (m.5178C.A in the MT-ND2 gene and m.8414C.T in the MT-ATP6 gene), synonymous variants (m.4883C.T in the MT-ND2 gene, m.14668C.T in the MTND6 gene) and one variant (m.3010G.A) in the MT-RNR2 gene. It is most likely that the non-synonymous variants of haplogroup D4 might be responsible for the potentially functional effect. Indeed, variant m.5178C.A had been reported to be associated with a variety of human disorders [39] and longevity [40], despite the fact that there was controversy regarding its role in longevity [41], and the functional assay did not support the notion that this variant could alter mitochondrial matrix pH and intracellular calcium dynamics [42]. The exact role of haplogroup D4 in LHON needs further study. Haplogroup F was very rare in the LHON patients (for 304 new patients, 3/304 = 0.99%; for pooled 479 patients, 6/ 479 = 1.25%), compared with its occurrence in the pooled Han Chinese from the general populations (274/1689 = 16.22%) and in the suspected LHON patients (143/843 = 16.96%). This result again confirmed the observation in our previous study [15] and a recent study in LHON patients from Thailand [10], in which an extremely low frequency of haplogroup F (including its subhaplogroups) was observed in LHON patients with m.11778G.A. Although the confounding effect of population stratification cannot be fully disregarded, the clear-cut different frequency of haplogroup F in two independent Chinese LHON patient cohorts (patients newly determined in this study and reported in our previous study [15]; Tables 2 and 3, Tables S6 and S7) with respect to the two independent control groups (suspected LHON patients plus the general Han Chinese) is so pronounced that it is reasonable to consider an alternative hypothesis that would involve haplogroup F as a protective factor in LHON patients. Because only six patients with m.11778G.A belong to haplogroup F in the LHON patients and half of them were sporadic, we could not estimate its potential effect on the clinical expression of LHON and analysis of the complete mtDNA sequence of some of these subjects yielded no insightful information [15]. Intriguingly, we also noticed a very low prevalence of F lineages (1.92%; 1/52) in LHON subjects with m.14484T.C in our recent study [23]. The exact reason for the extremely low frequency of F lineages in LHON patients with m.11778G.A or m.14484T.C remains enigmatic. Notably, haplogroup F is one of the sub-clades of haplogroup R9, and R9 showed evidently lower frequency in LHON patients than in the two control samples (Tables 2 and 3, Tables S4, S5, S6, S7), so the ancient genetic variants of haplogroup R9 might contribute to the pathogenesis of LHON patients. Variant m.13928G.Cwas the only non-synonymous nucleotide change at the basal branch level of haplogroup R9 and caused serine to threonine at position 531 of the MT-ND5 protein, but this variant did not alter the hydrophobicity significantly and occurred multiple times in different populations and haplogroup backgrounds (cf. www. phylotree.org). Similar to mtDNA variant m.12811T.Cwhich has been considered as the cause for the increased risk of M7b192 [15] and occurred in worldwide regions, the conservation and the hydrophobicity of m.13928G.C did not prove itself to be the possible pathogenic mutation, although recent studies showed that ancient variation may influence mtDNA replication and transcription [43] or contribute to cellular physiological changes in the presence of the primary mutations [44]. The deletion of adenine at position 249 is a characteristic variant of haplogroup F and other Asian haplogroups CZ, M31a1 and M36d1 [31]. Variant m.249delA was located in the mtTF1 binding site (MTTFX or TFAM) which binds with mtTF and controls mtDNA transcription [45]. Some single base changes within this region have been identified to alter protein binding efficiency [43]. Whether m.249delA could affect the efficiency of mtDNA transcription remains unclear. Population stratification could in part explain the results obtained in the present study. Monitoring population stratification is complex in mtDNA studies, but the use of two control groups, as performed in this study, helps to prevent its undesirable consequences in case-control studies. On the other hand, whether treating the