Abstract
Purpose
To investigate the role of mitochondrial haplotypes in the development of Leber’s hereditary optic neuropathy (LHON) associated with the ND4 G11778A mutation in Chinese families.
Design
Eight Han Chinese families with maternally transmitted LHON were studied using clinical, genetic, and molecular evaluations.
Participants
One hundred sixty-seven subjects from 8 Chinese families with a wide age range and severity of visual impairment.
Methods
All subjects underwent the clinical and genetic evaluation, as well as molecular analysis of mitochondrial DNA (mtDNA).
Main Outcome Measures
The ophthalmologic examinations included visual acuity, visual field examination, visual evoked potentials, and fundus photography. Mitochondrial DNA analysis included the polymerase chain reaction amplification of the entire mtDNA and subsequent sequence determination.
Results
Eight families exhibited extremely low penetrance of visual impairment, with the average of 13%. In particular, 14 (12 males and 2 females) of 119 matrilineal relatives in these families exhibited the variable severity and age at onset in visual dysfunction. The average age of onset of vision loss was 17 years. Molecular analysis of mtDNA identified the homoplasimic ND4 G11778A mutation and distinct sets of variants belonging to the Asian haplogroups M8a2, D4g2, B4a1c, B5b, N9a1, D4b2b, C, and M7b1. However, there was an absence of secondary LHON-associated mtDNA mutations in these 8 Chinese families.
Conclusions
The extremely low penetrance of vision loss in these 8 Chinese pedigrees strongly indicates that the G11778A mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the G11778A mutation in those Chinese families with very low penentrace of vision loss. However, nuclear backgrounds and environmental factors seem to be modifying factors for the phenotypic manifestation of the G11778A mutation in these Chinese families.
Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder leading to the rapid, painless, and bilateral loss of central vision.1–4 Mutations in mitochondrial DNA (mtDNA) are the molecular bases for this disorder.5–7 Of these, the ND1 G3460A, ND4 G11778A, and ND6 T14484C mutations, which involve genes encoding the subunits of respiratory chain complex I, account for more than 50% of LHON pedigrees worldwide.8–10 Those LHON-associated mtDNA mutations, unlike other pathogenic mtDNA mutations such as the Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS)-associated tRNALeu(UUR) A3243G mutation present in heteroplasmy (mixture of mutated and wild-type molecules),11 often occur nearly homoplasmy or homoplasmy. Such mtDNA mutations as the G11778A mutation often exhibit incomplete penetrance because some individuals carrying the mutations have normal vision.4,8,10,12 In addition, matrilineal relatives of intrafamilies or interfamilies, despite carrying the same LHON-associated mtDNA mutations, exhibited a wide range of severity, age at onset, and penetrance in visual impairment. Thus, other modifying factors, including environmental factors and nuclear and mitochondrial genetic modifiers, should modulate the phenotypic manifestation of visual impairment associated with those primary mtDNA mutations.3,4,13 In particular, a group of secondary LHON-associated mtDNA mutations such as T4216C, A4917G, G13708A, and haplogroup J were implicated in influencing the phenotypic manifestation of the primary mtDNA mutations, including the G11778A and T14484C mutations.14–17
However, these modifier factors remain poorly defined. With the aim of investigating the role of mitochondrial modifiers in the phenotypic expression of LHON, a systematic and extended mutational screening of mtDNA was been initiated in the large cohort at the Ophthalmology Clinic at the Wenzhou Medical College, Wenzhou, China.12,18–21 In a previous investigation, the authors showed that the ND4 G11696A, tRNAMet A4435G, and tRNAThr A15951G mutations contribute to the high penetrance and expressivity of vision loss in Chinese families.19–21 The present study undertook the clinical, genetic, and molecular characterization of another 8 Chinese families with maternally transmitted LHON. Fourteen (12 males and 2 females) of 119 matrilineal relatives in these families exhibited the variable severity and age at onset in visual dysfunction. Mutational analysis identified the ND4 G1177A mutation in these Chinese families. To elucidate the role of mitochondrial haplotype in the phenotypic manifestation of the G11778A mutation, polymerase chain reaction (PCR) amplification of the fragments spanning the entire mitochondrial genome and subsequent DNA sequence analysis in the matrilineal relatives of these families was performed.
