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

Abstract

Purpose.: To investigate the role of mitochondrial DNA (mt DNA) background on the expression of Leber hereditary optic neuropathy (LHON) in Southeast Asian carriers of the G11778A mutation.

Methods.: Complete mtDNA sequences were analyzed from 53 unrelated Southeast Asian G11778A LHON pedigrees in Thailand and 105 normal Thai controls, and mtDNA haplogroups were determined. Clinical phenotypes were tested for association with mtDNA haplogroup, with adjustment for potential confounders such as sex and age at onset.

Results.: mtDNA subhaplogroup B was significantly associated with LHON. Follow-up analysis narrowed the association down to subhaplogroup B5a1 (P = 0.008). Survival analyses with Cox's proportional hazards modeling on 469 samples (91 affected and 378 unaffected), adjusted for sex and heteroplasmy, revealed that haplogroup B5a1 tended to increase the risk of visual loss, but the trend was not statistically significant. Conversely, haplogroup F, the second most common haplogroup in the control population, was the least frequent haplogroup in LHON. This negative association was narrowed down to subhaplogroup F1 (P = 0.00043), suggesting that haplogroup F1 confers a protective effect. The distributions of sex, age at onset and heteroplasmy were not significantly different among haplogroups.

Conclusions.: The specific mtDNA background B5a1 was significantly associated with Southeast Asian G11778A LHON and appeared to modify the risk of visual loss.

Leber hereditary optic neuropathy (LHON, OMIM 535000) is a maternally inherited optic neuropathy, predominantly affecting young men. ¹ The mitochondrial DNA (mtDNA) LHON mutation is necessary but not sufficient for disease expression, as reflected by the strict maternal inheritance and markedly incomplete penetrance. The three most common primary LHON mutations, G3460A in ND1, G11778A in ND4, and T14484C in ND6, account for more than 90% of LHON cases worldwide ² with G11778A being the most common. In Thailand ³ and other Asian countries, ^{4 –6} G11778A is responsible for approximately 90% of LHON families. 

The sex bias and the marked incomplete penetrance of LHON indicate that there must be other factors that modify disease expression. Mitochondrial background, ^7–8 nuclear background, ^{9 –11} and environmental factors ¹² have been implicated in disease expression, although the precise mechanisms of pathogenesis are largely undefined. 

It is well-known that European haplogroup J is associated with LHON, ^{13 –15} and recently a large pan-European study showed that the risk of visual loss was increased in haplogroup J2 for LHON cases with G11778A and in haplogroup J1 for cases with T14484C. ⁸ It is believed that interactions between multiple alleles on haplogroup J and the primary LHON mutation increase susceptibility to visual loss, although no clear evidence has been demonstrated in this regard. However, LHON with the G11778A mutation is also found in different mtDNA lineages in other populations, including in Southeast Asia where there is essentially no haplogroup J. ^{16 –19} Yet, G11778A LHON in Southeast Asians as prevalent as in Europeans. A recent study of LHON in the Chinese also provided evidence that haplogroups M7b1′2 and M8a influence the clinical expression of LHON. ⁷ Given the different ethnic origins of Southeast Asians and Chinese, it is likely that there are also different mtDNA backgrounds modulating G11778A LHON expression in Southeast Asia. 

In the present study, mtDNA haplogroups were determined by complete mtDNA sequencing, which allows more accurate and more specific haplogroup determination. Haplogroups were tested for association with a variety of clinical phenotypes to identify potential mtDNA variants that influence LHON in Southeast Asian carriers of the G11778A mutation. 

Methods

Blood Samples

Blood samples from patients with optic neuropathy who had clinically suspected LHON were sent to our laboratory for diagnostic work-up. From 1994 to 2007, individuals from 60 LHON pedigrees with the G11778A mutation were identified and recruited into the study. All these pedigrees are of Thai or Chinese-Thai ethnic origin, except for one pedigree of Indian ethnic origin. The pedigrees were accessed through the probands, and they were scattered across different regions of Thailand. We performed field investigations of the families, and blood samples were collected from other family members. In each field investigation, details of familial relationships were confirmed, and eye examinations were performed by a neuro-ophthalmologist (WLC). These included Snellen's visual acuity test, Ishihara's color vision test, and a funduscopic examination. Nine unexamined maternal relatives were classified as affected, on the basis of a reliable history of acute visual loss without other known cause. G11778A mutation status was determined in all collected blood samples, and one sample from each family was selected for sequencing the entire mtDNA genome. 

