Genetics
Pathogenic Mitochondrial DNA Mutations and Associated Clinical Features in Korean Patients With Leber’s Hereditary Optic Neuropathy Hae Ri Yum,1 Hyojin Chae,2 Sun Young Shin,1 Yonggoo Kim,2 Myungshin Kim,2 and Shin Hae Park1 1Department
of Ophthalmology & Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Correspondence: Shin Hae Park, Department of Ophthalmology & Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-Gu, Seoul, 137701, Korea; [email protected]. Myungshin Kim, Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-Gu, Seoul, 137701, Korea; [email protected]. HRY and HC contributed equally to the work presented here and should therefore be regarded as equivalent authors. Submitted: July 24, 2014 Accepted: October 10, 2014 Citation: Yum HR, Chae H, Shin SY, Kim Y, Kim M, Park SH. Pathogenic mitochondrial DNA mutations and associated clinical features in Korean patients with Leber’s hereditary optic neuropathy. Invest Ophthalmol Vis Sci. 2014;55:8095–8101. DOI: 10.1167/iovs.14-15311
PURPOSE. To identify the spectrum of pathogenic mitochondrial DNA (mtDNA) mutations and clinical features in Korean patients with genetically confirmed Leber’s hereditary optic neuropathy (LHON). METHODS. The medical records of 34 unrelated, genetically confirmed LHON patients were reviewed. Total genomic DNA was isolated from the peripheral blood leukocytes of the patients with suspected LHON, and primary or secondary mtDNA mutations were identified by direct sequencing. We analyzed the visual acuity (VA), color vision, RNFL thickness, and visual field (VF) at the final visit from 20 patients who were followed-up for more than 6 months after the onset of LHON. RESULTS. Among 34 patients, 21 (61.8%) had the homoplasmic primary mutation, 11 (32.4%) had the homoplasmic secondary mutation, and 2 (5.9%) had the heteroplasmic primary mutation along with the homoplasmic secondary mutation. Analysis of mtDNA sequences revealed six different types of LHON-associated mutations: two primary LHON-associated primary mutations, m.11778G>A (20 patients, 58.8%) and m.14484T>C (3 patients, 8.8%), and four secondary LHON-associated mutations, which were m.3394T>C (3 patients, 8.8%), m.3497C>T (4 patients, 11.8%), m.11696G>A (4 patients, 11.8%), and m.14502T>C (2 patients, 5.9%). Secondary mutation-carrying patients demonstrated a decreased in RNFL thickness, similar to those in primary mutation–carrying LHON patients. These patients had a higher female ratio (P ¼ 0.019), better VA (P ¼ 0.043) and color vision (P ¼ 0.005), as well as better VF. CONCLUSIONS. In addition to common primary LHON-associated mutations, our study identified secondary mtDNA mutations, which should be considered when evaluating patients with optic atrophy. Keywords: Leber’s hereditary optic neuropathy, mitochondrial DNA, mutation
eber’s hereditary optic neuropathy (LHON) is typically characterized by bilateral acute or subacute loss of central vision, owing to focal degeneration of the retinal ganglion cell layer and optic nerve.1–3 It is a mitochondrial genetic disease inherited through maternal transmission that mainly affects young adult males.1–3 To date, over 30 different types of mitochondrial DNA (mtDNA) mutations, including 14 primary mutations, have been identified in patients with LHON (in the public domain http://www.mitomap.org/MITOMAP). Based on the genetic, biochemical, and clinical features, three mtDNA point mutations, ND1 m.3460G>A, ND4 m.11778G>A, and ND6 m.14484T>C, in the genes encoding the subunits of respiratory chain complex I have been traditionally considered as high-risk LHON mutations.4–7 These mutations represent approximately 80% to 95% of all LHON cases occurring in patients with different ethnic backgrounds.5–9 However, no primary mutations have been identified in a small minority of
diagnosed LHON cases. In one study, no primary mutations were found in 21% of LHON-affected patients from Finland.3 Recently, a growing number of mtDNA variants that are suspected to have a role in the disease expression of LHON have been reported.10–13 The pathological significance of mtDNA mutations other than primary mutations is uncertain and considered to be intermediate or low risk. Therefore, these have been proposed as secondary mutations. Secondary mutations that are relevant for the phenotypic expression of LHON appear to have a synergistic and deleterious effect together with the primary mutations.10–13 These secondary LHON mutations are homoplasmic, often occurring in combination with primary LHON mutations.10–13 They are found at a lower frequency in healthy subjects compared with the LHON population; however, they may have an additional role in the pathogenesis of LHON and increase the risk of disease expression.11,14,15
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
8095
L
IOVS j December 2014 j Vol. 55 j No. 12 j 8096
mtDNA Mutations in Korean Patients With LHON The distribution of LHON-associated mtDNA mutations is population-specific. However, little is known about the mutation and clinical characterization for Korean patients with LHON. Only five types of mtDNA mutations have been reported in Korean patients. Restriction fragment length polymorphism-PCR (RFLP-PCR) has revealed m.11778G>A (56.1%), m.14484T>C (15.9%), and m.3460G>A (1.2%) mutations in Korean LHON patients.16,17 In addition, secondary mtDNA mutations at nucleotide position (np) 4216 and 15257 were also reported in Korean LHON patients with or without primary mutation.18,19 In this study, we have used DNA sequencing to analyze LHON-associated mtDNA mutations together with the clinical characterization of Korean LHON patients.
METHODS This study was performed according to the principles of the Declaration of Helsinki, after approval by the institutional review board of Seoul St. Mary’s Hospital, College of Medicine, T h e C a t h o l i c U n i ve r s i t y o f Ko r e a , S e o u l , Ko r e a (KC14RISE0241).
Subjects The medical records of 34 unrelated genetically confirmed LHON patients at Seoul St. Mary’s Hospital from January 2009 to May 2014 were reviewed. The age, sex, family history, age at LHON onset, and initial/final visual acuity (VA) were evaluated for each patient. Acute or subacute visual loss with typical peripapillary microangiopathy were classified as type I clinical features. Bilateral optic atrophy with temporal retinal nerve fiber layer (RNFL) thinning and central scotoma were classified as type II clinical features. After thorough ophthalmologic evaluations, subjects were excluded on the basis of any of the following criteria: a history of retinal disease, including diabetic or hypertensive retinopathy, a history of eye trauma or surgery, a history of systemic or neurologic disease that may affect the optical coherence tomography (OCT) and visual field (VF) results, and any relevant drug history.
biomicroscopy, a dilated stereoscopic examination of the optic nerve head and fundus, a VF test, and optical coherence tomography (OCT). Clinical examinations were performed by an experienced neuro-ophthalmologist. We analyzed the ophthalmologic results at the final visit from 20 patients who were followed-up for more than 6 months after the onset of LHON. When both eyes were eligible for the study, we analyzed a worse-seeing eye in each patient. We used the 24-plate edition from the Ishihara test book published in 1997 by Kanehara & Co., Ltd. (Tokyo, Japan), and the score was set as the number of plates identified out of the first 12 screening plates and five diagnostic plates.22 The peripapillary RNFL thickness was measured using a spectral-domain Cirrus HD-OCT (software version 6.0, Carl Zeiss Meditec, Dublin, CA). Spectral-domain OCT imaging was obtained from a 3-dimensional data set from an optic cube scan, which was composed of 200 A-scans from each of 200 Bscans that covered a 6-mm2 area centered on the optic disc. Following the creation of the RNFL thickness map from the cube data set, the software then automatically determines the center of the disc and extracts a circumpapillary circle (1.73mm radius) from the cube data set, which it then uses to determine RNFL thickness. To avoid confusion, clock-hour sectors were named according to their location as either superior (S), inferior (I), temporal (T), or nasal (N). The following OCT parameters were used in the analysis: S, I, T, N, and overall RNFL thickness. Image quality was assessed by an experienced examiner who was unaware of the patient’s identity and condition. We performed at least two standard automated perimetry tests (Humphrey Visual Field Analyzer; Zeiss/Humphrey Systems, Dublin, CA, USA) 24–2, using the Swedish interactive threshold algorithm (SITA) strategy. The VF defect had to be present at the same location in at least two consecutive, reliable VFs for the result to be included in the study. The results from the VF tests were considered reliable, if the fixation losses were less than 20% and false positive and false negative rates were less than 15%.
