Genetics Mitochondrial Background May Influence Southeast Asian G11778A Leber Hereditary Optic Neuropathy

Supannee Kaewsutthi,1,2 Nopasak Phasukkijwatana,2,3 Yutthana Joyjinda,1 Wanicha Chuenkongkaew,3,4 Bussaraporn Kunhapan,1 Aung Win Tun,1 Bhoom Suktitipat,1 and Patcharee Lertrit1,4

PURPOSE. To investigate the role of mitochondrial DNA markedly incomplete penetrance. The three most common (mt DNA) background on the expression of Leber hereditary primary LHON mutations, G3460A in ND1, G11778A in ND4, optic neuropathy (LHON) in Southeast Asian carriers of the and T14484C in ND6, account for more than 90% of LHON G11778A mutation. cases worldwide2 with G11778A being the most common. In 3 4–6 METHODS. Complete mtDNA sequences were analyzed from 53 and other Asian countries, G11778A is responsi- unrelated Southeast Asian G11778A LHON pedigrees in Thai- ble for approximately 90% of LHON families. land and 105 normal Thai controls, and mtDNA The sex bias and the marked incomplete penetrance of were determined. Clinical phenotypes were tested for associ- LHON indicate that there must be other factors that modify disease expression. Mitochondrial background,7–8 nuclear ation with mtDNA haplogroup, with adjustment for potential 9–11 12 confounders such as sex and age at onset. background, and environmental factors have been impli- cated in disease expression, although the precise mechanisms RESULTS. mtDNA subhaplogroup B was significantly associated of pathogenesis are largely undefined. with LHON. Follow-up analysis narrowed the association down ϭ It is well-known that European haplogroup J is associated to subhaplogroup B5a1 (P 0.008). Survival analyses with with LHON,13–15 and recently a large pan-European study Cox’s proportional hazards modeling on 469 samples (91 af- showed that the risk of visual loss was increased in haplogroup fected and 378 unaffected), adjusted for sex and heteroplasmy, J2 for LHON cases with G11778A and in haplogroup J1 for revealed that haplogroup B5a1 tended to increase the risk of cases with T14484C.8 It is believed that interactions between visual loss, but the trend was not statistically significant. Con- multiple alleles on haplogroup J and the primary LHON muta- versely, haplogroup F, the second most common haplogroup tion increase susceptibility to visual loss, although no clear in the control population, was the least frequent haplogroup in evidence has been demonstrated in this regard. However, LHON. This negative association was narrowed down to sub- ϭ LHON with the G11778A mutation is also found in different haplogroup F1 (P 0.00043), suggesting that haplogroup F1 mtDNA lineages in other populations, including in Southeast confers a protective effect. The distributions of sex, age at where there is essentially no haplogroup J.16–19 Yet, onset and heteroplasmy were not significantly different among G11778A LHON in Southeast Asians as prevalent as in - haplogroups. ans. A recent study of LHON in the Chinese also provided CONCLUSIONS. The specific mtDNA background B5a1 was signif- evidence that haplogroups M7b1Ј2 and M8a influence the icantly associated with Southeast Asian G11778A LHON and clinical expression of LHON.7 Given the different ethnic ori- appeared to modify the risk of visual loss. (Invest Ophthalmol gins of Southeast Asians and Chinese, it is likely that there are Vis Sci. 2011;52:4742–4748) DOI:10.1167/iovs.10-5816 also different mtDNA backgrounds modulating G11778A LHON expression in . eber hereditary optic neuropathy (LHON, OMIM 535000) In the present study, mtDNA haplogroups were determined L is a maternally inherited optic neuropathy, predominantly by complete mtDNA sequencing, which allows more accurate affecting young men.1 The mitochondrial DNA (mtDNA) and more specific haplogroup determination. Haplogroups LHON mutation is necessary but not sufficient for disease were tested for association with a variety of clinical pheno- expression, as reflected by the strict maternal inheritance and types to identify potential mtDNA variants that influence LHON in Southeast Asian carriers of the G11778A mutation.