Blood samples were also obtained from 105 unrelated normal control subjects across Thailand. The normal controls were healthy and were recruited from five different regions of Thailand: the northern, the southern, the western, the eastern, and the central. To ensure that the control subjects represented region-specific modern Thai samples, they were required to have maternal ancestors who had resided in the same region for at least two generations. The whole mtDNA genome of these samples was sequenced to assign mtDNA haplogroups. The study adhered to the tenets of the Declaration of Helsinki and was approved by the Siriaj Institutional Review Board (SIRB), Mahidol University, Bangkok, Thailand, and all blood samples were obtained with informed consent. 

Determination of the G11778A Mutation

mtDNA was isolated from the leukocytes in a whole-blood sample of each individual, by using the standard phenol-chloroform protocol. The primary G11778A mutation was detected by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analyses, as described in Sudoyo et al. ²⁰ The heteroplasmy of the G11778A mutation was quantitated in the first 30 pedigrees of our series by a method of radioactive restriction analysis modified from that described by Moraes et al., ²¹ In brief, ³⁵S-dATP was added to the PCR reaction at the last amplification cycle. Then, the PCR product was digested with 7.5 units of the restriction endonuclease BclI (New England Biolabs [NEB], Ipswich, MA) at 50°C for 12 to 16 hours. The restriction products were separated in 8% acrylamide gel electrophoresis at 80 V for 90 minutes. The gel was dried (model 583 gel dryer; Bio-Rad, Hercules, CA) at 80°C for 120 minutes and exposed to autoradiograph film. The intensity of the bands in the autoradiogram were analyzed (ImageMaster 1D Prime ver. 0.51; GE Healthcare, Piscataway, NJ). Samples with a mutation load greater than or equal to 95% were considered to be homoplasmic. For the 30 pedigrees identified later in the study, after the first 30, heteroplasmic status was measured as a dichotomous variable (either heteroplasmic or homoplasmic) by PCR-RFLP and visualization of the restriction products in ethidium bromide–stained agarose gel electrophoresis. Excessive restriction enzyme was used to ensure complete digestion, and a known homoplasmic sample was used as a positive control. 

Mitochondrial Genome Sequencing

The entire mitochondrial genome covering 16,569 bp was amplified into 16 overlapping PCR fragments using 16 pairs of light-strand and heavy-strand oligonucleotide primers. The PCR reaction mixture consisted of 10× buffer, 25 mM MgCl₂, 10mM dNTP, 20 picomoles of each primer, and 2.5 units of Taq DNA polymerase (NEB) in a final volume of 50 μL. Reaction conditions were 94°C for 5 minutes, 94°C for 1 minute, the appropriate annealing temperature for 1 minute, and 72°C for 1.5 minutes, 30 cycles. The final extension continued for 8 minutes. Details of all the PCR primers are shown in Supplementary Table S1. The PCR product sizes ranged in length from 900 to 2000 bp and were verified by agarose gel electrophoresis. From the purified PCR products, 36 sequencing primers were used to generate overlapping sequences to cover the entire mitochondrial genome. The sequencing products were then analyzed (model 3730XL DNA Sequencer; Applied Biosystems, Inc. [ABI], Foster City CA). Details of all the sequencing primers are shown in Supplementary Table S2. 

Haplogroup Assignment

Complete mtDNA sequences of the samples were aligned with the revised Cambridge Reference Sequence (rCRS) ²² using Clustal W multiple alignment. ²³ Nucleotide sequences differing from the rCRS were manually rechecked from the electropherograms. Nucleotide variants were used to assign mtDNA haplogroups according to a Phylotree.org-Global human mtDNA Phylogenetic Tree Build 7. ²⁴  