Statistical Analysis Mutational Analysis After obtaining informed consent from each patient, blood samples were taken for mtDNA extraction and analysis. Total genomic DNA was isolated from peripheral blood leukocytes using the QIAmp DNA Mini Kit (Qiagen, Hamburg, Germany). The presence of the m.3460G>A, m.11778G>A, and m.14484T>C mutations and secondary mutations in genes mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), and mitochondrially encoded cytochrome B (MT-CYB) were screened by direct sequencing using an ABI prism Big Dye Terminator cycle sequencing Ready Reaction kit V 3.1 (Applied Biosystems, Foster City, CA, USA) and analyzed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). All sequences were analyzed against mitochondrial reference sequence NC_012920. We performed in silico analysis using the software Polyphen-2 (in the public domain http://genetics.bwh.harvard. edu/pph2/) and Sorting Intolerant from Tolerant (SIFT; in the public domain http://sift.jcvi.org/) to assess if the substitutions were predicted as potentially pathogenic.20,21
Ophthalmologic Examination Each participant underwent a comprehensive ophthalmologic assessment, which included the measurement of best-corrected visual acuity (BCVA), a color vision test, slit-lamp
Statistical analysis was performed using SPSS software (version 19.0; SPSS, Inc., Chicago, IL, USA). The v2 test and MannWhitney U test were used to compare data from the primary and secondary mutation groups. A P value of less than 0.05 was regarded as statistically significant.
RESULTS Genetic Analysis Among 34 patients, 21 (61.8%) had the homoplasmic primary mutation, 11 (32.4%) had the homoplasmic secondary mutation, and 2 (5.9%) had the heteroplasmic primary mutation along with the homoplasmic secondary mutation. The mtDNA mutations identified in each patient are summarized in Table 1. Six different kinds of mtDNA mutations were detected in 34 unrelated patients with bilateral optic atrophy. In addition to the two common LHON-associated primary mutations, m.11778G>A (20 patients, 58.8%) and m.14484T>C (3 patients, 8.8%), four secondary mutations were also identified, m.3394T>C (3 patients, 8.8%), m.3497C>T (4 patients, 11.8%), m.11696G>A (4 patients, 11.8%), and m.14502T>C (2 patients, 5.9%). In silico analysis of the secondary mutations generated differing results. Polyphen-2 predicted that all four substitutions would be benign and SIFT predicted that two of the secondary mutations
IOVS j December 2014 j Vol. 55 j No. 12 j 8097
13, 40 (Chinese)
Benign
The male:female ratio in this study was 21:13. The overall proportion of LHON patients with a family history of the disease was 38.2%. In 10 patients who had type I clinical features, with acute or subacute visual loss and peripapillary telangiectatic microangiopathy, the mean age of LHON onset was 25.2 years. Both Tables 2 and 3 summarize the clinical findings from patients carrying homoplasmic secondary LHON mutations and combined primary and secondary LHON mutations. We analyzed the ophthalmologic results at the final visit from 20 patients who were followed-up for more than 6 months after the onset of LHON. We then compared the clinical data from patients with primary and secondary LHON mutations (Table 4). In the secondary mutation group, there was a higher proportion of type II clinical features (P ¼ 0.020) and lower male to female ratio (P ¼ 0.019). No significant difference was found in the OCT parameters, including those in the temporal area, between the two groups. However, the BCVA was significantly better in patients with the secondary mutation than in those with the primary mutation (P ¼ 0.043). The color vision deficiency was also less prominent in patients with the secondary mtDNA mutation compared with those with the primary mtDNA mutation (P ¼ 0.005). Both the VF index and MD of the VF test were significantly different between the two groups (P ¼ 0.013 and P ¼ 0.024).
Confirmed þ reported/ possibly synergistic
Reported/possibly synergistic Confirmed þ reported/secondary
Homoplasmic Heteroplasmic þ homoplasmic Heteroplasmic þ homoplasmic
Tolerated
34
1
MT-ND6 MT-ND4 MT-ND1 MT-ND6 Combined
1 1
m.14502T>C m.11778G>A m.3497C>T m.14484T>C þ m.14502T>C
p.Ile58Val p.Arg340His p.Ala64Val p.Met64Val p.Ile58Val
DISCUSSION
Total
Benign Reported/possibly synergistic MT-ND4 4
m.11696G>A
p.Val313Ile
Homoplasmic
Tolerated
Benign Reported/secondary MT-ND1
Secondary
3
m.3497C>T
p.Ala64Val
Homoplasmic
Not tolerated
Probably Damaging Benign Benign Deleterious Deleterious Not tolerated Confirmed Confirmed Reported/unclear Homoplasmic Homoplasmic Homoplasmic p.Arg340His p.Met64Val p.Tyr30His MT-ND4 MT-ND6 MT-ND1
m.11778G>A m.14484T>C m.3394T>C
(m.3394T>C, m.3497C>T) would not be tolerated (Table 1). We found no patients carrying the primary mutation m.3460G>A. Patients with homoplasmic secondary mutations had common ocular features, with a pale optic disc and decreased peripapillary RNFL thickness in both eyes (Table 2; Fig. A–D). Two patients were shown to have homoplasmic secondary mutations and heteroplasmic primary mutations. The coexistence of mutations MT-ND1 m.3497C>T/MT-ND4 m.11778G>A and MT-ND6 m.14502T>C/MT-ND6 m.14484T>C was detected in each patient (Table 3, cases 12 and 13).
Clinical Features
19 2 3 Primary
Codon Mutation Gene Locus Numbers
TABLE 1. Sequence Analysis of Mutations in Korean Patients With LHON
Mutation Type
MITOMAP Status
SIFT
Polyphen-2
References
7 (American) 11 (Japanese) 12, 15 (Chinese) 37 (Finnish) 11 (Japanese) 38 (Thai) 10, 32, 33 (Chinese) 31 (Dutch) 39 (Chinese) 38 (Thai)
mtDNA Mutations in Korean Patients With LHON
In this study, we report genetic and clinical characterization of LHON in 34 unrelated Korean patients. Sequence analysis of the mitochondrial genome revealed six different kinds of LHON-associated mtDNA mutations in patients with bilateral visual impairment due to optic atrophy as a sole clinical manifestation. The two common primary LHON-associated mutations, m.11778G>A and m.14484T>C were identified in 58.8% (20 patients) and 8.8% (3 patients) of the patients, respectively. The four secondary mtDNA mutations, m.3394T>C, m.3497C>T, m.11696G>A, and m.14502T>C, which may be associated with LHON, were found in 8.8% (3 patients), 11.8% (4 patients), 11.8% (4 patients), and 5.9% (2 patients) of patients, respectively. These secondary mtDNA mutations have been previously reported in other populations, but reported here for the first time in Korean LHON patients.10–13 The seven complex I genes form a large proportion of the mitochondrial genome, comprising 38% of the total mtDNA. Complex I defects are increasingly being recognized as important causes of respiratory chain disease.23,24 The most common biochemical abnormalities seen in patients with LHON are complex I defects. Mutations in mtDNA may cause alterations of the electron transport components, which can compromise the normal electron flow.
IOVS j December 2014 j Vol. 55 j No. 12 j 8098
mtDNA Mutations in Korean Patients With LHON
TABLE 2. Clinical Findings of Korean Patients Carrying Homoplasmic Secondary LHON Mutations Final BCVA Case No. 1 2 3 4 5 6 7 8 9 10 11
Age
Sex
59 57 26 42 59 7 66 64 30 14 51
F F M F F F F F M F M
Mutation
Mutation Type
Age of Onset, y
m.3394T>C m.3497C>T m.3394T>C m.11696G>A m.11696G>A m.11696G>A m.3394T>C m.14502T>C m.3497C>T m.3497C>T m.11696G>A
Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic
58 19 26 42 Unclear 6 Unclear 4 Unclear 14 Unclear
Type I or II
Family History
Right
Left
Spontaneous Recovery
II II I I II II II II II I II
þ þ þ
20/32 20/200 20/80 20/200 20/32 20/160 20/40 20/32 20/40 20/25 20/100
20/25 20/1000 20/160 20/125 20/25 20/80 20/32 20/32 20/32 20/32 20/200
þ þ þ þ þ þ
M, male; F, female. TABLE 3. Clinical Findings of Korean Patients Carrying Combined Primary and Secondary LHON Mutations Final BCVA Case No.