1 3 From the Departments of Biochemistry and Ophthalmology and METHODS the 4Siriraj Neurogenetics Network, Faculty of Medicine Siriraj Hospi- tal, Mahidol University, Bangkok, Thailand. Blood Samples 2These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors. Blood samples from patients with optic neuropathy who had clinically Supported by two Siriraj Research Development Fund Grants suspected LHON were sent to our laboratory for diagnostic work-up. 001(III)/50 and R015233001. From 1994 to 2007, individuals from 60 LHON pedigrees with the Submitted for publication May 3, 2010; revised January 6, 2011; G11778A mutation were identified and recruited into the study. All accepted February 18, 2011. these pedigrees are of Thai or Chinese-Thai ethnic origin, except for Disclosure: S. Kaewsutthi, None; N. Phasukkijwatana, None; one pedigree of Indian ethnic origin. The pedigrees were accessed Y. Joyjinda, None; W. Chuenkongkaew, None; B. Kunhapan, None; A.W. Tun, None; B. Suktitipat, None; P. Lertrit, None through the probands, and they were scattered across different regions Corresponding author: Patcharee Lertrit, Department of Biochem- of Thailand. We performed field investigations of the families, and istry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok blood samples were collected from other family members. In each field 10700, Thailand; [email protected]. investigation, details of familial relationships were confirmed, and eye

Investigative Ophthalmology & Visual Science, June 2011, Vol. 52, No. 7 4742 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.

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examinations were performed by a neuro-ophthalmologist (WLC). Haplogroup Assignment These included Snellen’s visual acuity test, Ishihara’s color vision test, Complete mtDNA sequences of the samples were aligned with the and a funduscopic examination. Nine unexamined maternal relatives revised Cambridge Reference Sequence (rCRS)22 using Clustal W mul- were classified as affected, on the basis of a reliable history of acute tiple alignment.23 Nucleotide sequences differing from the rCRS were visual loss without other known cause. G11778A mutation status was manually rechecked from the electropherograms. Nucleotide variants determined in all collected blood samples, and one sample from each were used to assign mtDNA haplogroups according to a Phylotree.org- family was selected for sequencing the entire mtDNA genome. Global human mtDNA Phylogenetic Tree Build 7.24 Blood samples were also obtained from 105 unrelated normal control subjects across Thailand. The normal controls were healthy Statistical Analysis and were recruited from five different regions of Thailand: the north- ern, the southern, the western, the eastern, and the central. To ensure For comparing between proportions of individuals in the control and that the control subjects represented region-specific modern Thai LHON groups, ␹2 tests were used (or Fisher’s exact test when appro- samples, they were required to have maternal ancestors who had priate). The following criteria were applied to minimize ascertainment resided in the same region for at least two generations. The whole bias. First, 13 families with only one affected proband and little pedi- mtDNA genome of these samples was sequenced to assign mtDNA gree information were excluded. To avoid uncertainty about G11778A haplogroups. The study adhered to the tenets of the Declaration of status in distantly related, unexamined individuals, we included only Helsinki and was approved by the Siriaj Institutional Review Board first-degree maternal relatives (parents, siblings, or offspring) of indi- (SIRB), Mahidol University, Bangkok, Thailand, and all blood samples viduals whose mtDNA was tested and first-degree maternal relatives of were obtained with informed consent. unexamined affected individuals. To reduce selection bias in favor of affected persons, affected individuals and their siblings were included only if there was complete information on pedigree structure and Determination of the G11778A Mutation affection status for the whole sibship. Applying these filtering criteria mtDNA was isolated from the leukocytes in a whole-blood sample of resulted in 469 samples from 40 families, comprising 91 affected and each individual, by using the standard phenol-chloroform protocol. 378 unaffected and 229 males and 240 females. Since sex and hetero- The primary G11778A mutation was detected by polymerase chain plasmy appeared to be the predictors of disease expression, to study reaction–restriction fragment length polymorphism (PCR-RFLP) analy- the effects of mtDNA haplogroups on LHON expression, we per- ses, as described in Sudoyo et al.20 The heteroplasmy of the G11778A formed multivariate analyses to adjust for the confounding effects of mutation was quantitated in the first 30 pedigrees of our series by a those two predictors. We used survival analysis with Cox’s propor- method of radioactive restriction analysis modified from that described tional hazards modeling on our data set. The analysis was performed 25 by Moraes et al.,21 In brief, 35S-dATP was added to the PCR reaction at using the R statistical program. Survival time was the dependent the last amplification cycle. Then, the PCR product was digested with variable—that is, the age at onset of affected individuals or the current 7.5 units of the restriction endonuclease BclI (New England Biolabs age of unaffected individuals. The independent variables, sex and [NEB], Ipswich, MA) at 50°C for 12 to 16 hours. The restriction heteroplasmic status (homoplasmy or heteroplasmy), were first incor- products were separated in 8% acrylamide gel electrophoresis at 80 V porated into the Cox’s proportional hazards model. With the hypoth- for 90 minutes. The gel was dried (model 583 gel dryer; Bio-Rad, esis that neutral polymorphisms that constitute a mtDNA haplogroup Hercules, CA) at 80°C for 120 minutes and exposed to autoradiograph exert themselves as a single background for LHON expression, each film. The intensity of the bands in the autoradiogram were analyzed mtDNA haplogroup was regarded as independent. Each haplogroup (ImageMaster 1D Prime ver. 0.51; GE Healthcare, Piscataway, NJ). was then introduced separately into the model that had been adjusted Samples with a mutation load greater than or equal to 95% were for sex and heteroplasmy. considered to be homoplasmic. For the 30 pedigrees identified later in Analysis of variance (ANOVA) was used to compare age at onset in the study, after the first 30, heteroplasmic status was measured as a the different haplogroups. The Mann-Whitney test was used to com- dichotomous variable (either heteroplasmic or homoplasmic) by PCR- pare age at onset between the males and the females. The individuals RFLP and visualization of the restriction products in ethidium bro- whose ages at onset were recorded comprised 95 affected from 47 mide–stained agarose gel electrophoresis. Excessive restriction en- LHON pedigrees. P Ͻ 0.05 indicated significant results. zyme was used to ensure complete digestion, and a known homoplas- mic sample was used as a positive control. RESULTS