Statistical Analysis

For comparing between proportions of individuals in the control and LHON groups, χ² tests were used (or Fisher's exact test when appropriate). The following criteria were applied to minimize ascertainment bias. First, 13 families with only one affected proband and little pedigree information were excluded. To avoid uncertainty about G11778A status in distantly related, unexamined individuals, we included only first-degree maternal relatives (parents, siblings, or offspring) of individuals whose mtDNA was tested and first-degree maternal relatives of 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 affection status for the whole sibship. Applying these filtering criteria resulted in 469 samples from 40 families, comprising 91 affected and 378 unaffected and 229 males and 240 females. Since sex and heteroplasmy appeared to be the predictors of disease expression, to study the effects of mtDNA haplogroups on LHON expression, we performed multivariate analyses to adjust for the confounding effects of those two predictors. We used survival analysis with Cox's proportional hazards modeling on our data set. The analysis was performed using the R statistical program. ²⁵ Survival time was the dependent variable—that is, the age at onset of affected individuals or the current age of unaffected individuals. The independent variables, sex and heteroplasmic status (homoplasmy or heteroplasmy), were first incorporated into the Cox's proportional hazards model. With the hypothesis that neutral polymorphisms that constitute a mtDNA haplogroup exert themselves as a single background for LHON expression, each mtDNA haplogroup was regarded as independent. Each haplogroup was then introduced separately into the model that had been adjusted for sex and heteroplasmy. 

Analysis of variance (ANOVA) was used to compare age at onset in the different haplogroups. The Mann-Whitney test was used to compare age at onset between the males and the females. The individuals whose ages at onset were recorded comprised 95 affected from 47 LHON pedigrees. P < 0.05 indicated significant results. 

Results

Independent LHON Families

Initially, complete mitochondrial genomes were sequenced for 60 LHON index cases with the G11778A mutation. Of these, there were 53 distinct mitochondrial sequences or, in other words, there were seven pairs of identical mitochondrial genomes indicating relatedness through maternal lineages that people were unaware of. As a consequence, only 53 mtDNA sequences from LHON families (containing 106 affected) and 105 sequences from control subjects were included in further analyses. 

Distribution of mtDNA Haplogroups in G11778A Southeast Asian LHON

Table 1 shows the distribution of haplogroups in 53 independent G11778A LHON families compared with 105 control subjects. None of the 53 LHON families were closely related, as confirmed by the distinct mtDNA sequences in each family. There were six major haplogroups in the control group: M (43%), F (19%), B (17%), N (9%), R (8%), and D (5%). In the LHON group, six major haplogroups, M (51%), B (32%), R (8%), N (4%), D (4%), and F (2%) were found. Subhaplogroup B5a1 was found significantly more frequently in the LHON affected than in the control subjects (P = 0.008 after Bonferroni correction). Interestingly, haplogroup F, which is the second most common haplogroup in the control subjects, was the least frequent haplogroup in the LHON group (19% vs. 2%, P = 0.002). The difference was attributable to the F1 subhaplogroup (P = 0.00043). Specifically, most haplogroup F in the control sample was F1 (17%); however, this subhaplogroup was not found in the G11778A LHON families at all. The only LHON family with haplogroup F in our data set was classified as F3a (2%). 

Table 1.

 

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

	Mitochondrial Genomes, n (%)		P
	Control	LHON
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 < 0.0083).

mtDNA Haplogroups and Heteroplasmy

Of the 53 G11778A LHON pedigrees of Southeast Asian ethnic origin, 47% (25/53) had at least one heteroplasmic individual (i.e., were heteroplasmic families). The distribution of major haplogroups among heteroplasmic and homoplasmic families was similar (Table 2). It was observed that the single haplogroup F LHON family was heteroplasmic, although the proband was homoplasmic for the G11778A mutation. 

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)

Haplogroup and the Age at Onset

From the 47 LHON pedigrees with the G11778A mutation whose age at onset was recorded, the mean age at onset in 95 affected individuals was 24.9 ± 11.9 years (median, 21; range, 4–59): 22.4 ± 9.5 years (median, 20; range, 4–44; n = 75) for the males and 34 ± 15.5 years (median, 32.5; range, 10–59; n = 20) for the females. The difference in age at onset between the males and females was statistically significant (P = 0.002, Mann-Whitney test). However, age at onset did not vary significantly between the major haplogroups (P = 0.356, ANOVA). It was noted that haplogroup F, which was negatively associated with G11778A Southeast Asian LHON, displayed the highest mean age at onset (Fig. 1). 

Figure 1.

 

Boxplots of ages at onset of the G11778A LHON study patients categorized by mtDNA haplogroups.

