Leber hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by late-onset visual failure resulting from bilateral optic nerve atrophy (1-3). The disease is associated with three primary mitochondrial DNA (mtDNA) mutations: G11778A (4), T14484C (5,6), and G3460A (7,8), in the ND4, ND6, and ND1 genes, respectively. However, less than 50% of male and 10% of female carriers of LHON will develop optic neuropathy (9,10). This marked incomplete penetrance and gender bias indicate that additional genetic and/or environmental factors are required for the phenotypic expression of the pathogenic mtDNA mutations in LHON.
In European LHON, the G11778A and T14484C mutations show an association with the European-specific mtDNA haplogroup J (11-15). This leads to the suggestion that a combination of polymorphisms specific to this haplogroup increase the penetrance of these two primary mutations (14).
Although mtDNA haplogroup analyses have been performed in patients with LHON from European, Scandinavian, and North American populations, few studies have been carried out on patients with LHON in other parts of the world. Genetic information about Asian patients with LHON, especially when compared with that of European, Scandinavian, and North American patients with LHON, could facilitate the understanding of the molecular mechanism and the pathogenesis of LHON in general. To investigate the role of mitochondrial background in the expression of LHON, mtDNA haplogroup analysis was performed in 40 LHON individuals of Southeast Asian origin.
PATIENTS AND METHODS
All 40 patients with LHON were from the Department of Ophthalmology, Faculty of Medicine, at Siriraj, Ramathibodhi, and Chulalongkorn Hospitals, Bangkok, Thailand. All had developed acute loss of vision. Complete eye examinations were performed by WLC. After informed consent was obtained, a 5 mL venous blood sample from each patient was sent to our laboratory for mtDNA analysis. All samples were found to have the 11778 mutation, resulting in a definite diagnosis of LHON. All pedigrees were of Thai, Chinese-Thai, or Indian ethnic origin. All patients were born in Thailand.
As control subjects, we recruited 100 healthy individuals of Thai nationality who were seen for routine health examinations at Faculty of Medicine Siriraj Hospital in Bangkok between May 1999 and September 2002. Five milliliters of venous blood were drawn from each control subject with written informed consent. In all subjects, results of blood chemistry analyses and physical examination, including routine eye examination, were within normal limits.
Detection of G11778A LHON Mutation
DNA was extracted from venous blood samples using standard phenol-chloroform extraction. The G11778A mutation was detected by the mismatched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method as described previously (16).
MtDNA Haplogroup Analysis
The mtDNA haplogroup was determined by high-resolution RFLPs (17).
The χ2 test was used to compare the difference in haplogroup frequency among control subjects and patients of each haplogroup with LHON. For multiple comparison testing, the Bonferroni procedure was adopted. The Fisher exact test was used to compare the difference in haplogroup frequency between haplogroup F and non-F in control subjects and patients with LHON. All analyses were performed using the MedCalc software program (MedCalc Software, Mariakerke, Belgium) and SPSS software, version 13.0 (SPSS Inc., Chicago, IL).
The 40 patients with LHON were from provinces located in the central, northern, northeastern, and southern parts of Thailand. These 40 patients were not maternally related, as confirmed by RFLP and mtDNA sequences in the D-loop region.
In our 40 patients with the G11778A LHON mutation, 22 were classified as haplogroup M (55%), 8 were haplogroup B* (20%), and 4 were haplogroup B (12.5%) (Table 1). The mtDNA haplogroup could not be classified in 5 patients (12.5%). In the control subjects, the frequency of mtDNA haplogroup was 43%, 11%, and 9% for haplogroups M, B, and B*, respectively. Haplogroup F was not detected in any of our patients but was found in 15% of the control subjects. Neither patients with LHON nor our control subjects carried mitochondrial DNA haplogroup J.
To test whether the mtDNA haplogroup distribution in the G11778A LHON patients and control subjects was significantly different, the χ2 test, Bonferroni procedure, and Fisher exact test were performed. The P-values of the frequency difference among the patients with LHON and the control subjects in mtDNA haplogroups M, B*, B, and F were 0.272, 0.130, 0.967, and 0.022, respectively. These results indicated no significant difference in the frequencies of haplogroups M, B, and B* among the patients with LHON and the control subjects (P > 0.05) (Table 1). The P-value calculated from the frequency difference of haplogroup F in patients with LHON and control subjects was 0.022. When the Bonferroni method is applied, the significant P-value should be less than 0.005. Because the number of comparisons increased, the procedure became overly conservative. It was increasingly difficult to detect significant differences between the pairs of means; often, it was more difficult than really necessary. The Fisher exact test comparison of the haplogroup F and non-F frequencies in LHON patients and control subjects yielded a P-value of 0.005984 (Table 2).
The preferential association of LHON with haplogroup J in Caucasian patients has suggested a pathogenic role of the subset of mtDNA variants associated with this haplogroup in promoting the clinical expression of the primary LHON mutations. However, the combination of polymorphisms within haplogroup J that increases the risk of LHON expression has not been not yet identified.