Mitochondrial Genome Sequencing Independent LHON Families Initially, complete mitochondrial genomes were sequenced for The entire mitochondrial genome covering 16,569 bp was amplified 60 LHON index cases with the G11778A mutation. Of these, into 16 overlapping PCR fragments using 16 pairs of light-strand and there were 53 distinct mitochondrial sequences or, in other heavy-strand oligonucleotide primers. The PCR reaction mixture con- words, there were seven pairs of identical mitochondrial ge- sisted of 10ϫ buffer, 25 mM MgCl , 10mM dNTP, 20 picomoles of each 2 nomes indicating relatedness through maternal lineages that primer, and 2.5 units of Taq DNA polymerase (NEB) in a final volume people were unaware of. As a consequence, only 53 mtDNA of 50 ␮L. Reaction conditions were 94°C for 5 minutes, 94°C for 1 sequences from LHON families (containing 106 affected) and minute, the appropriate annealing temperature for 1 minute, and 72°C 105 sequences from control subjects were included in further for 1.5 minutes, 30 cycles. The final extension continued for 8 minutes. analyses. Details of all the PCR primers are shown in Supplementary Table S1 (http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5816/-/ Distribution of mtDNA Haplogroups in G11778A DCSupplemental). The PCR product sizes ranged in length from 900 Southeast Asian LHON to 2000 bp and were verified by agarose gel electrophoresis. From the purified PCR products, 36 sequencing primers were used to Table 1 shows the distribution of haplogroups in 53 indepen- generate overlapping sequences to cover the entire mitochondrial dent G11778A LHON families compared with 105 control genome. The sequencing products were then analyzed (model subjects. None of the 53 LHON families were closely related, as 3730XL DNA Sequencer; Applied Biosystems, Inc. [ABI], Foster City confirmed by the distinct mtDNA sequences in each family. CA). Details of all the sequencing primers are shown in Supplemen- There were six major haplogroups in the control group: M tary Table S2 (http://www.iovs.org/lookup/suppl/doi:10.1167/ (43%), F (19%), B (17%), N (9%), R (8%), and D (5%). In the iovs.10-5816/-/DCSupplemental). LHON group, six major haplogroups, M (51%), B (32%), R (8%),

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TABLE 1. The Distribution of mtDNA Haplogroups in 53 Independent G11778A Southeast Asian LHON and Normal Controls in Thailand

Mitochondrial Genomes, n (%)

Control LHON P

Haplogroup M 45 (43) 27 (51) 0.398 Haplogroup D 5 (5) 2 (4) 1.000 Haplogroup B 18 (17) 17 (32) 0.042 B5a1 5 (5) 10 (19) 0.008* Haplogroup F 20 (19) 1 (2) 0.002* F1 18 (17) 0 (0) 0.00043* Haplogroup N 9 (9) 2 (4) 0.337 Haplogroup R 8 (8) 4 (8) 1.000 Total 105 53 —

* Statistically significant after Bonferroni correction (P Ͻ 0.0083).