Patients and Methods
Patients
Eight Han Chinese families (Fig 1) were identified through the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, and Dongfang Hospital, Beijing. Informed consent, blood samples, and clinical evaluations were obtained from all participating family members, under protocols approved by the Cincinnati Children’s Hospital Medical Center Institutional Review Board and the Wenzhou Medical College Ethics Committee. Members of these pedigrees were interviewed at length to identify both personal or family medical histories of visual impairments, and other clinical abnormalities.
Figure 1.
Eight Chinese pedigrees with Leber’s hereditary optic neuropathy. Visually impaired individuals are indicated by filled symbols. Arrows denote the probands.
Ophthalmologic Examinations
The ophthalmologic examinations of proband and other members of this family were conducted, including visual acuity, visual field examination (Humphrey Visual Field Analyzer IIi, Swedish Interactive Thresholding Algorithm [SITA] Standard; Humphrey Instruments, San Leadro, CA), visual evoked potentials (VEP) (Roland Consult RETI port gamma, flash VEP; Wiesbaden, Germany), and fundus photography (Canon CR6-45NM fundus camera; Tokyo, Japan). The degree of visual impairment was defined according to the visual acuity as follows: normal, >0.3; mild, 0.3 to 0.1; moderate, <0.1 to 0.05; severe, <0.05 to 0.02; and profound, <0.02.
Mutational Analysis of the Mitochondrial Genome
Genomic DNA was isolated from whole blood of participants using the Puregene DNA Isolation Kits (Gentra Systems). For the examination of the ND4 G11778A mutation, the first PCR segments (803 bp) were amplified using genomic DNA as template and oligodeoxynucleotides corresponding to mtDNA at positions 11295 through 1209822 to rule out the coamplification of possible nuclear pseudogenes.23 Then, the second PCR product (212 bp) was amplified using the first PCR fragment as template and oligodeoxynucleotides corresponding to mtDNA at positions 11654 through 11865 and subsequently was digested with restriction enzymes Tsp45I, because the G11778A mutation creates the site for this restriction enzyme.12 Equal amounts of various digested samples then were analyzed by electrophoresis through 7% polyacrylamide gel. The proportions of digested and undigested PCR product were determined by the Image-Quant program after ethidium bromide staining to determine if the G11778A mutation is in the homoplasmy in these subjects. The entire mitochondrial genome of 8 probands was PCR amplified in 24 overlapping fragments using sets of the light strand and the heavy strand oligonucleotide primers as described previously.24 Each fragment was purified and subsequently was analyzed by direct sequencing in an ABI 3700 automated DNA sequencer using the Big Dye Terminator Cycle sequencing reaction kit. These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_001807).22 DNA and protein sequence alignments were carried out using seqweb program GAP (GCG).
Results
Screening of ND4 G11778A Mutation in Chinese Subjects with Leber’s Hereditary Optic Neuropathy
To elucidate further the molecular basis of vision loss, a mutational screening of the mitochondrial ND4 gene was performed in a cohort of Han Chinese subjects who were diagnosed with LHON by the Eye Clinic at the Wenzhou Medical College. First, DNA fragments spanning the ND4 G11778A mutation were PCR amplified from each affected subject. Each fragment was digested by a restriction enzyme Tsp45I and subsequent electrophoresis analysis. Of those, 8 subjects harbored the homoplasmic G11778A mutation (data not shown). The presence of the homoplasmic G11778A mutation in those subjects was confirmed by PCR amplification of fragments spanning the G11778A mutation and subsequent DNA sequence analysis (data not shown).
Clinical and Genetic Evaluation of 8 Chinese Pedigrees
A comprehensive history and physical examination as well as ophthalmologic examination were performed to identify both personal and family medical histories of visual impairments and other clinical abnormalities in all available members of 8 Han Chinese pedigrees carrying the G11778A mutation. In fact, comprehensive family medical histories of those probands and other available members of these Chinese families showed no other clinical abnormalities, including diabetes, muscular diseases, hearing dysfunction, and neurologic disorders.
In family WZ41, the proband (III-2), as shown in Table 1, came to the Ophthalmology Clinic of Wenzhou Medical College at the age of 18 years after experiencing painless, progressive deterioration of bilateral visual impairment for 2 weeks. He saw a dark cloud in the center of his vision and had problems appreciating colors, which all seemed a dark gray. His visual acuity was 0.06 and 0.08 in his right and left eyes, respectively. Visual field testing demonstrated large centrocaecal scotomata in both of his eyes. Fundus examination showed that both his optic discs were abnormal: vascular tortuosity of the central retinal vessels, a circumpapillary telangiectatic microangiopathy, and swelling of the retinal nerve fiber layer. Therefore, he exhibited typical clinical features of LHON. However, none of other 6 matrilineal relatives in this family had visual loss.