Haplogroup and Sex Bias

Twenty-three affected females in our data set were distributed in just two major haplogroups: B and M (Table 2). It was observed that more of the affected females than males carried haplogroup M. Haplogroup M was found in 78% (18/23) of the affected females and in 59% (49/83) of the affected males. However, the association was not statistically significant (P = 0.142, Fisher's exact test). 

mtDNA Haplogroup Effect on Visual Loss

The effect of mitochondrial haplogroups on the expression of G11778A LHON, adjusted for sex and heteroplasmy, was evaluated using survival analysis with Cox's proportional hazards model. The analysis was performed on the data set of 469 individuals from 40 pedigrees, comprising 91 affected and 378 unaffected (Table 3), which were included according to the criteria mentioned in the Statistical Analysis section. Of these, 68 (30%) of 229 males and 23 (10%) of 240 females had LHON. Unexamined individuals were assumed to be homoplasmic when their direct maternal ancestors were homoplasmic or when maternal relatives were tested, and all were found to be homoplasmic, whereas they were assumed to be heteroplasmic if at least one of the individuals in the same nuclear family was heteroplasmic. 

Table 3.

 

Haplogroup Distribution of Affected and Unaffected individuals from 40 Pedigrees

	Individuals with G11778A		Total (n)
	Unaffected (n)	Affected n (%)
B4a'g	5	1 (16.7)	6
B4c1b2	2	6 (75.0)	8
B4c2	27	4 (12.9)	31
B4g	16	1 (5.9)	17
B5a	49	14 (22.2)	63
B5a1a	3	1 (25.0)	4
D4a	2	1 (33.3)	3
D5b	2	1 (33.3)	3
F3a	10	1 (9.1)	11
M	46	14 (23.3)	60
M13	14	6 (30.0)	20
M17	46	11 (19.3)	57
M17a	3	1 (25.0)	4
M17c	13	6 (31.6)	19
M4b1	18	3 (14.3)	21
M7b'd	5	1 (16.7)	6
M7b1	89	13 (12.7)	102
M7e	7	1 (12.5)	8
N22	9	1 (10.0)	10
N9a	2	1 (33.3)	3
R22	10	3 (23.1)	13
Total	378	91 (19.4)	469

Most of the LHON families were classified into haplogroups M (51%) and B (32%), which enabled us to analyze these haplogroups in more specific subhaplogroups (Table 4). In the survival analyses, LHON families in haplogroup M were classified into subhaplogroup M7 (five families), including M7b1 (three families) and M17 (five families), and the rest was grouped as other M (six families). Haplogroup B LHON families were subdivided into B4 (5 families) and B5a1 (10 families). 

Table 4.

 

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

Haplogroup	Female (n)		Male (n)		Total (n)
	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	3	0	1	2	4	2
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
N	3	0	8	2	11	2
R22	6	0	4	3	10	3
Total	217	23	161	68	378	91

*  B4c1b2 excluded.

We initially evaluated the effect of sex and heteroplasmy on LHON expression. As expected, both were significant predictors of visual loss (Table 5). The males carried a 4.77-fold higher risk of LHON than did the females (P = 1.5 × 10⁻⁹). Homoplasmy of the G11778A mutation was associated with higher risk of LHON by a factor of 1.98, when compared with heteroplasmy (P = 0.01). There was no significant interaction between sex and heteroplasmy (data not shown). 

Table 5.

 

Effects of Sex and Heteroplasmic Status on the Risk of Visual Loss

Variable	HR	P
Sex	4.77	1.5 × 10−9
Homoplasmy	1.98	0.01

 HR, hazard ratio.

Each of the major haplogroups in Southeast Asian LHON, haplogroups B, D, F, M, N, and R were then introduced separately into the survival model adjusted for sex and heteroplasmy. None of them was found to significantly modify the risk of visual loss (Table 6). Since haplogroup B was found significantly more frequently in LHON than in the control group and the association was narrowed down to subhaplogroup B5a1 (Table 1), we then analyzed subhaplogroup B5a1 separately. We found a 1.61-fold increased risk of visual loss for B5a1 relative to non-B5a1 after adjustment for sex and heteroplasmy, but this increase was not statistically significant (P = 0.11). The analyses of subhaplogroups M7, M7b1, and M17 did not reveal significant results. 