newly analyzed 304 LHON patients with m.11778G.A and the reported LHON patients in our previous study [15] as independent samples, or pooled these new patients with the previously reported LHON patients [15] as an enlarged sample, we could get consistent findings for a lower frequency of the nested haplogroups R9, F, F1, and F1a. Therefore, we believe that different distribution frequencies of these haplogroups in LHON patients and control populations were biologically meaningful and might be shaped by the selection effect during the past. One limitation of the current study is that we lacked detailed clinical information for these patients newly sequenced in this study (most of these patients were collected more than five years ago and there was no follow-up information), we could not perform a systematic estimation for LHON penetrance, disease severity and propensity to spontaneous recovery of visual acuity, as well as, their correlation with mtDNA genetic background. In future study, we will attempt to collect all detailed clinical data for each newly recruited patient and fulfill the task. In summary, analysis of more newly collected LHON patients with m.11778G.A further supports the pattern that was observed in our previous studies [15], in that a significantly higher prevalence of haplogroups M7b and a strikingly low occurrence of haplogroup F have been confirmed. The higher frequency of M7b in patients with m.11778G.A than in control samples provides evidence for the internal consistency of association of haplogroup M7b192 with clinical expression of LHON. There was no evident effect of mtDNA background on Chinese patients with suspected LHON. Future studies could assist in providing further support to the present findings and provide new clues on the molecular mechanism by which a particular mtDNA background could influence clinical expression of LHON. mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27750 Supporting Information Figure S1 Pedigree information for three Chinese LHON families with m.11778G . A that were sequenced for the entire mtDNA sequence. Patient Le95 was regarded as sporadic according to our definition for sporadic patient in our recent study [23]. Affected individuals are marked by filled symbols. The proband that was sequenced for the entire mtDNA sequence in each family was marked by an arrow. The probands in these families had been analyzed for the mtDNA control region sequence variation in our previous study [15]. (DOC) Table S1 Information for the reported Han Chinese from the general populations. (DOC) Table S2 mtDNA sequence variation and haplogroup classification of 304 patients with LHON and m.11778G . A. (XLS) Table S3 mtDNA sequence variation and haplogroup classification of 843 patients with suspected LHON. (XLS) Table S4 Haplogroup frequencies and Pearson’s chisquare test in 479 LHON patients with m.11778G . A and 843 patients with suspected LHON. (DOC) Table S5 Haplogroup frequencies and Pearson’s chisquare test in 479 LHON patients with m.11778G . A and 1,689 Han Chinese from the general populations. (DOC) Table S6 Haplogroup frequencies and Pearson’s chisquare test in 175 LHON patients with m.11778G . A and 843 patients with suspected LHON. (DOC) Table S7 Haplogroup frequencies and Pearson’s chisquare test in 175 LHON patients with m.11778G . A and 1,689 Han Chinese from general populations. (DOC) Table S8 Comparison of the non-synonymous (NS) and synonymous (S) substitutions at the terminal branch level in the phylogenetic tree between 12 LHON patients belonging to haplogroup M7b192 and 69 reported Chinese complete mtDNAs from the general populations. (DOC) Methods S1 Supplementary methods for power calculations. (DOC) Acknowledgments We thank the patients who participated in this study. Author Contributions Conceived and designed the experiments: Y-GY A-MZ QZ. Performed the experiments: A-MZ XJ RB. Analyzed the data: A-MZ RB AS Q-PK YGY. Contributed reagents/materials/analysis tools: QZ XJ SL XX PW XG. Wrote the paper: Y-GY A-MZ AS. References 1. Yu-Wai-Man P, Griffiths PG, Hudson G, Chinnery PF (2009) Inherited mitochondrial optic neuropathies. J Med Genet 46: 145–158. 2. Yen M-Y, Wang A-G, Wei Y-H (2006) Leber’s hereditary optic neuropathy: a multifactorial disease. Prog Retin Eye Res 25: 381–396. 