Table 1.
Summary of Clinical Data for 14 Subjects of 8 Chinese Pedigrees Carrying the G11778A Mutation
| Subject | Gender | Age at Test (yrs) | Age at Onset (yrs) | Visual Acuity |
Level of Visual Impairment | |
|---|---|---|---|---|---|---|
| Right Eye | Left Eye | |||||
| WZ41-III-2 | M | 18 | 18 | 0.06 | 0.08 | Moderate |
| WZ42-III-11 | M | 18 | 14 | 0.044 | 0.06 | Severe |
| WZ42-III-14 | M | 17 | 15 | 0.03 | 1.5 | Mild |
| WZ43-III-5 | M | 16 | 15 | 0.02 | 0.02 | Profound |
| WZ44-III-1 | M | 20 | 15 | 0.02 | 0.06 | Severe |
| WZ45-III-1 | M | 31 | 15 | <0.01 | <0.01 | Profound |
| WZ45-III-2 | M | 28 | 17 | 0.03 | 0.5 | Severe |
| WZ46-II-2 | F | 40 | 20 | 0.02 | 0.02 | Severe |
| WZ46-III-7 | M | 17 | 16 | <0.01 | <0.01 | Profound |
| WZ46-III-9 | M | 7 | 7 | 0.4 | 0.25 | Mild |
| WZ47-II-1 | M | 58 | 30 | 0.1 | 0.1 | Mild |
| WZ47-II-3 | M | 52 | 36 | <0.01 | <0.01 | Profound |
| WZ47-III-9 | F | 18 | 15 | 0.1 | 0.1 | Mild |
| WZ48-III-10 | M | 22 | 16 | 0.01 | 0.05 | Severe |
F = female; M = male.
In family WZ42, the proband (III-11) was diagnosed with LHON by the Ophthalmology Clinic at the Dongfang Hospital at the age of 18 years. He began experiencing bilateral visual impairment at the age of 14 years. His visual acuity was 0.04 and 0.06 in the right and left eyes, respectively. Intraocular pressure (IOP) was 18.3 and 14.7 mmHg in the right and left eyes, respectively. Of other 24 matrilineal relatives in this pedigree, subject III-14 exhibited visual impairment at the age of 15 years. His visual acuity was 0.03 and 1.5 in his right and left eyes, respectively. Other members of this family exhibited normal vision.
In family WZ43, the proband (III-5) was a 16-year-old boy. He exhibited bilateral vision loss at the age of 15 years. He was diagnosed with LHON by the Ophthalmology Clinic at the Wenzhou Medical College. His visual acuity was 0.02 and 0.02 in the right and left eyes, respectively. His IOP was 16.3 mmHg in the right eye and 15.4 mmHg in the left eye. None of other 8 matrilineal relatives in this pedigree exhibited vision failure.
In family WZ44, the proband (III-1) reported painless, progressive deterioration of bilateral visual impairment at the age of 15 years and came to the Eye Clinic at the Dongfang Hospital at the age of 21 years. His visual acuity was 0.02 and 0.6 in the right and left eyes, respectively. The IOP was 16 mmHg in the right eye and 13 mmHg in the left eye. Ophthalmologic and other clinical evaluations showed that he had a typical clinical feature of LHON. However, the other 16 matrilineal relatives in this pedigree had normal vision.
In family WZ45, the proband (III-1) came to Ophthalmology Clinic at the Wenzhou Medical College at the age of 31 years. He experienced painless, progressive deterioration of bilateral vision loss at the age of 15 years. His visual acuity was <0.01 at both eyes. His sibling (III-2) lost central vision at the age of 17 years. His visual acuity was 0.03 and 0.05 in the right and left eyes, respectively. Both subjects demonstrated typical clinical features of LHON. None of other 10 matrilineal relatives in this family had vision deficit.
In family WZ46, the proband (III-9) came to Ophthalmology Clinic at the Dongfang Hospital at the age of 7 years after experiencing painless, progressive, deterioration of bilateral visual impairment 1 month previously. His visual acuity was 0.4 and 0.25 in the right and left eyes, respectively. Of other matrilineal relatives, subjects II-2 and III-7 exhibited clinical features of LHON at the age of 20 and 16 years, respectively. Visual acuity of II-2 and III-7 was 0.02 and <0.01 in both eyes, respectively. The other 16 matrilineal relatives in this pedigree had normal vision.