Age
Sex
Mutation
Mutation Type
12
43
M
13
21
M
m.11778G>A þ m.3497C>T m.14484T>C þ m.14502T>C
Heteroplasmic þ homoplasmic Heteroplasmic þ homoplasmic
Age of Onset, y
Type I or II
Family History
Right
Left
Spontaneous Recovery
23
II
20/1000
20/20000
5
II
20/50
20/50
This study found that the m.11778G>A mutation in the MTND4 gene was the most prevalent LHON-associated mutation responsible for 58.8% of Korean LHON patients, which was consistent with studies from other countries.5,6,14 The m.14484T>C mutation in the MT-ND6 gene, an another common primary LHON-associated mutation, had been reported to be the second most prevalent mtDNA mutation type, with an incidence of 15.9% (13/82) in a previous Korean LHON study.16 In this study, only two patients carried the homoTABLE 4. Comparisons of Clinical Data of Patients With Primary and Secondary Mutation of LHON
Type I : Type II Sex ratio (M:F) BCVA, logMAR Color vision (mean Ishihara score) RNFL, lm Superior RNFL Nasal RNFL Inferior RNFL Temporal RNFL Overall RNFL
Primary Mutation, N ¼ 12
Secondary Mutation, N¼8
P Value
3:1 10:2 0.72 6 0.54
1:7 2:6 0.41 6 0.48
0.020* 0.019* 0.043†
3.00 6 4.20
12.11 6 6.66
0.005†
6 6 6 6 6
32.41 14.20 45.49 8.88 19.84
0.487† 0.231† 0.375† 0.097† 0.512†
7.57 6 10.96 79.33 6 32.43
0.024† 0.013†
112.17 64.58 109.67 49.75 84.00
6 6 6 6 6
39.65 11.65 44.79 16.06 25.94
98.38 62.38 93.13 40.38 73.25
VF MD, dbs VFI
18.11 6 11.06 45.44 6 33.45
VFI, visual field index. * v2 test (Fisher exact test in the case of presence of cell with frequency of lower than 5). † Mann-Whitney U test. Data are expressed as the mean 6 SD.
plasmic m.14484T>C mutations. We found no patients carrying the primary mutation m.3460G>A. The frequencies of three primary mtDNA mutations for LHON in the Asian populations were demonstrated in Table 5.8,16,25–30 The prevalences have been reported to be 77% to 95% for the m.11778G>A mutation, 5% to 22% for the m.14484T>C mutation, and 0% to 4% for the m.3460G>A mutation. The relatively lower contribution of the m.11778G>A mutation to LHON was an interesting finding in our study. Four secondary mtDNA mutations, which were all listed on MITOMAP, were identified in this study. Interestingly, the homoplasmic m.11696G>A mutation was the second most prevalent LHON-associated mtDNA mutation identified in our study (4 unrelated patients, 11.8%). The homoplasmic m.11696G>A mutation in the MT-ND4 gene associated with LHON has been identified in a large Dutch family and in some Chinese pedigrees.10,31–33 This mutation, which is located within a predicted transmembrane region, results in the replacement of an arginine with a histidine and is currently classified as provisional according to the MITOMAP website.34 The two types of secondary mtDNA mutations in the MT-ND1 gene were detected in seven patients. The homoplasmic m.3394T>C and m.3497C>T mutation were found in three unrelated patients, respectively. The ND1 protein is involved in the first step of the electron transport chain of oxidative phosphorylation. The m.3394T>C and m.3497C>T mutations resulted in the substitution of a histidine for a tyrosine at position 30 and a valine for an alanine at position 64 of the ND1 protein. These two mutations occur within a highly conserved region in the human ND1 protein.34–36 Mutations in this highly conserved region are assumed to alter the structure and function of the ND1 protein. The m.3394T > C mutation has been reported to be associated with LHON in American, Japanese, Finnish, and Chinese populations.7,11,12,37 This mutation is currently considered to have an unclear status on the MITOMAP resource. The m.3497C>T mutation was first reported to be associated with Japanese LHON patients in
IOVS j December 2014 j Vol. 55 j No. 12 j 8099
mtDNA Mutations in Korean Patients With LHON TABLE 5. Frequencies of the Three Primary mtDNA Mutations for LHON in Asia Mutation
References
Ethnic Population
Mashima et al.8
Japanese
Yamada et al.25 Jia et al.26 Yen et al.27 Ji et al. 28 Liang et al. 29 Sudoyo et al.30 Kim et al.16 Present study
Japanese Chinese Chinese Chinese Chinese Southeast Asian Korean Korean
No. of LHON Proband
m.11778G>A Number (%)
m.14484T>C Number (%)
m.3460G>A Number (%)
68
59 (86.8)
6 (8.8)
3 (4.4)
72 346 25 1164 1218 19 60 34
63 (88) 312 (90.2) 23 (92) 18 (94.7) 46 (76.7) 20 (58.8)
6 (8) 30 (8.7) 2 (8) 59 (4.8) 1 (5.3) 13 (21.7) 3 (8.8)
combination with the m.11778G>A mutation.11 The homoplasmic m.3497C>T LHON-associated mutation is categorized as provisional on the MITOMAP resource. 11,38 The m.14502T>C mutation in the MT-ND6 gene was also identified in our study. The m.14502T>C mutation causes substitution of a highly conserved isoleucine to valine, at position 58 in the ND6 protein.34 The homoplasmic m.14502T>C mutation, which was first detected in three unrelated Chinese families, has been suggested as a possible pathogenic target.39 The coexistence of a homoplasmic m.14502T>C mutation with a heteroplasmic m.14484T>C mutation was present in one patient (Table 3). The m.14502T>C mutation may exert a
3 4 0 16
(4) (1.1) (0) (1.4) 0 (0) 1 (1.6) 0 (0)
Other Reported Mutations m.3394T>C, m.4216T>C, m.7444G>A, m.9438G>A, m.13708G>A
m.3394T>C, m.3497C>T, m.4216T>C m.14502T>C, m.14459G>A m.3316G>A m.3394T>C, m.3497C>T, m.11696G>A, m.14502T>C
synergistic effect with the nearby primary m.14484T>C mutation.13,40,41 Interestingly, a considerable number of Korean LHON patients carried secondary LHON-associated mtDNA mutations. In silico analysis using SIFT, predicted two of the secondary mutations (m.3394T>C, m.3497C>T) detected in our study as not tolerated. Whether these mutations are pathogenic LHON mutations is unclear. However, the occurrence of homoplasmic m.3394T>C and m.3497C>T in the MTND1 gene and the MT-ND4 m.11696G>A mutations in several genetically unrelated patients with bilateral optic atrophy strongly suggested their involvement in the pathogenesis of
FIGURE. (A) Patient number 1 was a 59-year-old female with a homoplasmic m.3394T>C mutation. Her final visual acuity was 20/32 in the right eye and 20/25 in the left eye. (B) Patient number 2 was a 57-year-old female with a homoplasmic m.3497C>T mutation. Her final visual acuity was 20/ 200 in the right eye and 20/1000 in the left eye. (C) Patient number 4 was a 42-year-old female with a homoplasmic m.11696G>A mutation. Her final visual acuity was 20/200 in the right eye and 20/125 in the left eye. (D) Patient number 8 was a 64-year-old female with a homoplasmic m.14502T>C mutation. Her final visual acuity was 20/32 in both eyes.
IOVS j December 2014 j Vol. 55 j No. 12 j 8100
mtDNA Mutations in Korean Patients With LHON visual impairment (Table 2). Our findings support the notion that the homoplasmic m.11696G>A, m.3394T>C, and m.14502T>C mutations could be the causative mutations of LHON.10,12,39 We did not observe any significant differences in the RNFL thickness between patients with primary and secondary mutations (Table 4). Patients carrying a secondary mutation demonstrated a decreased RNFL thickness, compatible with optic atrophy, similar to those in primary mutation–carrying LHON patients (Fig., Table 4). These patients had a higher female ratio, better VA and color vision as well as better VF (Table 4). The pathophysiology of LHON is complex, and incomplete penetrance and variable expressivity, in terms of severity and age at onset of visual impairment, are typical features of LHON, found even in matrilineal relatives carrying an identical LHON mtDNA mutation. They implicate that secondary modulating factors such as nuclear modifier genes, environmental factors and mitochondrial variants/haplotypes are necessary in the phenotypic manifestation of the optic neuropathy.15,28,42–46 In our study, 13 (38.2%) patients had a family history of visual loss related to optic neuropathy, the remaining 21 (61.8%) patients were sporadic. However, extended pedigree analyses for maternally-transmitted LHON patients have not been performed enough to demonstrate the phenotypic manifestations, such as the penetrance, age at onset, and disease severity. In addition, sequence analysis of the entire mitochondrial genomes is necessary to delineate the phenotypic manifestation of mitochondrial variants in its full aspect. Further research of pedigree analyses of the penetrance and expressivity in the context of mitochondrial variants/haplotypes will conclusively demonstrate the influence of the mtDNA mutations in the phenotypic manifestation of LHON. Based on our results, Korean patients with LHON exhibited a wide spectrum of mtDNA mutations responsible for visual impairment. This research suggests that the evaluation of patients with optic atrophy should include both the primary and secondary LHON-associated mtDNA mutations. Bidirectional DNA sequencing is recommended for the genetic confirmation of suspected LHON patients.