Table 6.

 

Effects of mtDNA Haplogroups on Visual Loss in G11778A LHON after Being Adjusted for Sex and Heteroplasmy

Haplogroup	HR	P
B	1.30	0.26
B4*	0.55	0.16
B4c1b2	3.47	4.1 × 10−3
B5a1	1.61	0.11
D	1.97	0.34
F	0.42	0.39
M	0.88	0.55
M7	0.70	0.2
M7b1	0.70	0.24
M17	1.17	0.59
Other M	1.04	0.89
N	0.48	0.3
R	1.21	0.75

 HR, hazard ratio.

*  B4c1b2 excluded.

Comparison of Complete mtDNA Sequences between 53 G11778A LHON and 105 Controls

The complete mtDNA sequences of 53 independent G11778A Southeast Asian LHON families and 105 control subjects were compared in each nucleotide position. There were 32 variants that were found significantly more frequently in the LHON sample (P < 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 variants A10679G and G11914A. The G709A and A10398G variants, although associated with B5a, also occurred frequently in other haplogroups. However, only G11778A and G11914A were significant after Bonferroni correction (P < 0.005). 

Table 7.

 

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

Position	Gene	rCRS	Variant	Amino Acid Alteration	Individuals with Variants, n (%)		P	Haplogroups with LHON Families Carrying Variants
					LHON (n = 53)	Control (n = 105)
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 × 10−43 †	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 < 0.005).

Discussion

We studied mtDNA haplogroups in a data set of 53 unrelated Southeast Asian G11778A LHON pedigrees in Thailand. Using information from complete mtDNA sequences, we found that haplogroup B, in particular subhaplogroup B5a1, occurred significantly more frequently in G11778A LHON families than in the control subjects, while haplogroup F was significantly less frequent in the LHON sample than in the controls. This negative association between haplogroup F and LHON was also reported in a recent large-scale study of G11778A Chinese LHON, with very similar frequencies of the haplogroup to our study (17% in Han Chinese controls and 2% in LHON), ⁷ but those investigators found no effect of haplogroup F on disease penetrance. Interestingly, our data showed that LHON with haplogroup F was associated with low disease penetrance (Table 3) and correspondingly delayed age at onset (Fig. 1). From the survival analyses adjusted for sex and heteroplasmy, haplogroup F showed a 0.416-fold decreased risk of visual loss relative to non–haplogroup F; however, the difference was not statistically significant (P = 0.39). The rarity of haplogroup F in our G11778A LHON families (only one family with this haplogroup) limited the statistical power necessary to interpret results confidently. Overall, given the consistently low frequency of haplogroup F in both Southeast Asian and Chinese G11778A LHON families, it is possible that haplogroup F confers protection from visual failure in G11778A LHON carriers and thus would become less detected in clinical practice. Alternatively, it may be more difficult for the G11778A mutation to occur on a haplogroup F background. 

Our survival analyses showed that haplogroup B5a1 seems to increase risk of visual failure (hazards ratio [HR] = 1.61, P = 0.11), which would explain why it was found significantly more frequently in the LHON than in the control group (P = 0.008). Although this was a large cohort of Southeast Asian G11778A LHON (67 maternal relatives from 10 families), a large sample of B5a1 carriers with LHON may be needed to confirm the increased risk conferred by this haplogroup. 

In an attempt to explain the association between haplogroup B5a1 and Southeast Asian G11778A LHON and the possible increase in risk of visual loss associated with this haplogroup, the variants in B5a1 were investigated (Fig. 2). From macrohaplogroup R to haplogroup B5a1, there were only two nonsynonymous variants in this lineage, which are G8584A (A20T in ATP6) and A10398G (T114A in ND3). We compared complete mtDNA sequences of the 10 B5a1 LHON families and 105 controls in all haplogroups, and found no variants significantly more frequent in LHON other than those variants characterizing haplogroup B5a1. The variant A10398G is a common variant that is found in many branches of the world mtDNA phylogeny. ²⁴ It is also one of the variants that characterize haplogroup J, which is associated with increased penetrance of G11778A and T14484 LHON in Europeans. ⁸ Apart from haplogroup 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 observed significantly more frequently (P = 0.01) in our LHON families (75%, 40/53) than in the control subjects (54%, 57/105; Table 7). In addition, A10398G has been implicated in a wide range of diseases, such as breast cancer, ²⁶ Parkinson's disease, ²⁷ and bipolar disorder. ²⁸ All evidence suggests that A10398G, despite being a common variant, may be a modifier for G11778A LHON expression, especially when it occurs in conjunction with other variants in haplogroup B5a1 in our population or with variants in haplotype J in Caucasians. 