In family WZ47, the proband (III-9) began experiencing painless, progressive deterioration of bilateral visual impairment at the age of 15 years and came to the Ophthalmology Clinic at the Dongfang Hospital at the age of 18 years. Her visual impairment occurred within 3 years, first in the right eye, and then in left eye. She saw a dark cloud in the center of her vision and had problems appreciating colors, which all seemed dark gray. Her visual acuity was 0.1 in both eyes. The IOP was 15 and 18 mmHg in the right and left eyes, respectively. Of other matrilineal relatives, subjects II-1 and II-3 experienced loss of vision at the age of 30 and 36 years, respectively. Visual acuity of II-1 and II-3 was 0.1 and <0.01 in both eyes, respectively. Eight other matrilineal relatives in this pedigree had normal vision.
In family WZ48, the proband (III-10) came to eye clinic at the age of 22 years after experiencing painless, progressive deterioration of bilateral visual impairment 5 years previously. His visual acuity was 0.01 and 0.05 in the right and left eyes, respectively. The IOP was 19 mmHg in the right eye and 17 mmHg in the left eye. Ophthalmologic clinical evaluations showed that he had typical clinical features of LHON. However, none of other 18 matrilineal relatives in this family had vision loss.
Mitochondrial DNA Analysis
To assess the role of mtDNA variants in the phenotypic expression of the G11778A mutation, PCR amplification was performed of fragments spanning entire mitochondrial genome as well as subsequent DNA sequence analysis in 8 probands. In addition to the identical G11778A mutation, as shown in Table 2 (available at http://aaojournal.org), these subjects exhibited distinct sets of mtDNA polymorphism. Of other nucleotide changes in these mitochondrial genomes, there are 48 (1 novel and 47 known) variants in the D-loop, 6 known variants in the 12S rRNA gene, 4 known variants in the 16S rRNA gene, the previous identified C02/tRNALys intergenic 9-bp deletion corresponding to mtDNA at positions 8271 through 8279,25 the T4386C mutation in the tRNAGln gene, the G5821A mutation in the tRNACys gene, the A12172G mutation in the tRNAHis gene, the G15927G mutation in the tRNAThr gene,26 53 known and 6 novel silent variants in the protein encoding genes, as well as 25 missense mutations (4 novel and 21 known) in the protein encoding genes.27 These missense mutations are the G4048A (D248N) in the ND1 gene; the G4959A (A164P), C5178A (L237M), and G5460A (A331T) in the ND2 gene; the G7853A (V190A) and G8027A (A148T) in the C02 gene; the C8414T (L17F) in the A8 gene; the G8584A (A20T), C8684T (T53I), A8701G (T59A), A8860G (T112A), and AG9073G (T183A) in A6 gene; the T10084C (I8T) and A10398G (T114A) in the ND3 gene; the A12358G (T8A), A12361G (T9A), T12811C (Y159H), and T13866C (L517P) in the ND5 gene; the T14318C (N119S) in the ND6 gene; and the C14766T (T7I), A14978G (I78V), A15326G (T194A), A15662G (I306V), and A15671G (M309V) in the Cytb gene. These variants in RNAs and polypeptides were evaluated further by phylogenetic analysis of these variants and sequences from other organisms including mouse,28 bovine,29 and Xenopus laevis.30 Of these, the Y159H in ND5 variant located at a highly conserved residue, whereas the G15927A variant located at a highly conserved nucleotide (G28) in the tRNAThr.26 However, none of other variants showed evolutionary conservation.
Table 2.