Acknowledgments Supported by grants from Basic Science Research Program through the National Research Foundation of Korea (NRF; Daejeon, Korea) funded by the Ministry of Education (MOE; Sejong, Korea) (NRF2013R1A1A2006801). Disclosure: H.R. Yum, None; H. Chae, None; S.Y. Shin, None; Y. Kim, None; M. Kim, None; S.H. Park, None
References 1. Newman NJ. Leber’s hereditary optic neuropathy. New genetic considerations. Arch Neurol. 1993;50:540–548. 2. Nikoskelainen EK. Clinical feature of LHON. Clin Neurosci. 1994;2:115–120. 3. Nikoskelainen EK, Huoponen K, Juvonen V, Lamminen T, Nummelin K, Savontaus ML. Ophthalmologic findings in Leber hereditary optic neuropathy, with special reference to mtDNA mutations. Ophthalmology. 1996;103:504–514. 4. Howell N. Primary LHON mutations: trying to separate ‘fruyt’ from ‘chaf.’ Clin Neurosci. 1994;2:130–137. 5. Brown MD, Wallace DC. Spectrum of mitochondrial DNA mutations in Leber’s hereditary optic neuropathy. Clin Neurosci. 1994;2:138–145. 6. Mackey DA, Oostra RJ, Rosenberg T, et al. Primary pathogenic mtDNA mutations in multigeneration pedigrees with Leber
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
hereditary optic neuropathy. Am J Hum Genet. 1996;59:481– 485. Johns DR, Neufeld MJ, Park RD. An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. Biochem Biophys Res Commun. 1992;187:1551–1557. Mashima Y, Yamada K, Wakakura M, et al. Spectrum of pathogenic mitochondrial DNA mutations and clinical features in Japanese families with Leber’s hereditary optic neuropathy. Curr Eye Res. 1998;17:403–408. Oostra RJ, Bolhuis PA, Wijburg FA, Zorn-Ende G, BleekerWagemakers EM. Leber’s hereditary optic neuropathy: correlations between mitochondrial genotype and visual outcome. J Med Genet. 1994;31:280–286. Zhou X, Wei Q, Yang L, et al. Leber’s hereditary optic neuropathy is associated with the mitochondrial ND4 G11696A mutation in five Chinese families. Biochem Biophys Res Commun. 2006;340:69–75. Matsumoto M, Hayasaka S, Kadoi C, et al. Secondary mutations of mitochondrial DNA in Japanese patients with Leber’s hereditary optic neuropathy. Ophthalmic Genet. 1999;20: 153–160. Liang M, Guan M, Zhao F, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND1 T3394C mutation. Biochem Biophys Res Commun. 2009;383:286– 292. Zhang S, Wang L, Hao Y, et al. T14484C and 14502C in the mitochondrial ND6 gene are associated with Leber’s hereditary optic neuropathy in a Chinese family. Mitochondrion. 2008;8:205–210. Vergani L, Martinuzzi A, Carelli V, et al. MtDNA mutations associated with Leber’s hereditary optic neuropathy: studies on cytoplasmic hybrid (cybrid) cells. Biochem Biophys Res Commun. 1995;210:880–888. Zhang M, Zhou X, Li C, et al. Mitochondrial haplogroup M9a specific variant ND1 T3394C may have a modifying role in the phenotypic expression of the LHON-associated ND4 G11778A mutation. Mol Genet Metab. 2010;101:192–199. Kim JY, Hwang JM, Chang BL, Park SS. Spectrum of the mitochondrial DNA mutations of Leber’s hereditary optic neuropathy in Koreans. J Neurol. 2003;250:278–281. Hwang JM, Chang BL, Koh HJ, Kim JY, Park SS. Leber’s hereditary optic neuropathy with 3460 mitochondrial DNA mutation. J Korean Med Sci. 2002;17:283–286. Hwang JM. A family with Leber’s hereditary optic neuropathy with mitochondrial 11778/ND4 and 4216/ND1 mutations. Korean J Ophthalmol. 2000;14:45–48. Hwang JM, Chang BL, Park SS. Leber’s hereditary optic neuropathy mutations in Korean patients with multiple sclerosis. Ophthalmologica. 2001;215:398–400. Sunyaev S, Ramensky V, Koch I, Lathe W III, Kondrashov AS, Bork P. Prediction of deleterious human alleles. Hum Mol Genet. 2001;10:591–597. Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res. 2002;12: 436–446. Birch J. A practical guide for colour-vision examination: report of the Standardization Committee of the International Research Group on Colour-Vision Deficiencies. Ophthalmic Physiol Opt. 1985;5:265–285. Johns DR, Berman J. Alternative, simultaneous complex I mitochondrial DNA mutations in Leber’s hereditary optic neuropathy. Biochem Biophys Res Commun. 1991;174:1324– 1330. Mitchell AL, Elson JL, Howell N, Taylor RW, Turnbull DM. Sequence variation in mitochondrial complex I genes: mutation or polymorphism? J Med Genet. 2006;43:175–179.
mtDNA Mutations in Korean Patients With LHON 25. Yamada K, Mashima Y, Hiida Y, Oguchi Y. DNA diagnosis of Leber’s hereditary optic neuropathy performed at Keio University Hospital. Nihon Ganka Gakkai Zasshi. 2001;105: 608–613. 26. Jia X, Li S, Xiao X, Guo X, Zhang Q. Molecular epidemiology of mtDNA mutations in 903 Chinese families suspected with Leber hereditary optic neuropathy. J Hum Genet. 2006;51: 851–856. 27. Yen MY, Wang AG, Chang WL, Hsu WM, Liu JH, Wei YH. Leber’s hereditary optic neuropathy-the spectrum of mitochondrial DNA mutations in Chinese patients. Jpn J Ophthalmol. 2002;46:45–51. 28. Ji Y, Liang M, Zhang M, et al. Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families. J Hum Genet. 2014;59:134–140. 29. Liang M, Jiang P, Li F, et al. Frequency and spectrum of mitochondrial ND6 mutations in 1218 Han Chinese subjects with Leber’s hereditary optic neuropathy. Invest Ophthalmol Vis Sci. 2014;55:1321–1331. 30. Sudoyo H, Suryadi H, Lertrit P, Pramoonjago P, Lyrawati D, Marzuki S. Asian-specific mtDNA backgrounds associated with the primary G11778A mutation of Leber’s hereditary optic neuropathy. J Hum Genet. 2002;47:594–604. 31. De Vries DD, Went LN, Bruyn GW, et al. Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia. Am J Hum Genet. 1996;58:703– 711. 32. Qu J, Li R, Zhou X, et al. Cosegregation of the ND4 G11696A mutation with the LHON-associated ND4 G11778A mutation in a four generation Chinese family. Mitochondrion. 2007;7: 140–146. 33. Zhao FX, Zhou XT, Qu J, et al. Leber’s hereditary optic neuropathy is associated with the mitochondrial G11696A mutation in two Chinese families. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2007;24:556–559. 34. Fearnley IM, Walker JE. Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim Biophys Acta. 1992;1140:105–134. 35. Dupuis A. Identification of two genes of Rhodobacter capsulatus coding for proteins homologous to the ND1 and 23 kDa subunits of the mitochondrial Complex I. FEBS Lett. 1992;301:215–218.
IOVS j December 2014 j Vol. 55 j No. 12 j 8101 36. Yusnita Y, Norsiah MD, Rahman AJ. Mutations in mitochondrial NADH dehydrogenase subunit 1 (mtND1) gene in colorectal carcinoma. Malays J Pathol. 2010;32:103–110. 37. Puomila A, H¨am¨al¨ainen P, Kivioja S, et al. Epidemiology and penetrance of Leber hereditary optic neuropathy in Finland. Eur J Hum Genet. 2007;15:1079–1089. 38. Phasukkijwatana N, Chuenkongkaew WL, Suphavilai R, et al. The unique characteristics of Thai Leber hereditary optic neuropathy: analysis of 30 G11778A pedigrees. J Hum Genet. 2006;51:298–304. 39. Zhao F, Guan M, Zhou X, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation. Biochem Biophys Res Commun. 2009;389:466– 472. 40. Shu L, Zhang YM, Huang XX, Chen CY, Zhang XN. Complete mitochondrial DNA sequence analysis in two southern Chinese pedigrees with Leber hereditary optic neuropathy revealed secondary mutations along with the primary mutation. Int J Ophthalmol. 2012;5:28–31. 41. Qian Y, Zhou X, Hu Y, et al. Clinical evaluation and mitochondrial DNA sequence analysis in three Chinese families with Leber’s hereditary optic neuropathy. Biochem Biophys Res Commun. 2005;332:614–621. 42. Zhou X, Zhang H, Zhao F, et al. Very high penetrance and occurrence of Leber’s herediary optic neuropathy in a large Han Chinese pedigree carrying the ND4 G11778A mutation. Mol Genet Metab. 2010;100:379–384. 43. Tong Y, Sun YH, Zhou X, et al. Very low penetrance of Leber’s hereditary optic neuropathy in five Han Chinese families carrying the ND1 G3460A mutation. Mol Genet Metab. 2010; 99:417–424. 44. Hudson G, Carelli V, Spruijt L, et al. Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background. Am J Hum Genet. 2007;81:228–233. 45. Qu J, Zhou X, Zhang J, et al. Extremely low penetrance of Leber’s hereditary optic neuropathy in 8 Han Chinese families carrying the ND4 G11778A mutation. Ophthalmology. 2009; 116:558–564. 46. Zhang J, Zhou X, Zhou J, et al. Mitochondrial ND6 T14502C variant may modulate the phenotypic expression of LHONassociated G11778A mutation in four Chinese families. Biochem Biophys Res Commun. 2010;399:647–653.