3. Man PYW, Turnbull DM, Chinnery PF (2002) Leber hereditary optic neuropathy. J Med Genet 39: 162–169. 4. Carelli V, Ross-Cisneros FN, Sadun AA (2004) Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 23: 53–89. 5. Hudson G, Carelli V, Spruijt L, Gerards M, Mowbray C, et al. (2007) Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background. Am J Hum Genet 81: 228–233. 6. Kerrison JB, Miller NR, Hsu F, Beaty TH, Maumenee IH, et al. (2000) A casecontrol study of tobacco and alcohol consumption in Leber hereditary optic neuropathy. Am J Ophthalmol 130: 803–812. 7. Hudson G, Keers S, Man PYW, Griffiths P, Huoponen K, et al. (2005) Identification of an X-chromosomal locus and haplotype modulating the phenotype of a mitochondrial DNA disorder. Am J Hum Genet 77: 1086–1091. 8. Kirkman MA, Yu-Wai-Man P, Korsten A, Leonhardt M, Dimitriadis K, et al. (2009) Gene-environment interactions in Leber hereditary optic neuropathy. Brain 132: 2317–2326. 9. Zhang A-M, Jia X, Zhang Q, Yao Y-G (2010) No association between the SNPs (rs3749446 and rs1402000) in the PARL gene and LHON in Chinese patients with m.11778G.A. Hum Genet 128: 465–468. 10.KaewsutthiS,PhasukkijwatanaN,JoyjindaY,ChuenkongkaewW, Kunhapan B, et al. (2011) Mitochondrial haplogroup background may influence Southeast Asian G11778A Leber hereditary optic neuropathy. Invest Ophthalmol Vis Sci 52: 4742–4748. 11. Phasukkijwatana N, Kunhapan B, Stankovich J, Chuenkongkaew WL, Thomson R, et al. (2010) Genome-wide linkage scan and association study of PARL to the expression of LHON families in Thailand. Hum Genet 128: 37–49. 12. Brown MD, Sun F, Wallace DC (1997) Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage. Am J Hum Genet 60: 381–387. 13. Torroni A, Petrozzi M, D’Urbano L, Sellitto D, Zeviani M, et al. (1997) Haplotype and phylogenetic analyses suggest that one European-specific mtDNA background plays a role in the expression of Leber hereditary optic neuropathy by increasing the penetrance of the primary mutations 11778 and 14484. Am J Hum Genet 60: 1107–1121. 14. Carelli V, Achilli A, Valentino ML, Rengo C, Semino O, et al. (2006) Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees. Am J Hum Genet 78: 564–574. 15. Ji Y, Zhang A-M, Jia X, Zhang Y-P, Xiao X, et al. (2008) Mitochondrial DNA haplogroups M7b192 and M8a affect clinical expression of leber hereditary optic neuropathy in Chinese families with the m.11778G.A mutation. Am J Hum Genet 83: 760–768. 16. Newman NJ (2005) Hereditary optic neuropathies: from the mitochondria to the optic nerve. Am J Ophthalmol 140: 517–523. 17. Jia X, Li S, Wang P, Guo X, Zhang Q (2010) mtDNA m.3635G.Amaybe classified as a common primary mutation for Leber hereditary optic neuropathy in the Chinese population. Biochem Biophys Res Commun 403: 237–241. 18. Zhang A-M, Zou Y, Guo X, Jia X, Zhang Q, et al. (2009) Mitochondrial DNA mutation m.3635G.A may be associated with Leber hereditary optic neuropathy in Chinese. Biochem Biophys Res Commun 386: 392–395. 19. Valentino ML, Barboni P, Ghelli A, Bucchi L, Rengo C, et al. (2004) The ND1 gene of complex I is a mutational hot spot for Leber’s hereditary optic neuropathy. Ann Neurol 56: 631–641. 20. Chinnery PF, Brown DT, Andrews RM, Singh-Kler R, Riordan-Eva P, et al. (2001) The mitochondrial ND6 gene is a hot spot for mutations that cause Leber’s hereditary optic neuropathy. Brain 124: 209–218. 21. Zou Y, Jia X, Zhang A-M, Wang W-Z, Li S, et al. (2010) The MT-ND1 and MT-ND5 genes are mutational hotspots for Chinese families with clinical features of LHON but lacking the three primary mutations. Biochem Biophys Res Commun 399: 179–185. 22. Yu D, Jia X, Zhang A-M, Guo X, Zhang Y-P, et al. (2010) Molecular characterization of six Chinese families with m.3460G.A and Leber hereditary optic neuropathy. Neurogenetics 11: 349–356. 23. Yu D, Jia X, Zhang A-M, Li S, Zou Y, et al. (2010) Mitochondrial DNA sequence variation and haplogroup distribution in Chinese patients with LHON and m.14484T.C. PLoS ONE 5: e13426. 24. Zhang A-M, Bandelt H-J, Jia X, Zhang W, Li S, et al. (2011) Is mitochondrial tRNAphe variant m.593T.C a synergistically pathogenic mutation in Chinese LHON families with m.11778G.A? PLoS ONE 6: e26511. mtDNA Haplogroup and LHON PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27750