mtDNA Variants in Eight Chinese Pedigrees with Leber’s hereditary Optic Neuropathy
| Gene | Position | Replacement | Conservation (H/B/M/X)a | CRSb | WZ41 | WZ42 | WZ43 | WZ44 | WZ45 | WZ46 | WZ47 | WZ48 | Previously Reportedc |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D-loop | 73 | A to G | A | G | G | G | G | G | G | G | G | Yes | |
| 94 | G to A | G | A | Yes | |||||||||
| 103 | G to A | G | A | Yes | |||||||||
| 131 | T to C | T | C | Yes | |||||||||
| 146 | T to C | T | C | C | Yes | ||||||||
| 150 | C to T | C | T | T | Yes | ||||||||
| 194 | C to T | C | T | Yes | |||||||||
| 199 | T to C | T | C | Yes | |||||||||
| 228 | G to T | G | T | Yes | |||||||||
| 249 | Del A | A | Del A | Yes | |||||||||
| 263 | A to G | A | G | G | G | G | G | G | G | G | Yes | ||
| 298 | C to T | C | T | Yes | |||||||||
| 309 | C to CC | C | CC | CC | Yes | ||||||||
| 310 | T to CTC | T | TC | TC | CTC | CTC | CTC | TC | CTC | CTC | Yes | ||
| 481 | C to T | C | T | No | |||||||||
| 489 | T to C | T | C | C | C | C | C | Yes | |||||
| 514 | Del C | C | Del C | Del C | Del C | Yes | |||||||
| 515 | Del A | A | Del A | Del A | Del A | Yes | |||||||
| 573 | C to CCC | C | CCC | Yes | |||||||||
| 16093 | T to C | T | C | C | Yes | ||||||||
| 16111 | G to A | G | A | Yes | |||||||||
| 16129 | G to A | G | A | A | Yes | ||||||||
| 16140 | T to C | T | C | Yes | |||||||||
| 16167 | C to T | C | T | Yes | |||||||||
| 16172 | T to C | T | C | Yes | |||||||||
| 16182 | A to C | A | C | Yes | |||||||||
| 16183 | A to C | A | C | C | Yes | ||||||||
| 16184 | C to T | C | T | Yes | |||||||||
| 16189 | T to C | T | C | C | C | Yes | |||||||
| 16192 | C to T | C | T | Yes | |||||||||
| 16217 | T to C | T | C | Yes | |||||||||
| 16223 | C to T | C | T | T | T | T | T | Yes | |||||
| 16234 | C to T | C | T | Yes | |||||||||
| 16243 | T to C | T | C | Yes | |||||||||
| 16257 | C to A | C | A | Yes | |||||||||
| 16261 | T to C | T | C | Yes | |||||||||
| 16261 | C to T | C | T | Yes | |||||||||
| 16271 | T to C | T | C | Yes | |||||||||
| 16289 | T to C | T | C | Yes | |||||||||
| 16293 | A to C | A | C | Yes | |||||||||
| 16297 | T to C | T | C | Yes | |||||||||
| 16298 | T to C | T | C | C | Yes | ||||||||
| 16319 | G to A | G | A | Yes | |||||||||
| 16327 | C to T | C | T | Yes | |||||||||
| 16335 | A to G | A | G | Yes | |||||||||
| 16362 | T to C | T | C | C | Yes | ||||||||
| 16463 | A to G | A | G | Yes | |||||||||
| 16519 | T to C | T | C | C | C | C | C | Yes | |||||
| 12S rRNA | 709 | G to A | G/A/A/- | G | A | A | A | Yes | |||||
| 722 | C to T | C/T/A- | C | T | Yes | ||||||||
| 750 | A to G | A/A/A/- | A | G | G | G | G | G | G | G | G | Yes | |
| 1382 | A to C | A/A/A/G | A | C | Yes | ||||||||
| 1438 | A to G | A/A/A/G | A | G | G | G | G | G | G | G | G | Yes | |
| 1598 | G to A | G/A/T/T | G | A | Yes | ||||||||
| 16S rRNA | 1719 | G to A | G/G/T/C | G | A | Yes | |||||||
| 2706 | A to G | A/G/A/A | A | G | G | G | G | G | G | G | G | Yes | |
| 2835 | C to T | C/A/A/A | C | T | Yes | ||||||||
| 3010 | G to A | G/G/A/A | G | A | A | Yes | |||||||
| ND1 | 3552 | T to A | T | A | Yes | ||||||||
| 4048 | G to A (Asp to Asn) | D/N/Y/F | G | A | Yes | ||||||||
| 4071 | C to T | C | T | Yes | |||||||||
| 4161 | C to T | C | T | No | |||||||||
| 4164 | A to G | A | G | Yes | |||||||||