Pathogenic Mitochondrial DNA Mutations and Associated Clinical Features in Korean Patients With Leber’s Hereditary Optic Neuropathy Hae Ri Yum,1 Hyojin Chae,2 Sun Young Shin,1 Yonggoo Kim,2 Myungshin Kim,2 and Shin Hae Park1 1Department
of Ophthalmology & Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Correspondence: Shin Hae Park, Department of Ophthalmology & Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-Gu, Seoul, 137701, Korea; [email protected]. Myungshin Kim, Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-Gu, Seoul, 137701, Korea; [email protected]. HRY and HC contributed equally to the work presented here and should therefore be regarded as equivalent authors. Submitted: July 24, 2014 Accepted: October 10, 2014 Citation: Yum HR, Chae H, Shin SY, Kim Y, Kim M, Park SH. Pathogenic mitochondrial DNA mutations and associated clinical features in Korean patients with Leber’s hereditary optic neuropathy. Invest Ophthalmol Vis Sci. 2014;55:8095–8101. DOI: 10.1167/iovs.14-15311
PURPOSE. To identify the spectrum of pathogenic mitochondrial DNA (mtDNA) mutations and clinical features in Korean patients with genetically confirmed Leber’s hereditary optic neuropathy (LHON). METHODS. The medical records of 34 unrelated, genetically confirmed LHON patients were reviewed. Total genomic DNA was isolated from the peripheral blood leukocytes of the patients with suspected LHON, and primary or secondary mtDNA mutations were identified by direct sequencing. We analyzed the visual acuity (VA), color vision, RNFL thickness, and visual field (VF) at the final visit from 20 patients who were followed-up for more than 6 months after the onset of LHON. RESULTS. Among 34 patients, 21 (61.8%) had the homoplasmic primary mutation, 11 (32.4%) had the homoplasmic secondary mutation, and 2 (5.9%) had the heteroplasmic primary mutation along with the homoplasmic secondary mutation. Analysis of mtDNA sequences revealed six different types of LHON-associated mutations: two primary LHON-associated primary mutations, m.11778G>A (20 patients, 58.8%) and m.14484T>C (3 patients, 8.8%), and four secondary LHON-associated mutations, which were m.3394T>C (3 patients, 8.8%), m.3497C>T (4 patients, 11.8%), m.11696G>A (4 patients, 11.8%), and m.14502T>C (2 patients, 5.9%). Secondary mutation-carrying patients demonstrated a decreased in RNFL thickness, similar to those in primary mutation–carrying LHON patients. These patients had a higher female ratio (P ¼ 0.019), better VA (P ¼ 0.043) and color vision (P ¼ 0.005), as well as better VF. CONCLUSIONS. In addition to common primary LHON-associated mutations, our study identified secondary mtDNA mutations, which should be considered when evaluating patients with optic atrophy. Keywords: Leber’s hereditary optic neuropathy, mitochondrial DNA, mutation
eber’s hereditary optic neuropathy (LHON) is typically characterized by bilateral acute or subacute loss of central vision, owing to focal degeneration of the retinal ganglion cell layer and optic nerve.1–3 It is a mitochondrial genetic disease inherited through maternal transmission that mainly affects young adult males.1–3 To date, over 30 different types of mitochondrial DNA (mtDNA) mutations, including 14 primary mutations, have been identified in patients with LHON (in the public domain http://www.mitomap.org/MITOMAP). Based on the genetic, biochemical, and clinical features, three mtDNA point mutations, ND1 m.3460G>A, ND4 m.11778G>A, and ND6 m.14484T>C, in the genes encoding the subunits of respiratory chain complex I have been traditionally considered as high-risk LHON mutations.4–7 These mutations represent approximately 80% to 95% of all LHON cases occurring in patients with different ethnic backgrounds.5–9 However, no primary mutations have been identified in a small minority of
diagnosed LHON cases. In one study, no primary mutations were found in 21% of LHON-affected patients from Finland.3 Recently, a growing number of mtDNA variants that are suspected to have a role in the disease expression of LHON have been reported.10–13 The pathological significance of mtDNA mutations other than primary mutations is uncertain and considered to be intermediate or low risk. Therefore, these have been proposed as secondary mutations. Secondary mutations that are relevant for the phenotypic expression of LHON appear to have a synergistic and deleterious effect together with the primary mutations.10–13 These secondary LHON mutations are homoplasmic, often occurring in combination with primary LHON mutations.10–13 They are found at a lower frequency in healthy subjects compared with the LHON population; however, they may have an additional role in the pathogenesis of LHON and increase the risk of disease expression.11,14,15
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
8095
L
IOVS j December 2014 j Vol. 55 j No. 12 j 8096
mtDNA Mutations in Korean Patients With LHON The distribution of LHON-associated mtDNA mutations is population-specific. However, little is known about the mutation and clinical characterization for Korean patients with LHON. Only five types of mtDNA mutations have been reported in Korean patients. Restriction fragment length polymorphism-PCR (RFLP-PCR) has revealed m.11778G>A (56.1%), m.14484T>C (15.9%), and m.3460G>A (1.2%) mutations in Korean LHON patients.16,17 In addition, secondary mtDNA mutations at nucleotide position (np) 4216 and 15257 were also reported in Korean LHON patients with or without primary mutation.18,19 In this study, we have used DNA sequencing to analyze LHON-associated mtDNA mutations together with the clinical characterization of Korean LHON patients.
METHODS This study was performed according to the principles of the Declaration of Helsinki, after approval by the institutional review board of Seoul St. Mary’s Hospital, College of Medicine, T h e C a t h o l i c U n i ve r s i t y o f Ko r e a , S e o u l , Ko r e a (KC14RISE0241).
Subjects The medical records of 34 unrelated genetically confirmed LHON patients at Seoul St. Mary’s Hospital from January 2009 to May 2014 were reviewed. The age, sex, family history, age at LHON onset, and initial/final visual acuity (VA) were evaluated for each patient. Acute or subacute visual loss with typical peripapillary microangiopathy were classified as type I clinical features. Bilateral optic atrophy with temporal retinal nerve fiber layer (RNFL) thinning and central scotoma were classified as type II clinical features. After thorough ophthalmologic evaluations, subjects were excluded on the basis of any of the following criteria: a history of retinal disease, including diabetic or hypertensive retinopathy, a history of eye trauma or surgery, a history of systemic or neurologic disease that may affect the optical coherence tomography (OCT) and visual field (VF) results, and any relevant drug history.
biomicroscopy, a dilated stereoscopic examination of the optic nerve head and fundus, a VF test, and optical coherence tomography (OCT). Clinical examinations were performed by an experienced neuro-ophthalmologist. We analyzed the ophthalmologic results at the final visit from 20 patients who were followed-up for more than 6 months after the onset of LHON. When both eyes were eligible for the study, we analyzed a worse-seeing eye in each patient. We used the 24-plate edition from the Ishihara test book published in 1997 by Kanehara & Co., Ltd. (Tokyo, Japan), and the score was set as the number of plates identified out of the first 12 screening plates and five diagnostic plates.22 The peripapillary RNFL thickness was measured using a spectral-domain Cirrus HD-OCT (software version 6.0, Carl Zeiss Meditec, Dublin, CA). Spectral-domain OCT imaging was obtained from a 3-dimensional data set from an optic cube scan, which was composed of 200 A-scans from each of 200 Bscans that covered a 6-mm2 area centered on the optic disc. Following the creation of the RNFL thickness map from the cube data set, the software then automatically determines the center of the disc and extracts a circumpapillary circle (1.73mm radius) from the cube data set, which it then uses to determine RNFL thickness. To avoid confusion, clock-hour sectors were named according to their location as either superior (S), inferior (I), temporal (T), or nasal (N). The following OCT parameters were used in the analysis: S, I, T, N, and overall RNFL thickness. Image quality was assessed by an experienced examiner who was unaware of the patient’s identity and condition. We performed at least two standard automated perimetry tests (Humphrey Visual Field Analyzer; Zeiss/Humphrey Systems, Dublin, CA, USA) 24–2, using the Swedish interactive threshold algorithm (SITA) strategy. The VF defect had to be present at the same location in at least two consecutive, reliable VFs for the result to be included in the study. The results from the VF tests were considered reliable, if the fixation losses were less than 20% and false positive and false negative rates were less than 15%.
