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Show ORIGINAL CONTRIBUTION Mitochondrial DNA Haplogroup Distribution in Pedigrees of Southeast Asian G11 778A Leber Hereditary Optic Neuropathy Pattamon Tharaphan, MSc, Wanicha L. Chuenkongkaew, MD, Komon Luangtrakool, MSc, Thitima Sanpachudayan, MSc, Bhoom Suktitipat, MD, Rungnapa Suphavilai, MSc, Chatchawan Srisawat, MD, PhD, Thanyachai Sura, MD, and Patcharee Lertrit, MD, PhD Abstract: To investigate the association of mitochondrial DNA ( mtDNA) haplogroups and Leber hereditary optic neuropathy ( LHON) in the Southeast Asian population, mtDNA haplogroup determination was performed by high- resolution restriction fragment length polymorphism in 42 patients with LHON who were carrying the G11778A mutation and in control subjects drawn from a Thai urban population unaffected by LHON. The patients with LHON were of Thai, Thai- Chinese, and Indian origin. Three mtDNA haplogroups, M, B*, and B, were found in LHON patients in a frequency similar to that in control subjects. mtDNA haplogroup F was found in none of the patients with LHON but was the second most common haplogroup in control subjects. The G11778A mutation must have arisen in our population independently from the mutation in Caucasians. In contrast to Caucasians, no specific mtDNA haplotype was associated with the patients with LHON in the Southeast Asian population. The mitochondrial polymorphisms that modify the expression of LHON in Southeast Asians could not be identified in this study. The lack of haplogroup F in our patients with LHON may indicate the protective effect of this haplogroup in the expression of this disorder. (/ Neuro- Ophthalmol 2006; 26: 264- 267) Department of Biochemistry ( PT, KL, TSanpachudayan, BS, RS, CS, PL), Department of Ophthalmology ( WLC), Siriraj Neurogenetics Network ( WLC, PL), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkoknoi, Bangkok, Thailand; and Department of Medicine ( TSura), Faculty of Medicine, Ramathibodhi Hospital, Mahidol University, Bangkok, Thailand. This study was supported financially by Siriraj Research Development Fund, Faculty of Medicine Siriraj Hospital, Mahidol 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. 3L), University ( Grant 004( III)/ 45), and from the Ministry of University Affairs LC, and Faculty of Graduate Studies, Mahidol University to Pattamon noi, Tharaphan. Address correspondence to Patcharee Lertrit, MD, PhD, Dead, partment of Biochemistry, Faculty of Medicine Siriraj Hospital, and Mahidol University, Bangkok 10700, Thailand; E- mail: sipwy@ idol mahidol. ac. th 264 J Neuro- Ophthalmol, Vol. 26, No. 4, 2006 LHON in Southeast Asia J Neuro- Ophthalmol, Vol. 26, No. 4, 2006 PATIENTS AND METHODS Patients 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 DNAwas extracted from venous blood samples using standard phenol- chloroform extraction. The Gil 778 A 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). Statistical Analysis The x2 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). RESULTS 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 x2 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). TABLE 1. Distribution of the mtDNA haplogroups in 100 normal subject: RFLPs in this Haplogroup A B B* F M C D G Others Unclassified s and 40 patients with LHON determined by study using the x2 Control subjects ( 100 total) 3 11 9 15 43 1 1 1 1 15 test LHON ( 40 total) 0 5 ( 12.5%) 8 ( 20%) 0 22 ( 55%) 0 0 0 0 5 ( 12.5%) P value 0.645 0.967 0.130 0.022 0.272 0.634 0.634 0.634 0.634 0.901 265 J Neuro- Ophthalmol, Vol. 26, No. 4, 2006 Tharaphan et al TABLE 2. Comparison of the haplogroup frequency between haplogroup F and non- F in control subjects and patients with LHON Haplogroup Non F F Control Subjects ( N = 100) 85 15 LHON ( N = 40) 40 0 P value 0.005984 0.005984 DISCUSSION 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%) andB* ( 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. 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