| tRNAGln | 4386 | T to C | T/T/A/C | T | C | Yes | |||||||
| ND2 | 4715 | A to G | A | G | G | Yes | |||||||
| 4769 | A to G | A | G | G | G | G | G | G | G | G | Yes | ||
| 4883 | C to T | C | T | Yes | |||||||||
| 4895 | A to G | A | G | Yes | |||||||||
| 4959 | G to A (Ala to Pro) | A/I/M/I | G | A | Yes | ||||||||
| 5178 | C to A (Leu to Met) | L/T/T/T | C | A | A | Yes | |||||||
| 5231 | G to A | G | A | A | Yes | ||||||||
| 5351 | A to G | A | G | Yes | |||||||||
| 5417 | G to A | G | A | Yes | |||||||||
| 5460 | G to A (Ala to Thr) | A/V/I/I | G | A | Yes | ||||||||
| 5465 | T to C | T | C | Yes | |||||||||
| tRNACys | 5821 | G to A | G/A/A/A | G | A | Yes | |||||||
| CO1 | 6053 | C to T | C | T | No | ||||||||
| 6179 | G to A | G | A | Yes | |||||||||
| 6338 | A to G | A | G | Yes | |||||||||
| 6357 | C to T | C | T | Yes | |||||||||
| 6455 | C to T | C | T | Yes | |||||||||
| 6680 | T to C | T | C | Yes | |||||||||
| 7028 | C to T | C | T | T | T | T | T | T | T | T | Yes | ||
| 7043 | C to T | C | T | Yes | |||||||||
| 7052 | A to G | A | G | Yes | |||||||||
| 7196 | C to A | C | A | A | Yes | ||||||||
| 7271 | A to G | A | G | Yes | |||||||||
| CO2 | 7684 | T to C | T | C | Yes | ||||||||
| 7853 | G to A (Val to Ala) | V/I/I/V | G | A | A | Yes | |||||||
| 8020 | G to A | G | A | Yes | |||||||||
| 8027 | G to A (Ala148Thr) | A/M/L/S | G | A | Yes | ||||||||
| NC7 | 8271_9 | 9bpDel | 9bp | 9bpDel | 9bpDel | Yes | |||||||
| ATP8 | 8414 | C to T (Leu to Phe) | L/F/M/W | C | T | T | Yes | ||||||
| ATP6 | 8584 | G to A (Ala to Thr) | A/V/V/I | G | A | A | A | Yes | |||||
| 8684 | C to T (Thr to Ile) | T/V/I/F | C | T | No | ||||||||
| 8701 | A to G (Thr to Ala) | T/S/L/Q | A | G | G | G | G | G | Yes | ||||
| 8829 | C to T | C | T | Yes | |||||||||
| 8860 | A to G (Thr to Ala) | T/A/A/T | A | G | G | G | G | G | G | G | G | Yes | |
| 8964 | C to T | C | T | Yes | |||||||||
| 9073 | A to G (Thr to Ala) | T/S/N/S | A | G | No | ||||||||
| 9123 | G to A | G | A | Yes | |||||||||
| CO3 | 9296 | C to T | C | T | Yes | ||||||||
| 9540 | T to C | T | C | C | C | C | C | Yes | |||||
| 9545 | A to G | A | G | Yes | |||||||||
| 9824 | T to C | T | C | C | Yes | ||||||||
| 9950 | T to C | T | C | Yes | |||||||||
| ND3 | 10084 | T to C (Ile to Thr) | I/T/I/I | T | C | Yes | |||||||
| 10238 | T to C | T | C | Yes | |||||||||
| 10398 | A to G (Thr to Ala) | T/T/T/A | A | G | G | G | G | G | G | Yes | |||
| 10400 | C to T | C | T | T | T | T | T | Yes | |||||
| ND4 | 10873 | T to C | T | C | C | C | C | C | Yes | ||||
| 11101 | A to G | A | G | No | |||||||||
| 11296 | C to T | C | T | Yes | |||||||||
| 11719 | G to A | G | A | A | A | A | A | A | A | A | Yes | ||
| 11778 | G to A (Arg to His) | R/R/R/R | G | A | A | A | A | A | A | A | A | Yes | |
| 11914 | G to A | G | A | Yes | |||||||||
| 11935 | T to C | T | C | Yes | |||||||||
| 12007 | G to A | G | A | Yes | |||||||||
| tRNAHis | 12172 | A to G | A/A/A/T | A | G | Yes | |||||||
| ND5 | 12358 | A to G (Thr to Ala) | T/S/I/M | A | G | G | Yes | ||||||
| 12361 | A to G (Thr to Ala) | T/L/L/L | A | G | Yes | ||||||||
| 12372 | G to A | G | A | Yes | |||||||||
| 12405 | C to T | C | T | Yes | |||||||||
| 12705 | C to T | C | T | T | T | T | T | T | Yes | ||||
| 12811 | T to C (Tyr to His) | Y/Y/Y/Y | T | C | Yes | ||||||||
| 12957 | T to C | T | C | Yes | |||||||||