Figure 2.

 

Map of mtDNA haplogroups constructed from complete mtDNA sequences of Southeast Asian G11778A LHON families using the phylogenetic tree of global mtDNA mutation ²⁴ as a scaffold. The map was constructed from the 53 LHON and 105 control mitochondrial genomes using the median-joining approach ³³ 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; +2C, insertion of two cytosines; !, back mutation; A, transversion to adenine. The G11778A mutation was omitted from the map.

Our group previously reported, in 30 LHON families with the G11778A mutation, a high percentage (37%) of heteroplasmic families (families with at least one heteroplasmic individual) in the Thai population, compared with ∼15% in other G11778A LHON–affected groups in the literature, ³ with the highest (33%) found in England. ²⁹ With almost two times more families in the present study, we observed an even higher proportion of heteroplasmic families (47%, 25/53 families) in the Thai population. We did not find any association between mitochondrial haplogroups and heteroplasmic families. The explanation for this high prevalence of G11778A heteroplasmy in our sample remains unclear. The G11778A mutational event may have recently occurred multiple times in our group, or there may be factors that maintain heteroplasmy of the G11778A mutation in Thai LHON families. 

There have been conflicting reports as to whether leukocyte heteroplasmy of the primary LHON mutation is associated with risk of visual loss. ^{30 –32} Our survival analyses indicated that leukocyte heteroplasmy is a significant predictor of visual failure and that being homoplasmic for the G11778A mutation increases the risk of visual failure 1.98 fold relative to being heteroplasmic (P = 0.01), consistent with findings from a recent pan-European study. ⁸ In that study, the degree of heteroplasmy was not quantified accurately, and a few assumptions (based on the principle of mitochondrial genetics) were made regarding heteroplasmic status in unexamined individuals (see the Haplogroup Effect section in Results). However, analyses without taking heteroplasmy into consideration yielded the same conclusions as the results reported here. 

A recent study in Chinese G11778A LHON reported an association between haplogroup M7b1′2 and penetrance of LHON. ⁷ Our survival analysis in 102 individuals from three families with haplogroup M7b1 did not replicate this result. In contrast, the penetrance in M7b1 carriers was reduced relative to that in non-M7b1 carriers in our population. This finding may indicate genetic heterogeneity between Southeast Asian and Chinese LHON, a possibility that is supported by the different mtDNA haplogroups associated with LHON in the two populations (B5a1 in Southeast Asia but D4 and M7c in China). Alternatively, the small number of M7b1 families in our data set may account for this discrepancy. 

Despite considerable clinical, molecular, and biochemical investigations of LHON, the exact pathogenesis is still poorly defined. Several factors such as mitochondrial background, nuclear background, and environment are believed to influence disease expression. ² There may also be complex interactions between these factors. Identification of these susceptibility factors, however, is critically important to understanding the pathophysiology of LHON and to find therapeutic interventions for this currently incurable, devastating disease. 

Supplementary Materials

Table st01, DOC - Table st01, DOC 

Table st02, DOC - Table st02, DOC 

Footnotes

 Supported by two Siriraj Research Development Fund Grants 001(III)/50 and R015233001.

Footnotes

 Disclosure: S. Kaewsutthi, None; N. Phasukkijwatana, None; Y. Joyjinda, None; W. Chuenkongkaew, None; B. Kunhapan, None; A.W. Tun, None; B. Suktitipat, None; P. Lertrit, None

The authors thank James Stankovich for the proofreading of the manuscript. 

Appendix

Internet Resources

Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/  

The Entrez Nucleotide database, http://www.ncbi.nlm.nih.gov/nucleotide  

MITOMAP database, http://www.mitomap.org/mitoseq.html  

Phylotree.org-Global human mtDNA phylogenetic tree, http://www.phylotree.org  

Network 4.5.1.6 Program, http://www.fluxus_engineering.com