N (4%), D (4%), and F (2%) were found. Subhaplogroup B5a1 FIGURE 1. Boxplots of ages at onset of the G11778A LHON study patients categorized by mtDNA haplogroups. was found significantly more frequently in the LHON affected than in the control subjects (P ϭ 0.008 after Bonferroni cor- rection). Interestingly, haplogroup F, which is the second most ated with G11778A Southeast Asian LHON, displayed the high- common haplogroup in the control subjects, was the least est mean age at onset (Fig. 1). frequent haplogroup in the LHON group (19% vs. 2%, P ϭ 0.002). The difference was attributable to the F1 subhaplo- Haplogroup and Sex Bias group (P ϭ 0.00043). Specifically, most haplogroup F in the control sample was F1 (17%); however, this subhaplogroup Twenty-three affected females in our data set were distributed was not found in the G11778A LHON families at all. The only in just two major haplogroups: B and M (Table 2). It was LHON family with haplogroup F in our data set was classified observed that more of the affected females than males carried as F3a (2%). haplogroup M. Haplogroup M was found in 78% (18/23) of the affected females and in 59% (49/83) of the affected males. ϭ mtDNA Haplogroups and Heteroplasmy However, the association was not statistically significant (P 0.142, Fisher’s exact test). Of the 53 G11778A LHON pedigrees of Southeast Asian ethnic origin, 47% (25/53) had at least one heteroplasmic individual mtDNA Haplogroup Effect on Visual Loss (i.e., were heteroplasmic families). The distribution of major The effect of mitochondrial haplogroups on the expression of haplogroups among heteroplasmic and homoplasmic families G11778A LHON, adjusted for sex and heteroplasmy, was eval- was similar (Table 2). It was observed that the single haplo- uated using survival analysis with Cox’s proportional hazards group F LHON family was heteroplasmic, although the pro- model. The analysis was performed on the data set of 469 band was homoplasmic for the G11778A mutation. individuals from 40 pedigrees, comprising 91 affected and 378 Haplogroup and the Age at Onset unaffected (Table 3), which were included according to the criteria mentioned in the Statistical Analysis section. Of these, From the 47 LHON pedigrees with the G11778A mutation 68 (30%) of 229 males and 23 (10%) of 240 females had LHON. whose age at onset was recorded, the mean age at onset in 95 Unexamined individuals were assumed to be homoplasmic affected individuals was 24.9 Ϯ 11.9 years (median, 21; range, when their direct maternal ancestors were homoplasmic or 4–59): 22.4 Ϯ 9.5 years (median, 20; range, 4–44; n ϭ 75) for when maternal relatives were tested, and all were found to be the males and 34 Ϯ 15.5 years (median, 32.5; range, 10–59; homoplasmic, whereas they were assumed to be heteroplas- n ϭ 20) for the females. The difference in age at onset between mic if at least one of the individuals in the same nuclear family the males and females was statistically significant (P ϭ 0.002, was heteroplasmic. Mann-Whitney test). However, age at onset did not vary signif- Most of the LHON families were classified into haplogroups icantly between the major haplogroups (P ϭ 0.356, ANOVA). M (51%) and B (32%), which enabled us to analyze these It was noted that haplogroup F, which was negatively associ- haplogroups in more specific subhaplogroups (Table 4). In the

TABLE 2. Sex Bias and Heteroplasmy of G11778A LHON Classified by mtDNA Haplogroups

Characteristics Frequency in Each Haplogroup, n (%)

B D F M N R Total

Sex of affected individuals Females 5 (22) 0 (0) 0 (0) 18 (78) 0 (0) 0 (0) 23 (100) Males 25 (30) 2 (2) 1 (1) 49 (59) 2 (2) 4 (5) 83 (100) Heteroplasmy Heteroplasmic families 9 (36) 1 (4) 1 (4) 12 (48) 1 (4) 1 (4) 25 (100) Homoplasmic families 8 (29) 1 (4) 0 (0) 15 (54) 1 (4) 3 (11) 28 (100)

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TABLE 3. Haplogroup Distribution of Affected and Unaffected TABLE 5. Effects of Sex and Heteroplasmic Status on the Risk of individuals from 40 Pedigrees Visual Loss