| 12972 | A to G | A | G | No | |||||||||
| 13104 | A to G | A | G | Yes | |||||||||
| 13263 | A to G | A | G | G | Yes | ||||||||
| 13886 | T to C (Leu to Pro) | L/S/N/T | T | C | No | ||||||||
| ND6 | 14318 | T to C (Asn to Ser) | N/N/D/S | T | C | Yes | |||||||
| 14383 | C to T | C | T | No | |||||||||
| 14470 | T to C | C | T | Yes | |||||||||
| 14668 | C to T | C | T | T | Yes | ||||||||
| Cytb | 14766 | C to T (Thr to Ile) | T/S/T/S | C | T | T | T | T | T | T | T | T | Yes |
| 14783 | T to C | T | C | C | C | C | C | Yes | |||||
| 14978 | A to G (Ile to Val) | I/I/L/L | A | G | Yes | ||||||||
| 15043 | G to A | G | A | A | A | A | A | Yes | |||||
| 15223 | C to T | C | T | No | |||||||||
| 15301 | G to A | G | A | A | A | A | A | Yes | |||||
| 15326 | A to G (Thr to Ala) | T/M/I/I | A | G | G | G | G | G | G | G | G | Yes | |
| 15487 | A to T | A | T | T | Yes | ||||||||
| 15508 | C to T | C | T | Yes | |||||||||
| 15661 | C to T | C | T | Yes | |||||||||
| 15662 | A to G (Ile to Val) | I/L/F/L | A | G | Yes | ||||||||
| 15671 | A to G (Met to Val) | M/T/T/T | A | G | No | ||||||||
| tRNAThr | 15927 | G to A | G/G/G/G | G | A | Yes |
Conservation of amino acid for polypeptides or nucleotide for RNAs in human (H), bovine (B), mouse (M), and Xenopus laevis (X).
CRS, Cambridge reference sequence (Andrews et al., 1999).
See online mitochondrial genome database http://www.mitomap.org.
Discussion
In the present study, clinical, genetic, and molecular characterization was performed of 8 Han Chinese families with LHON. Visual impairment as a sole clinical phenotype was present only in the maternal lineage of these pedigrees carrying the homoplasmic ND4 G11778A mutation. Clinical and genetic evaluations revealed the variable severity and age at onset in visual impairment in these matrilineal relatives, although these subjects share some common features, specifically, the rapid, painless, bilateral loss of central vision. The average age at onset for visual impairment in matrilineal relatives in these families, as shown in Table 3, varied from 14 years (WZ46) to 27 years (WZ47), with an average of 17 years. As shown in Table 3, in 7 matrilineal relatives in another Chinese family, optic neuropathy developed at the average age of 14, 17, 18, 18, 19, 20, and 22 years, respectively,12,18–21 whereas the average ages at onset of visual loss were 24 and 28 years from matrilineal relatives of 66 and 49 white pedigrees carrying the G11778A mutation, respectively.31,32 Thus, matrilineal relatives in these families seemed to have an earlier age at onset than those in other families carrying the G11778A mutation. Furthermore, only 2 of 14 affected matrilineal relatives were female among these 8 families. As shown in Table 3, these Chinese families exhibited extremely low penetrance of visual impairment (affected matrilineal relatives/total matrilineal relatives), ranging from 5.3% to 27.3%, with the average of 13%, whereas the penetrance of visual impairment in another 7 Chinese pedigrees carrying the G11778A mutation were 16%, 33%, 33%, 36%, 36%, 57%, and 60%, respectively, with an average of 39%.12,18–21 However, in approximately 50% of white males and approximately 10% of white females carrying one of the following LHON-associated mutations, G3460A, G11778A, or T14484C, optic neuropathy indeed developed.2,33
Table 3.
