February 2006
Volume 47, Issue 2
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Biochemistry and Molecular Biology  |   February 2006
The Novel A4435G Mutation in the Mitochondrial tRNAMet May Modulate the Phenotypic Expression of the LHON-Associated ND4 G11778A Mutation
Author Affiliations
  • Jia Qu
    From the School of Ophthalmology and Optometry, and the
    Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, China; the Divisions of
    Contributed equally to the work and therefore should be considered equivalent authors.
  • Ronghua Li
    Human Genetics,
    Contributed equally to the work and therefore should be considered equivalent authors.
  • Xiangtian Zhou
    From the School of Ophthalmology and Optometry, and the
  • Yi Tong
    From the School of Ophthalmology and Optometry, and the
    The First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China; and the
  • Fan Lu
    From the School of Ophthalmology and Optometry, and the
  • Yaping Qian
    Human Genetics,
  • Yongwu Hu
    Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, China; the Divisions of
  • Jun Qin Mo
    Pathology, and
    Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio.
  • Constance E. West
    Ophthalmology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio;
    Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio.
  • Min-Xin Guan
    Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, China; the Divisions of
    Human Genetics,
    Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio.
Investigative Ophthalmology & Visual Science February 2006, Vol.47, 475-483. doi:10.1167/iovs.05-0665
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      Jia Qu, Ronghua Li, Xiangtian Zhou, Yi Tong, Fan Lu, Yaping Qian, Yongwu Hu, Jun Qin Mo, Constance E. West, Min-Xin Guan; The Novel A4435G Mutation in the Mitochondrial tRNAMet May Modulate the Phenotypic Expression of the LHON-Associated ND4 G11778A Mutation. Invest. Ophthalmol. Vis. Sci. 2006;47(2):475-483. doi: 10.1167/iovs.05-0665.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigating the role of mitochondrial haplotypes in the development of Leber’s hereditary optic neuropathy (LHON) associated with the ND4 G11778A mutation in Chinese families.

methods. A three-generation Chinese family with LHON was studied by clinical and genetic evaluation as well as molecular and biochemical analysis of mitochondrial (mt)DNA.

results. This family exhibits a high penetrance and expressivity of visual impairment. The average age at onset was 13.9 years in this family. Of the family members, 86% of the male and 29% of the female matrilineal relatives had visual loss, with a wide range of severity, from blindness to nearly normal vision. Molecular analysis of mtDNA identified the homoplasmic ND4 G11778A mutation and 35 other variants, belonging to the Asian haplogroup D5. Of other variants, the novel homoplasmic A4435G mutation absent in 164 Chinese controls is localized at 3′ end adjacent to the anticodon, at conventional position 37 (A37), of tRNAMet. The adenine (A37) at this position of tRNAMet is extraordinarily conserved from bacteria to human mitochondria. This modified A37 was shown to contribute to the high fidelity of codon recognition and to the structural formation and stabilization of functional tRNAs. In fact, the significant reduction of the steady state levels in tRNAMet was observed in cells carrying the both the A4435G and G11778A mutations but not cells carrying only the G11778A mutation. Thus, a failure in mitochondrial tRNA metabolism, caused by the A4435G mutation, may worsen the mitochondrial dysfunction associated with the primary G11778A mutation.

conclusions. The novel tRNAMet A4435G mutation has a potential modifier role in increasing the penetrance and expressivity of the primary LHON-associated G11778A mutation in this Chinese family.