Statistical Analysis Mutational Analysis After obtaining informed consent from each patient, blood samples were taken for mtDNA extraction and analysis. Total genomic DNA was isolated from peripheral blood leukocytes using the QIAmp DNA Mini Kit (Qiagen, Hamburg, Germany). The presence of the m.3460G>A, m.11778G>A, and m.14484T>C mutations and secondary mutations in genes mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), and mitochondrially encoded cytochrome B (MT-CYB) were screened by direct sequencing using an ABI prism Big Dye Terminator cycle sequencing Ready Reaction kit V 3.1 (Applied Biosystems, Foster City, CA, USA) and analyzed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). All sequences were analyzed against mitochondrial reference sequence NC_012920. We performed in silico analysis using the software Polyphen-2 (in the public domain http://genetics.bwh.harvard. edu/pph2/) and Sorting Intolerant from Tolerant (SIFT; in the public domain http://sift.jcvi.org/) to assess if the substitutions were predicted as potentially pathogenic.20,21
Ophthalmologic Examination Each participant underwent a comprehensive ophthalmologic assessment, which included the measurement of best-corrected visual acuity (BCVA), a color vision test, slit-lamp
Statistical analysis was performed using SPSS software (version 19.0; SPSS, Inc., Chicago, IL, USA). The v2 test and MannWhitney U test were used to compare data from the primary and secondary mutation groups. A P value of less than 0.05 was regarded as statistically significant.
RESULTS Genetic Analysis Among 34 patients, 21 (61.8%) had the homoplasmic primary mutation, 11 (32.4%) had the homoplasmic secondary mutation, and 2 (5.9%) had the heteroplasmic primary mutation along with the homoplasmic secondary mutation. The mtDNA mutations identified in each patient are summarized in Table 1. Six different kinds of mtDNA mutations were detected in 34 unrelated patients with bilateral optic atrophy. In addition to the two common LHON-associated primary mutations, m.11778G>A (20 patients, 58.8%) and m.14484T>C (3 patients, 8.8%), four secondary mutations were also identified, m.3394T>C (3 patients, 8.8%), m.3497C>T (4 patients, 11.8%), m.11696G>A (4 patients, 11.8%), and m.14502T>C (2 patients, 5.9%). In silico analysis of the secondary mutations generated differing results. Polyphen-2 predicted that all four substitutions would be benign and SIFT predicted that two of the secondary mutations
IOVS j December 2014 j Vol. 55 j No. 12 j 8097
13, 40 (Chinese)
Benign
The male:female ratio in this study was 21:13. The overall proportion of LHON patients with a family history of the disease was 38.2%. In 10 patients who had type I clinical features, with acute or subacute visual loss and peripapillary telangiectatic microangiopathy, the mean age of LHON onset was 25.2 years. Both Tables 2 and 3 summarize the clinical findings from patients carrying homoplasmic secondary LHON mutations and combined primary and secondary LHON mutations. We analyzed the ophthalmologic results at the final visit from 20 patients who were followed-up for more than 6 months after the onset of LHON. We then compared the clinical data from patients with primary and secondary LHON mutations (Table 4). In the secondary mutation group, there was a higher proportion of type II clinical features (P ¼ 0.020) and lower male to female ratio (P ¼ 0.019). No significant difference was found in the OCT parameters, including those in the temporal area, between the two groups. However, the BCVA was significantly better in patients with the secondary mutation than in those with the primary mutation (P ¼ 0.043). The color vision deficiency was also less prominent in patients with the secondary mtDNA mutation compared with those with the primary mtDNA mutation (P ¼ 0.005). Both the VF index and MD of the VF test were significantly different between the two groups (P ¼ 0.013 and P ¼ 0.024).
Confirmed þ reported/ possibly synergistic
Reported/possibly synergistic Confirmed þ reported/secondary
Homoplasmic Heteroplasmic þ homoplasmic Heteroplasmic þ homoplasmic
Tolerated
34
1
MT-ND6 MT-ND4 MT-ND1 MT-ND6 Combined
1 1
m.14502T>C m.11778G>A m.3497C>T m.14484T>C þ m.14502T>C
p.Ile58Val p.Arg340His p.Ala64Val p.Met64Val p.Ile58Val
DISCUSSION
Total
Benign Reported/possibly synergistic MT-ND4 4
m.11696G>A
p.Val313Ile
Homoplasmic
Tolerated
Benign Reported/secondary MT-ND1
Secondary
3
m.3497C>T
p.Ala64Val
Homoplasmic
Not tolerated
Probably Damaging Benign Benign Deleterious Deleterious Not tolerated Confirmed Confirmed Reported/unclear Homoplasmic Homoplasmic Homoplasmic p.Arg340His p.Met64Val p.Tyr30His MT-ND4 MT-ND6 MT-ND1
m.11778G>A m.14484T>C m.3394T>C
(m.3394T>C, m.3497C>T) would not be tolerated (Table 1). We found no patients carrying the primary mutation m.3460G>A. Patients with homoplasmic secondary mutations had common ocular features, with a pale optic disc and decreased peripapillary RNFL thickness in both eyes (Table 2; Fig. A–D). Two patients were shown to have homoplasmic secondary mutations and heteroplasmic primary mutations. The coexistence of mutations MT-ND1 m.3497C>T/MT-ND4 m.11778G>A and MT-ND6 m.14502T>C/MT-ND6 m.14484T>C was detected in each patient (Table 3, cases 12 and 13).
Clinical Features
19 2 3 Primary
Codon Mutation Gene Locus Numbers
TABLE 1. Sequence Analysis of Mutations in Korean Patients With LHON
Mutation Type
MITOMAP Status
SIFT
Polyphen-2
References
7 (American) 11 (Japanese) 12, 15 (Chinese) 37 (Finnish) 11 (Japanese) 38 (Thai) 10, 32, 33 (Chinese) 31 (Dutch) 39 (Chinese) 38 (Thai)
mtDNA Mutations in Korean Patients With LHON
In this study, we report genetic and clinical characterization of LHON in 34 unrelated Korean patients. Sequence analysis of the mitochondrial genome revealed six different kinds of LHON-associated mtDNA mutations in patients with bilateral visual impairment due to optic atrophy as a sole clinical manifestation. The two common primary LHON-associated mutations, m.11778G>A and m.14484T>C were identified in 58.8% (20 patients) and 8.8% (3 patients) of the patients, respectively. The four secondary mtDNA mutations, m.3394T>C, m.3497C>T, m.11696G>A, and m.14502T>C, which may be associated with LHON, were found in 8.8% (3 patients), 11.8% (4 patients), 11.8% (4 patients), and 5.9% (2 patients) of patients, respectively. These secondary mtDNA mutations have been previously reported in other populations, but reported here for the first time in Korean LHON patients.10–13 The seven complex I genes form a large proportion of the mitochondrial genome, comprising 38% of the total mtDNA. Complex I defects are increasingly being recognized as important causes of respiratory chain disease.23,24 The most common biochemical abnormalities seen in patients with LHON are complex I defects. Mutations in mtDNA may cause alterations of the electron transport components, which can compromise the normal electron flow.
IOVS j December 2014 j Vol. 55 j No. 12 j 8098
mtDNA Mutations in Korean Patients With LHON
TABLE 2. Clinical Findings of Korean Patients Carrying Homoplasmic Secondary LHON Mutations Final BCVA Case No. 1 2 3 4 5 6 7 8 9 10 11
Age
Sex
59 57 26 42 59 7 66 64 30 14 51
F F M F F F F F M F M
Mutation
Mutation Type
Age of Onset, y
m.3394T>C m.3497C>T m.3394T>C m.11696G>A m.11696G>A m.11696G>A m.3394T>C m.14502T>C m.3497C>T m.3497C>T m.11696G>A
Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic Homoplasmic
58 19 26 42 Unclear 6 Unclear 4 Unclear 14 Unclear
Type I or II
Family History
Right
Left
Spontaneous Recovery
II II I I II II II II II I II
þ þ þ
20/32 20/200 20/80 20/200 20/32 20/160 20/40 20/32 20/40 20/25 20/100
20/25 20/1000 20/160 20/125 20/25 20/80 20/32 20/32 20/32 20/32 20/200
þ þ þ þ þ þ
M, male; F, female. TABLE 3. Clinical Findings of Korean Patients Carrying Combined Primary and Secondary LHON Mutations Final BCVA Case No.