Haplogroup J is one of nine common European specific haplogroups; therefore, it is expected that LHON would be more prevalent in Europeans. This is not the case because the two primary mutations, G11778A and T14484C, were also found in distantly related Thai populations. Our 40 independent LHON pedigrees, whose ethnic origins are Southeast Asian, carried the G11778A mutation, the mutation found in most patients with LHON in other parts of the world, including Europe, North America, and Scandinavia (18-21). In our patients with LHON, only mtDNA haplogroups M, B*, and B were found. None had mtDNA haplogroup J. Even though mitochondrial DNA haplogroup J has been found in some Asian populations, this mitochondrial DNA haplogroup was detected neither in our control subjects nor in other Thai populations reported on previously (22). These three haplogroups are the major Asian haplogroups (17,23,24). Among our patients with G11778A LHON, 55% were classified as haplogroup M, a value not significantly different (P-value = 0.272) from the 43% in our control subjects. The other 5 and 8 patients were haplogroup B (12.5%) and B* (20%), respectively. The frequency of these two haplogroups in the patients with LHON and control subjects was not significantly different either (P-values = 0.967 and 0.130, respectively). Thus, the mitochondrial DNA haplogroup distribution of our LHON patients was not significantly different from those of a non-LHON Thai population (Table 1). Our results support the idea that the effect of nuclear haplotype is probably greater than that of mtDNA haplotype (25). Our results also showed that the G11778A mutation must have arisen in our population independently from this mutation in Caucasians.
Interestingly, one mitochondrial haplogroup-haplogroup F-was found at a high frequency (15%) in control subjects but not at all in our patients with LHON. We have also looked at mtDNA haplogroups in the other patients with G11778A LHON, who were Indian in origin, and in 2 of our patients with T14484C LHON. These patients were classified in haplogroup M or B* but not haplogroup F. Only macrohaplogroup M and haplogroups B, B*, and BM have been reported in Asian LHON so far (26,27). There has been no previous report of mtDNA haplogroup F in patients of Asian origin with LHON. However, whether the mitochondrial haplogroup F carries the variant(s) that can negatively affect or prevent the occurrence of mutations in this disorder needs further investigation.
The authors thank Dr. Chayanon Peerapittayamongkol and Ms. Bussaraporn Kunhapan for their assistance in the statistical analysis.
1. Leber T. Uber hereditare und congenital-angelegte Sehnervenleiden. Graefe's Arch Ophthalmol
2. Nikoskelainen EK, Savontaus ML, Wanne OP, et al. Leber's hereditary optic neuroretinopathy, a maternally inherited disease: a genealogic study in four pedigrees. Arch Ophthalmol
3. Newman NJ. Leber's hereditary optic neuropathy: new genetic considerations. Arch Neurol
4. Wallace DC, Singh G, Lott MT, et al. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science
5. Johns DR, Neufeld MJ, Park RD. An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. Biochem Biophys Res Commun
6. Mackey D, Howell N. A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology. Am J Hum Genet
7. Howell N, Kubacka I, Xu M, et al. Leber hereditary optic neuropathy: involvement of the mitochondrial ND1 gene and evidence for an intragenic suppressor mutation. Am J Hum Genet
8. Huoponen K, Vilkki J, Aula P, et al. A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy. Am J Hum Genet
9. Harding AE, Sweeney MG, Govan GG, et al. Pedigree analysis in Leber hereditary optic neuropathy families with a pathogenic mtDNA mutation. Am J Hum Genet
10. Riordan-Eva P, Harding AE. Leber's hereditary optic neuropathy: the clinical relevance of different mitochondrial DNA mutations. J Med Genet
11. Brown MD, Sun F, Wallace DC. Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage. Am J Hum Genet
12. Brown MD, Starikovskaya E, Derbeneva O, et al. The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup. J Hum Genet
13. Howell N, Kubacka I, Halvorson S, et al. Phylogenetic analysis of the mitochondrial genomes from Leber hereditary optic neuropathy pedigrees. Genetics
14. Torroni A, Petrozzi M, D'Urbano L, et al. 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
15. Man PY, Howell N, Mackey DA, et al. Mitochondrial DNA haplogroup distribution within Leber hereditary optic neuropathy pedigrees. J Med Genet
16. Lertrit P, Imsumran A, Trongpanich Y, et al. Mitochondrial genetics of mitochondrial diseases in Thailand. Siriraj Hosp Gaz
17. Ballinger SW, Schurr TG, Torroni A, et al. Southeast Asian mitochondrial DNA analysis reveals genetic continuity of ancient mongoloid migrations. Genetics
18. Chinnery PF, Brown DT, Andrews RM, et al. The mitochondrial ND6 gene is a hot spot for mutations that cause Leber's hereditary optic neuropathy. Brain
19. Hofmann S, Jaksch M, Bezold R, et al. Population genetics and disease susceptibility: characterization of central European haplogroups by mtDNA gene mutations, correlation with D loop variants and association with disease. Hum Mol Genet
20. Huoponen K, Vilkki J, Savontaus ML, et al. Analysis of mitochondrial ND4 gene DNA sequence in Finnish families with Leber hereditary optic neuroretinopathy. Genomics
21. Wallace DC, Brown MD, Lott MT. Mitochondrial DNA variation in human evolution and disease. Gene
22. Fucharoen G, Fucharoen S, Horai S. Mitochondrial DNA polymorphisms in Thailand. J Hum Genet
23. Schurr TG, Wallace DC. Mitochondrial DNA diversity in Southeast Asian populations. Hum Biol
24. Wallace DC, Lott MT. MITOMAP: a human mitochondrial genome database, 2003. Available at: http://www.mitomap.org
. Accessed May 19, 2005.
25. Hudson G, Keers S, Yu Wai Man P, et al. Identification of an X-chromosomal locus and haplotype modulating the phenotype of a mitochondrial DNA disorder. Am J Hum Genet
26. Sudoyo H, Suryadi H, Lertrit P, et al. Asian-specific mtDNA backgrounds associated with the primary G11778A mutation of Leber's hereditary optic neuropathy. J Hum Genet
27. Nishioka T, Tasaki M, Soemantri A, et al. Leber's hereditary optic neuropathy with 14484 mutation in Central Java, Indonesia. J Hum Genet