Summary of Clinical and Molecular Data for 15 Chinese Families Carrying the ND4 G11778A Mutation
| Pedigree | Ratio of Affected Males to Affected Females | Average Age at Onset (yrs) | No. of Matrilineal Relatives | Penetrance (%)* | Secondary Mutations | Mitochondrial DNA Haplogroup |
|---|---|---|---|---|---|---|
| WZ41 | 1:0 | 18 | 7 | 14.3 | M8a2 | |
| WZ42 | 2:0 | 14.5 | 25 | 8 | D4g2 | |
| WZ43 | 1:0 | 15 | 9 | 11.1 | B4a1c | |
| WZ44 | 1:0 | 15 | 17 | 5.9 | B5b | |
| WZ45 | 2:0 | 16 | 12 | 16.7 | N9a1 | |
| WZ46 | 2:1 | 14 | 19 | 15.8 | D4b2b | |
| WZ47 | 2:1 | 27 | 11 | 27.3 | C | |
| WZ48 | 1:0 | 16 | 19 | 5.3 | M7b1 | |
| WZ112 | 6:0 | 22 | 38 | 15.8 | B5 | |
| WZ219 | 3:1 | 14 | 14 | 57.1 | A4435G | D5 |
| WZ320 | 2:1 | 19 | 10 | 60 | A15951G | D4 |
| WZ418 | 3:0 | 17 | 9 | 33.3 | F1 | |
| WZ5 | 4:1 | 20 | 14 | 35.7 | D4b2b | |
| WZ6 | 1.2:1 | 18 | 31 | 35.5 | M10a | |
| WZ1221 | 3.5:1 | 18 | 30 | 33 | G11696A | D4 |
The extremely low penetrance of vision loss in these 8 Chinese pedigrees strongly indicated that the G11778A mutation was itself insufficient to produce a clinical phenotype. Nuclear backgrounds, environmental factors, and mitochondrial haplotypes may contribute to various penetrance and expressivities of visual impairment among families carrying the G11778A mutation. The phenotypic variability of matrilineal relatives from within and among families, including the variable severity of visual impairment, suggests a role of nuclear backgrounds in the phenotypic manifestation of the G11778A mutation as described other pedigrees.3,18,31 Furthermore, it is possible that environmental factors also may contribute to the phenotypic variability of visual loss in matrilineal relatives of these families. In fact, the mitochondrial variants and haplotypes have been shown to influence the penetrance and expressivity of visual loss associated with primary mtDNA mutations. In particular, secondary LHON mutations at positions 4216 and 13708 may increase the penetrance and expressivity of LHON associated with primary LHON mutations, including G11778A and T14484C.15,16,34 Furthermore, the G7444A mutation in the C01 and tRNASer(UCN) genes also has been implicated in influencing the penetrance and phenotypic expression of visual loss associated with the primary LHON mutations.6 In addition, the ND4 G11696A, tRNAMet A4435G, and tRNAThr A15951G mutations may increase the penetrance and expressivity of vision loss in Chinese families.19–21
These Chinese pedigrees showed distinct sets of sequence variations in their mitochondrial genomes. As shown in Table 3, their mtDNAs belong to Eastern Asian haplogroups M8a2, D4g2, B4a1c, B5b, N9a1, D4b2b, C, and M7b1,35 whereas the other 7 Chinese pedigrees carrying the G11778A mutation belong to haplogroups D4, B5, D5, D4, F1, D4b2b, and M10a.12,18–21 This suggested that the G11778A mutation, similar to the G111778A mutation in white and Japanese families,8,10,15,17 occurred sporadically and multiplied through evolution of the mtDNA in China. Of these mtDNA variants, only the ND5 Y159H variant located at a highly conserved residue and the G15927A variant located at a highly conserved nucleotide (G28) in the tRNAThr.26 However, these 8 Chinese families demonstrated an absence of secondary LHON mutations. By contrast, 3 Chinese pedigrees with higher penentrance of vision loss harbored functionally significant mtDNA variants: the ND4 G11696A, tRNAMet A4435G, and tRNAThr A15951G.19–21 The presences of these mtDNA variants in 3 Chinese pedigrees,19–21 but the absences of these mtDNA mutations in these 8 Chinese families, seem to account for different penetrances among these pedigrees. These data suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the G11778A mutation in those Chinese families with very low penentrace of vision loss.
Acknowledgments
Supported by the National Institutes of Health, Bethesda, Maryland (grant nos.: R01DC05230 and R01DC07696 [M.X.G.]); the National Science Foundation of China, Beijing, China (Chinese Young Scholar Award no.: 30628013); the Zhejiang Provincial Natural Science Foundation, Hangzhou, Zhejiang, China (grant nos.: Z204492 [M.X.G.] and ZB0202); and Zhejiang Province, Hangzhou, Zhejiang, China (Key Research and Development Program project grant no. 2004C14005 [J.Q.]).
Footnotes
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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