Age
Sex
Mutation
Mutation Type
12
43
M
13
21
M
m.11778G>A þ m.3497C>T m.14484T>C þ m.14502T>C
Heteroplasmic þ homoplasmic Heteroplasmic þ homoplasmic
Age of Onset, y
Type I or II
Family History
Right
Left
Spontaneous Recovery
23
II
20/1000
20/20000
5
II
20/50
20/50
This study found that the m.11778G>A mutation in the MTND4 gene was the most prevalent LHON-associated mutation responsible for 58.8% of Korean LHON patients, which was consistent with studies from other countries.5,6,14 The m.14484T>C mutation in the MT-ND6 gene, an another common primary LHON-associated mutation, had been reported to be the second most prevalent mtDNA mutation type, with an incidence of 15.9% (13/82) in a previous Korean LHON study.16 In this study, only two patients carried the homoTABLE 4. Comparisons of Clinical Data of Patients With Primary and Secondary Mutation of LHON
Type I : Type II Sex ratio (M:F) BCVA, logMAR Color vision (mean Ishihara score) RNFL, lm Superior RNFL Nasal RNFL Inferior RNFL Temporal RNFL Overall RNFL
Primary Mutation, N ¼ 12
Secondary Mutation, N¼8
P Value
3:1 10:2 0.72 6 0.54
1:7 2:6 0.41 6 0.48
0.020* 0.019* 0.043†
3.00 6 4.20
12.11 6 6.66
0.005†
6 6 6 6 6
32.41 14.20 45.49 8.88 19.84
0.487† 0.231† 0.375† 0.097† 0.512†
7.57 6 10.96 79.33 6 32.43
0.024† 0.013†
112.17 64.58 109.67 49.75 84.00
6 6 6 6 6
39.65 11.65 44.79 16.06 25.94
98.38 62.38 93.13 40.38 73.25
VF MD, dbs VFI
18.11 6 11.06 45.44 6 33.45
VFI, visual field index. * v2 test (Fisher exact test in the case of presence of cell with frequency of lower than 5). † Mann-Whitney U test. Data are expressed as the mean 6 SD.
plasmic m.14484T>C mutations. We found no patients carrying the primary mutation m.3460G>A. The frequencies of three primary mtDNA mutations for LHON in the Asian populations were demonstrated in Table 5.8,16,25–30 The prevalences have been reported to be 77% to 95% for the m.11778G>A mutation, 5% to 22% for the m.14484T>C mutation, and 0% to 4% for the m.3460G>A mutation. The relatively lower contribution of the m.11778G>A mutation to LHON was an interesting finding in our study. Four secondary mtDNA mutations, which were all listed on MITOMAP, were identified in this study. Interestingly, the homoplasmic m.11696G>A mutation was the second most prevalent LHON-associated mtDNA mutation identified in our study (4 unrelated patients, 11.8%). The homoplasmic m.11696G>A mutation in the MT-ND4 gene associated with LHON has been identified in a large Dutch family and in some Chinese pedigrees.10,31–33 This mutation, which is located within a predicted transmembrane region, results in the replacement of an arginine with a histidine and is currently classified as provisional according to the MITOMAP website.34 The two types of secondary mtDNA mutations in the MT-ND1 gene were detected in seven patients. The homoplasmic m.3394T>C and m.3497C>T mutation were found in three unrelated patients, respectively. The ND1 protein is involved in the first step of the electron transport chain of oxidative phosphorylation. The m.3394T>C and m.3497C>T mutations resulted in the substitution of a histidine for a tyrosine at position 30 and a valine for an alanine at position 64 of the ND1 protein. These two mutations occur within a highly conserved region in the human ND1 protein.34–36 Mutations in this highly conserved region are assumed to alter the structure and function of the ND1 protein. The m.3394T > C mutation has been reported to be associated with LHON in American, Japanese, Finnish, and Chinese populations.7,11,12,37 This mutation is currently considered to have an unclear status on the MITOMAP resource. The m.3497C>T mutation was first reported to be associated with Japanese LHON patients in
IOVS j December 2014 j Vol. 55 j No. 12 j 8099
mtDNA Mutations in Korean Patients With LHON TABLE 5. Frequencies of the Three Primary mtDNA Mutations for LHON in Asia Mutation
References
Ethnic Population
Mashima et al.8
Japanese
Yamada et al.25 Jia et al.26 Yen et al.27 Ji et al. 28 Liang et al. 29 Sudoyo et al.30 Kim et al.16 Present study
Japanese Chinese Chinese Chinese Chinese Southeast Asian Korean Korean
No. of LHON Proband
m.11778G>A Number (%)
m.14484T>C Number (%)
m.3460G>A Number (%)
68
59 (86.8)
6 (8.8)
3 (4.4)
72 346 25 1164 1218 19 60 34
63 (88) 312 (90.2) 23 (92) 18 (94.7) 46 (76.7) 20 (58.8)
6 (8) 30 (8.7) 2 (8) 59 (4.8) 1 (5.3) 13 (21.7) 3 (8.8)
combination with the m.11778G>A mutation.11 The homoplasmic m.3497C>T LHON-associated mutation is categorized as provisional on the MITOMAP resource. 11,38 The m.14502T>C mutation in the MT-ND6 gene was also identified in our study. The m.14502T>C mutation causes substitution of a highly conserved isoleucine to valine, at position 58 in the ND6 protein.34 The homoplasmic m.14502T>C mutation, which was first detected in three unrelated Chinese families, has been suggested as a possible pathogenic target.39 The coexistence of a homoplasmic m.14502T>C mutation with a heteroplasmic m.14484T>C mutation was present in one patient (Table 3). The m.14502T>C mutation may exert a
3 4 0 16
(4) (1.1) (0) (1.4) 0 (0) 1 (1.6) 0 (0)
Other Reported Mutations m.3394T>C, m.4216T>C, m.7444G>A, m.9438G>A, m.13708G>A
m.3394T>C, m.3497C>T, m.4216T>C m.14502T>C, m.14459G>A m.3316G>A m.3394T>C, m.3497C>T, m.11696G>A, m.14502T>C
synergistic effect with the nearby primary m.14484T>C mutation.13,40,41 Interestingly, a considerable number of Korean LHON patients carried secondary LHON-associated mtDNA mutations. In silico analysis using SIFT, predicted two of the secondary mutations (m.3394T>C, m.3497C>T) detected in our study as not tolerated. Whether these mutations are pathogenic LHON mutations is unclear. However, the occurrence of homoplasmic m.3394T>C and m.3497C>T in the MTND1 gene and the MT-ND4 m.11696G>A mutations in several genetically unrelated patients with bilateral optic atrophy strongly suggested their involvement in the pathogenesis of
FIGURE. (A) Patient number 1 was a 59-year-old female with a homoplasmic m.3394T>C mutation. Her final visual acuity was 20/32 in the right eye and 20/25 in the left eye. (B) Patient number 2 was a 57-year-old female with a homoplasmic m.3497C>T mutation. Her final visual acuity was 20/ 200 in the right eye and 20/1000 in the left eye. (C) Patient number 4 was a 42-year-old female with a homoplasmic m.11696G>A mutation. Her final visual acuity was 20/200 in the right eye and 20/125 in the left eye. (D) Patient number 8 was a 64-year-old female with a homoplasmic m.14502T>C mutation. Her final visual acuity was 20/32 in both eyes.
IOVS j December 2014 j Vol. 55 j No. 12 j 8100
mtDNA Mutations in Korean Patients With LHON visual impairment (Table 2). Our findings support the notion that the homoplasmic m.11696G>A, m.3394T>C, and m.14502T>C mutations could be the causative mutations of LHON.10,12,39 We did not observe any significant differences in the RNFL thickness between patients with primary and secondary mutations (Table 4). Patients carrying a secondary mutation demonstrated a decreased RNFL thickness, compatible with optic atrophy, similar to those in primary mutation–carrying LHON patients (Fig., Table 4). These patients had a higher female ratio, better VA and color vision as well as better VF (Table 4). The pathophysiology of LHON is complex, and incomplete penetrance and variable expressivity, in terms of severity and age at onset of visual impairment, are typical features of LHON, found even in matrilineal relatives carrying an identical LHON mtDNA mutation. They implicate that secondary modulating factors such as nuclear modifier genes, environmental factors and mitochondrial variants/haplotypes are necessary in the phenotypic manifestation of the optic neuropathy.15,28,42–46 In our study, 13 (38.2%) patients had a family history of visual loss related to optic neuropathy, the remaining 21 (61.8%) patients were sporadic. However, extended pedigree analyses for maternally-transmitted LHON patients have not been performed enough to demonstrate the phenotypic manifestations, such as the penetrance, age at onset, and disease severity. In addition, sequence analysis of the entire mitochondrial genomes is necessary to delineate the phenotypic manifestation of mitochondrial variants in its full aspect. Further research of pedigree analyses of the penetrance and expressivity in the context of mitochondrial variants/haplotypes will conclusively demonstrate the influence of the mtDNA mutations in the phenotypic manifestation of LHON. Based on our results, Korean patients with LHON exhibited a wide spectrum of mtDNA mutations responsible for visual impairment. This research suggests that the evaluation of patients with optic atrophy should include both the primary and secondary LHON-associated mtDNA mutations. Bidirectional DNA sequencing is recommended for the genetic confirmation of suspected LHON patients.