Individuals with G11778A Variable HR P

Ϫ9 Unaffected (n) Affected n (%) Total (n) Sex 4.77 1.5 ϫ 10 Homoplasmy 1.98 0.01 B4a’g 5 1 (16.7) 6 B4c1b2 2 6 (75.0) 8 HR, hazard ratio. B4c2 27 4 (12.9) 31 B4g 16 1 (5.9) 17 B5a 49 14 (22.2) 63 significantly more frequently in LHON than in the control B5a1a 3 1 (25.0) 4 group and the association was narrowed down to subhaplo- D4a 2 1 (33.3) 3 group B5a1 (Table 1), we then analyzed subhaplogroup B5a1 D5b 2 1 (33.3) 3 separately. We found a 1.61-fold increased risk of visual loss for F3a 10 1 (9.1) 11 B5a1 relative to non-B5a1 after adjustment for sex and hetero- M 46 14 (23.3) 60 ϭ M13 14 6 (30.0) 20 plasmy, but this increase was not statistically significant (P M17 46 11 (19.3) 57 0.11). The analyses of subhaplogroups M7, M7b1, and M17 did M17a 3 1 (25.0) 4 not reveal significant results. M17c 13 6 (31.6) 19 M4b1 18 3 (14.3) 21 Comparison of Complete mtDNA Sequences M7b’d 5 1 (16.7) 6 between 53 G11778A LHON and 105 Controls M7b1 89 13 (12.7) 102 M7e 7 1 (12.5) 8 The complete mtDNA sequences of 53 independent G11778A N22 9 1 (10.0) 10 Southeast Asian LHON families and 105 control subjects were N9a 2 1 (33.3) 3 compared in each nucleotide position. There were 32 variants R22 10 3 (23.1) 13 that were found significantly more frequently in the LHON sam- Total 378 91 (19.4) 469 ple (P Ͻ 0.05). Of these, only 10 variants were located in the coding region, as listed in Table 7. Most of the variants were associated with haplogroup B5a, except for the two synonymous survival analyses, LHON families in haplogroup M were classi- variants A10679G and G11914A. The G709A and A10398G vari- fied into subhaplogroup M7 (five families), including M7b1 ants, although associated with B5a, also occurred frequently in (three families) and M17 (five families), and the rest was other haplogroups. However, only G11778A and G11914A were grouped as other M (six families). Haplogroup B LHON families significant after Bonferroni correction (P Ͻ 0.005). were subdivided into B4 (5 families) and B5a1 (10 families). We initially evaluated the effect of sex and heteroplasmy on DISCUSSION LHON expression. As expected, both were significant predictors of visual loss (Table 5). The males carried a 4.77-fold higher risk of We studied mtDNA haplogroups in a data set of 53 unrelated LHON than did the females (P ϭ 1.5 ϫ 10Ϫ9). Homoplasmy of the Southeast Asian G11778A LHON pedigrees in Thailand. Using G11778A mutation was associated with higher risk of LHON by a information from complete mtDNA sequences, we found that factor of 1.98, when compared with heteroplasmy (P ϭ 0.01). haplogroup B, in particular subhaplogroup B5a1, occurred There was no significant interaction between sex and hetero- significantly more frequently in G11778A LHON families than plasmy (data not shown). in the control subjects, while haplogroup F was significantly Each of the major haplogroups in Southeast Asian LHON, less frequent in the LHON sample than in the controls. This haplogroups B, D, F, M, N, and R were then introduced sepa- negative association between haplogroup F and LHON was rately into the survival model adjusted for sex and hetero- also reported in a recent large-scale study of G11778A Chinese plasmy. None of them was found to significantly modify the LHON, with very similar frequencies of the haplogroup to our risk of visual loss (Table 6). Since haplogroup B was found study (17% in controls and 2% in LHON),7 but

TABLE 4. Individuals with the G11778A Mutation in Each mtDNA Haplogroup Included in the Survival Analyses

Female (n) Male (n) Total (n)

Haplogroup Unaffected Affected Unaffected Affected Unaffected Affected

B4* 30 2 18 4 48 6 B4c1b2 0 1 2 5 2 6 B5a1 34 2 18 13 52 15 D 301242 F3a 5 0 5 1 10 1 M17 35 4 27 14 62 18 M7 53 4 48 22 101 26 M7b1 47 4 42 9 89 13 Other M 48 10 30 13 78 23 N3082112 R22 6 0 4 3 10 3 Total 217 23 161 68 378 91

* B4c1b2 excluded.

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TABLE 6. Effects of mtDNA Haplogroups on Visual Loss in G11778A large sample of B5a1 carriers with LHON may be needed to LHON after Being Adjusted for Sex and Heteroplasmy confirm the increased risk conferred by this haplogroup. In an attempt to explain the association between haplo- Haplogroup HR P group B5a1 and Southeast Asian G11778A LHON and the pos- B 1.30 0.26 sible increase in risk of visual loss associated with this haplo- B4* 0.55 0.16 group, the variants in B5a1 were investigated (Fig. 2). From B4c1b2 3.47 4.1 ϫ 10Ϫ3 macrohaplogroup R to haplogroup B5a1, there were only two B5a1 1.61 0.11 nonsynonymous variants in this lineage, which are G8584A D 1.97 0.34 (A20T in ATP6) and A10398G (T114A in ND3). We compared F 0.42 0.39 complete mtDNA sequences of the 10 B5a1 LHON families and M 0.88 0.55 M7 0.70 0.2 105 controls in all haplogroups, and found no variants signifi- M7b1 0.70 0.24 cantly more frequent in LHON other than those variants char- M17 1.17 0.59 acterizing haplogroup B5a1. The variant A10398G is a common Other M 1.04 0.89 variant that is found in many branches of the world mtDNA N 0.48 0.3 phylogeny.24 It is also one of the variants that characterize R 1.21 0.75 haplogroup J, which is associated with increased penetrance of G11778A and T14484 LHON in Europeans.8 Apart from haplo- HR, hazard ratio. * B4c1b2 excluded. group B5a, in our population, A10398G was also found in haplogroups C, D, G, M, and R, none of which was significantly associated with LHON. Nonetheless, A10398G was still ob- those investigators found no effect of haplogroup F on disease served significantly more frequently (P ϭ 0.01) in our LHON penetrance. Interestingly, our data showed that LHON with families (75%, 40/53) than in the control subjects (54%, 57/ haplogroup F was associated with low disease penetrance 105; Table 7). In addition, A10398G has been implicated in a (Table 3) and correspondingly delayed age at onset (Fig. 1). wide range of diseases, such as breast cancer,26 Parkinson’s From the survival analyses adjusted for sex and heteroplasmy, disease,27 and bipolar disorder.28 All evidence suggests that haplogroup F showed a 0.416-fold decreased risk of visual loss A10398G, despite being a common variant, may be a modifier relative to non–haplogroup F; however, the difference was not for G11778A LHON expression, especially when it occurs in P ϭ statistically significant ( 0.39). The rarity of haplogroup F in conjunction with other variants in haplogroup B5a1 in our our G11778A LHON families (only one family with this haplo- population or with variants in haplotype J in Caucasians. group) limited the statistical power necessary to interpret re- Our group previously reported, in 30 LHON families with sults confidently. Overall, given the consistently low frequency of haplogroup F in both Southeast Asian and Chinese G11778A the G11778A mutation, a high percentage (37%) of heteroplas- LHON families, it is possible that haplogroup F confers protec- mic families (families with at least one heteroplasmic individ- ual) in the Thai population, compared with ϳ15% in other tion from visual failure in G11778A LHON carriers and thus 3 would become less detected in clinical practice. Alternatively, G11778A LHON–affected groups in the literature, with the 29 it may be more difficult for the G11778A mutation to occur on highest (33%) found in England. With almost two times more a haplogroup F background. families in the present study, we observed an even higher Our survival analyses showed that haplogroup B5a1 seems proportion of heteroplasmic families (47%, 25/53 families) in to increase risk of visual failure (hazards ratio [HR] ϭ 1.61, P ϭ the Thai population. We did not find any association between 0.11), which would explain why it was found significantly mitochondrial haplogroups and heteroplasmic families. The more frequently in the LHON than in the control group (P ϭ explanation for this high prevalence of G11778A heteroplasmy 0.008). Although this was a large cohort of Southeast Asian in our sample remains unclear. The G11778A mutational event G11778A LHON (67 maternal relatives from 10 families), a may have recently occurred multiple times in our group, or

TABLE 7. Analysis of Complete mtDNA Sequences in 53 G11778A LHON Pedigrees and 105 Control Subjects