Acknowledgments Supported by grants from Basic Science Research Program through the National Research Foundation of Korea (NRF; Daejeon, Korea) funded by the Ministry of Education (MOE; Sejong, Korea) (NRF2013R1A1A2006801). Disclosure: H.R. Yum, None; H. Chae, None; S.Y. Shin, None; Y. Kim, None; M. Kim, None; S.H. Park, None
References 1. Newman NJ. Leber’s hereditary optic neuropathy. New genetic considerations. Arch Neurol. 1993;50:540–548. 2. Nikoskelainen EK. Clinical feature of LHON. Clin Neurosci. 1994;2:115–120. 3. Nikoskelainen EK, Huoponen K, Juvonen V, Lamminen T, Nummelin K, Savontaus ML. Ophthalmologic findings in Leber hereditary optic neuropathy, with special reference to mtDNA mutations. Ophthalmology. 1996;103:504–514. 4. Howell N. Primary LHON mutations: trying to separate ‘fruyt’ from ‘chaf.’ Clin Neurosci. 1994;2:130–137. 5. Brown MD, Wallace DC. Spectrum of mitochondrial DNA mutations in Leber’s hereditary optic neuropathy. Clin Neurosci. 1994;2:138–145. 6. Mackey DA, Oostra RJ, Rosenberg T, et al. Primary pathogenic mtDNA mutations in multigeneration pedigrees with Leber
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
hereditary optic neuropathy. Am J Hum Genet. 1996;59:481– 485. Johns DR, Neufeld MJ, Park RD. An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. Biochem Biophys Res Commun. 1992;187:1551–1557. Mashima Y, Yamada K, Wakakura M, et al. Spectrum of pathogenic mitochondrial DNA mutations and clinical features in Japanese families with Leber’s hereditary optic neuropathy. Curr Eye Res. 1998;17:403–408. Oostra RJ, Bolhuis PA, Wijburg FA, Zorn-Ende G, BleekerWagemakers EM. Leber’s hereditary optic neuropathy: correlations between mitochondrial genotype and visual outcome. J Med Genet. 1994;31:280–286. Zhou X, Wei Q, Yang L, et al. Leber’s hereditary optic neuropathy is associated with the mitochondrial ND4 G11696A mutation in five Chinese families. Biochem Biophys Res Commun. 2006;340:69–75. Matsumoto M, Hayasaka S, Kadoi C, et al. Secondary mutations of mitochondrial DNA in Japanese patients with Leber’s hereditary optic neuropathy. Ophthalmic Genet. 1999;20: 153–160. Liang M, Guan M, Zhao F, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND1 T3394C mutation. Biochem Biophys Res Commun. 2009;383:286– 292. Zhang S, Wang L, Hao Y, et al. T14484C and 14502C in the mitochondrial ND6 gene are associated with Leber’s hereditary optic neuropathy in a Chinese family. Mitochondrion. 2008;8:205–210. Vergani L, Martinuzzi A, Carelli V, et al. MtDNA mutations associated with Leber’s hereditary optic neuropathy: studies on cytoplasmic hybrid (cybrid) cells. Biochem Biophys Res Commun. 1995;210:880–888. Zhang M, Zhou X, Li C, et al. Mitochondrial haplogroup M9a specific variant ND1 T3394C may have a modifying role in the phenotypic expression of the LHON-associated ND4 G11778A mutation. Mol Genet Metab. 2010;101:192–199. Kim JY, Hwang JM, Chang BL, Park SS. Spectrum of the mitochondrial DNA mutations of Leber’s hereditary optic neuropathy in Koreans. J Neurol. 2003;250:278–281. Hwang JM, Chang BL, Koh HJ, Kim JY, Park SS. Leber’s hereditary optic neuropathy with 3460 mitochondrial DNA mutation. J Korean Med Sci. 2002;17:283–286. Hwang JM. A family with Leber’s hereditary optic neuropathy with mitochondrial 11778/ND4 and 4216/ND1 mutations. Korean J Ophthalmol. 2000;14:45–48. Hwang JM, Chang BL, Park SS. Leber’s hereditary optic neuropathy mutations in Korean patients with multiple sclerosis. Ophthalmologica. 2001;215:398–400. Sunyaev S, Ramensky V, Koch I, Lathe W III, Kondrashov AS, Bork P. Prediction of deleterious human alleles. Hum Mol Genet. 2001;10:591–597. Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res. 2002;12: 436–446. Birch J. A practical guide for colour-vision examination: report of the Standardization Committee of the International Research Group on Colour-Vision Deficiencies. Ophthalmic Physiol Opt. 1985;5:265–285. Johns DR, Berman J. Alternative, simultaneous complex I mitochondrial DNA mutations in Leber’s hereditary optic neuropathy. Biochem Biophys Res Commun. 1991;174:1324– 1330. Mitchell AL, Elson JL, Howell N, Taylor RW, Turnbull DM. Sequence variation in mitochondrial complex I genes: mutation or polymorphism? J Med Genet. 2006;43:175–179.
mtDNA Mutations in Korean Patients With LHON 25. Yamada K, Mashima Y, Hiida Y, Oguchi Y. DNA diagnosis of Leber’s hereditary optic neuropathy performed at Keio University Hospital. Nihon Ganka Gakkai Zasshi. 2001;105: 608–613. 26. Jia X, Li S, Xiao X, Guo X, Zhang Q. Molecular epidemiology of mtDNA mutations in 903 Chinese families suspected with Leber hereditary optic neuropathy. J Hum Genet. 2006;51: 851–856. 27. Yen MY, Wang AG, Chang WL, Hsu WM, Liu JH, Wei YH. Leber’s hereditary optic neuropathy-the spectrum of mitochondrial DNA mutations in Chinese patients. Jpn J Ophthalmol. 2002;46:45–51. 28. Ji Y, Liang M, Zhang M, et al. Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families. J Hum Genet. 2014;59:134–140. 29. Liang M, Jiang P, Li F, et al. Frequency and spectrum of mitochondrial ND6 mutations in 1218 Han Chinese subjects with Leber’s hereditary optic neuropathy. Invest Ophthalmol Vis Sci. 2014;55:1321–1331. 30. Sudoyo H, Suryadi H, Lertrit P, Pramoonjago P, Lyrawati D, Marzuki S. Asian-specific mtDNA backgrounds associated with the primary G11778A mutation of Leber’s hereditary optic neuropathy. J Hum Genet. 2002;47:594–604. 31. De Vries DD, Went LN, Bruyn GW, et al. Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia. Am J Hum Genet. 1996;58:703– 711. 32. Qu J, Li R, Zhou X, et al. Cosegregation of the ND4 G11696A mutation with the LHON-associated ND4 G11778A mutation in a four generation Chinese family. Mitochondrion. 2007;7: 140–146. 33. Zhao FX, Zhou XT, Qu J, et al. Leber’s hereditary optic neuropathy is associated with the mitochondrial G11696A mutation in two Chinese families. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2007;24:556–559. 34. Fearnley IM, Walker JE. Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim Biophys Acta. 1992;1140:105–134. 35. Dupuis A. Identification of two genes of Rhodobacter capsulatus coding for proteins homologous to the ND1 and 23 kDa subunits of the mitochondrial Complex I. FEBS Lett. 1992;301:215–218.
IOVS j December 2014 j Vol. 55 j No. 12 j 8101 36. Yusnita Y, Norsiah MD, Rahman AJ. Mutations in mitochondrial NADH dehydrogenase subunit 1 (mtND1) gene in colorectal carcinoma. Malays J Pathol. 2010;32:103–110. 37. Puomila A, H¨am¨al¨ainen P, Kivioja S, et al. Epidemiology and penetrance of Leber hereditary optic neuropathy in Finland. Eur J Hum Genet. 2007;15:1079–1089. 38. Phasukkijwatana N, Chuenkongkaew WL, Suphavilai R, et al. The unique characteristics of Thai Leber hereditary optic neuropathy: analysis of 30 G11778A pedigrees. J Hum Genet. 2006;51:298–304. 39. Zhao F, Guan M, Zhou X, et al. Leber’s hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation. Biochem Biophys Res Commun. 2009;389:466– 472. 40. Shu L, Zhang YM, Huang XX, Chen CY, Zhang XN. Complete mitochondrial DNA sequence analysis in two southern Chinese pedigrees with Leber hereditary optic neuropathy revealed secondary mutations along with the primary mutation. Int J Ophthalmol. 2012;5:28–31. 41. Qian Y, Zhou X, Hu Y, et al. Clinical evaluation and mitochondrial DNA sequence analysis in three Chinese families with Leber’s hereditary optic neuropathy. Biochem Biophys Res Commun. 2005;332:614–621. 42. Zhou X, Zhang H, Zhao F, et al. Very high penetrance and occurrence of Leber’s herediary optic neuropathy in a large Han Chinese pedigree carrying the ND4 G11778A mutation. Mol Genet Metab. 2010;100:379–384. 43. Tong Y, Sun YH, Zhou X, et al. Very low penetrance of Leber’s hereditary optic neuropathy in five Han Chinese families carrying the ND1 G3460A mutation. Mol Genet Metab. 2010; 99:417–424. 44. Hudson G, Carelli V, Spruijt L, et al. Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background. Am J Hum Genet. 2007;81:228–233. 45. Qu J, Zhou X, Zhang J, et al. Extremely low penetrance of Leber’s hereditary optic neuropathy in 8 Han Chinese families carrying the ND4 G11778A mutation. Ophthalmology. 2009; 116:558–564. 46. Zhang J, Zhou X, Zhou J, et al. Mitochondrial ND6 T14502C variant may modulate the phenotypic expression of LHONassociated G11778A mutation in four Chinese families. Biochem Biophys Res Commun. 2010;399:647–653.