Individuals with Variants, n (%)

Amino Acid LHON Control Haplogroups with LHON Families P Carrying Variants (105 ؍ n) (53 ؍ Position Gene rCRS Variant Alteration (n

709 12S rRNA G A — 18 (34) 19 (18) 0.030 B4c1b2, B5a, F3a, M,* M17c, M10a1, N22, R22 3537 ND1 A G L 10 (19) 6 (6) 0.022 B5a 6960 COI C T L 10 (19) 5 (5) 0.008 B5a 8584 ATPase6 G A A to T 10 (19) 5 (5) 0.008 B5a 9950 COIII T C V 10 (19) 5 (5) 0.008 B5a 10398 ND3 A G T to A 40 (75) 57 (54) 0.010 B5a, D4a, D5b, M,* M4b1, M7b1, M7b’d, M7e, M9d, M10a1, M12, M13, M17, M22, M33a, M71, R22 10679 ND4L A G E 3 (6) 0 (0) 0.036 M17 11778 ND4 G A R to H 53 (100) 0 (0) 2.5 ϫ 10Ϫ43† All 11914 ND4 G A T 7 (13) 1 (1) 0.002† M,* M17, M7b’d 15235 Cytb A G W 10 (19) 5 (5) 0.008 B5a

Only the coding region nucleotide variants found significantly more frequently in LHON are shown. * Haplogroup M was assigned when the mtDNA could not be classified into a more specific subhaplogroup according to the published mtDNA World Phylogenetic Tree Build 7.24 † Statistically significant after Bonferroni correction (P Ͻ 0.005).

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FIGURE 2. Map of mtDNA haplogroups constructed from complete mtDNA sequences of Southeast Asian G11778A LHON families using the phylogenetic tree of global mtDNA mutation24 as a scaffold. The map was constructed from the 53 LHON and 105 control mitochondrial genomes using the median-joining approach33 implemented in the Network 4.5.1.6 program (Fluxus Technology Ltd., Suffolk, UK), and was then draw manually. Twelve of the LHON families with haplogroups B5a1, B4c1b2, and F3a are indicated by the family names in the rectangular boxes. Haplogroup names are shown in bold. Nucleotide variants belonging to each haplogroup or haplotype are shown along each branch. They are transitions (based on the rCRS), unless the base changes are specified. Their positions are shown with the following suffixes: /s, synonymous substitution; /ns, nonsynonymous substitution; /r, ribosomal RNA variant; /t, transfer RNA variant; d, deletion; ϩ2C, insertion of two cytosines; !, back mutation; A, transversion to adenine. The G11778A mutation was omitted from the map.

there may be factors that maintain heteroplasmy of the LHON.7 Our survival analysis in 102 individuals from three G11778A mutation in Thai LHON families. families with haplogroup M7b1 did not replicate this result. There have been conflicting reports as to whether leuko- In contrast, the penetrance in M7b1 carriers was reduced cyte heteroplasmy of the primary LHON mutation is associated relative to that in non-M7b1 carriers in our population. This with risk of visual loss.30–32 Our survival analyses indicated finding may indicate genetic heterogeneity between South- that leukocyte heteroplasmy is a significant predictor of visual east Asian and Chinese LHON, a possibility that is supported failure and that being homoplasmic for the G11778A mutation by the different mtDNA haplogroups associated with LHON increases the risk of visual failure 1.98 fold relative to being in the two populations (B5a1 in Southeast Asia but D4 and heteroplasmic (P ϭ 0.01), consistent with findings from a M7c in ). Alternatively, the small number of M7b1 recent pan-European study.8 In that study, the degree of het- families in our data set may account for this discrepancy. eroplasmy was not quantified accurately, and a few assump- Despite considerable clinical, molecular, and biochemi- tions (based on the principle of mitochondrial genetics) were cal investigations of LHON, the exact pathogenesis is still made regarding heteroplasmic status in unexamined individu- poorly defined. Several factors such as mitochondrial back- als (see the Haplogroup Effect section in Results). However, ground, nuclear background, and environment are believed analyses without taking heteroplasmy into consideration to influence disease expression.2 There may also be com- yielded the same conclusions as the results reported here. plex interactions between these factors. Identification of A recent study in Chinese G11778A LHON reported an these susceptibility factors, however, is critically important association between haplogroup M7b1Ј2 and penetrance of to understanding the pathophysiology of LHON and to find

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