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder leading to the rapid, painless, bilateral loss of central vision. 1 2 3 4 Since the landmark discovery of the ND4 G11778A mutation associated with LHON, 5 approximately 25 LHON-associated mitochondrial (mt)DNA (mtDNA) mutations have been identified in various ethnic populations. 6 7 Of these, the ND1 G3460A, ND4 G11778A, and ND6 T14484C mutations, which involve genes encoding the subunits of respiratory chain complex I, accounts for ∼80% to 95% of LHON pedigrees in different ethnic backgrounds. 8 9 10 In particular, the G11778A mutation has been shown to be responsible for ∼90% of total LHON cases in the Japanese population. 10 Those LHON-associated mtDNA mutations, unlike other pathogenic mtDNA mutations, such as the MELAS-associated tRNALeu(UUR) A3243G mutation, 11 often occur nearly or completely homoplasmically. Those mtDNA mutations, such as the G11778A mutation often exhibit incomplete penetrance, evidenced by the fact that some individuals carrying the mutations(s) have normal vision. 4 8 10 12 In addition, matrilineal intrafamily or interfamily relatives, despite carrying the same LHON-associated mtDNA mutation(s) exhibit the variable severity, age at onset, and progression in visual impairment. Thus, other modifier factors including environmental factors, nuclear modifier genes and mitochondrial haplotypes should modulate the phenotypic manifestation of visual impairment associated with those primary mtDNA mutations. 3 4 13 In particular, a group of so-called “secondary” LHON-associated mtDNA mutations, including T4216C, A4917G, and G13708A, 14 were implicated as acting in synergy with primary mtDNA mutations, including the G11778A and T14484C. 15 Furthermore, the mitochondrial haplogroup J can influence the phenotypic manifestation of the primary LHON G11778A and T14484C mutations in a very large cohort of families of European ancestry. 16 17  
However, these modifier factors remain poorly defined. With the purpose of investigating the role of mitochondrial haplotypes in the development of LHON, a systematic and extended mutational screening of mtDNA has been initiated in the large clinical population of Ophthalmology Clinic at the Wenzhou Medical College, China. 12 18 In the previous investigation, we showed that six affected matrilineal relatives were exclusively males in a large Chinese family. 12 Sequence analysis of mtDNA revealed the presence of the G11778A mutation, but other variants in mtDNA belonged to haplogroup B6b, which is unlikely to have a modifying role in the phenotypic manifestation of the G11778A mutation. 12 In the present study, we performed the clinical, genetic, and molecular characterization of another Chinese family with maternally transmitted LHON. Eight (six males/two female) of 14 matrilineal relatives in this three-generation family exhibited the variable severity and age at onset in visual dysfunction. Mutational analysis has led to identification of the ND4 G1177A mutation in this Chinese family. To elucidate the role of mitochondrial haplotype in the phenotypic manifestation of the G11778A mutation, we performed a PCR-amplification of the fragments spanning entire mitochondrial genome and subsequent DNA sequence analysis in the matrilineal relatives of this family. Of other mtDNA nucleotide changes, the novel A4435G mutation is localized at the 3′ end adjacent to the anticodon (position 37) of tRNAMet. The adenine at this position of tRNAMet is extraordinarily conserved from bacteria to human mitochondria. The functional significance of this A4435G mutation was evaluated by examining for the steady state levels of mitochondrial tRNAMet, and other tRNAs, including tRNALys, tRNALeu(UUR), and tRNAGln using lymphoblastoid cell lines derived from an affected matrilineal relative carrying both G11778A and A4435 mutations, from an affected Chinese subject carrying only the G11778A mutation, 18 and from a married-in control individual lacking those mtDNA mutations. 
Methods
Patients and Subjects
We ascertained a Chinese family (Fig. 1)through the School of Ophthalmology and Optometry, Wenzhou Medical College. 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 Institute Review Board and the Wenzhou Medical College ethics committee. Members of this pedigree were interviewed at length, to identify both personal and family medical histories of visual impairments and other clinical abnormalities. The 164 control DNA samples used for screening for the presence of mtDNA mutations were obtained from a panel of unaffected and unrelated subjects from the Chinese ancestry. 
Ophthalmic Examinations
The ophthalmic examinations of the proband and other members of this family were conducted, including visual acuity, visual field examination (Humphrey Visual Field Analyzer IIi, SITA Standard; Carl Zeiss Meditec, Oberkochen, Germany), visual evoked potentials (VEP; RETI port gamma, flash VEP; Roland Consult, Brandenberg, Germany), and fundus photography (CR6-45NM fundus camera; Canon, Lake Success, NY). The degree of visual impairment was defined according to 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 (Puregene DNA Isolation Kit; Gentra Systems, Minneapolis, MN). The presence of the G3460A, G11778A, and T14484C mutations were examined as detailed elsewhere. 8 Briefly, affected individuals’ DNA fragments spanning these mtDNA mutations were amplified by PCR using oligodeoxynucleotides corresponding to mtDNA at positions 3,108-3,717 for the G3460A mutation, 11,654-11,865 for the G11778A mutation, and 14,260-14,510 for the T14484C mutation. 19 For the detection of the G3460A mutation, the amplified PCR segments were digested with a restriction enzyme BsaHI, 8 whereas the presence of the T14484C mutation was examined by digesting PCR products with the restriction enzyme MvaI. 8 For the examination of the G11778A mutation, the first PCR segments (803 bp) were amplified using genomic DNA as the template and oligodeoxynucleotides corresponding to mtDNA at positions 11,295-12,098, to rule out the coamplification of possible nuclear pseudogenes. 20 Then, the second PCR product (212 bp) was amplified, using the first PCR fragment as the template and oligodeoxynucleotides corresponding to mtDNA at positions 11,654-11,865, and subsequently digested with the restriction enzyme Tsp45I as the G1178A mutation creates the site for this restriction enzyme. 12 For the quantification of the A4435G mutation, the PCR segments (700 bp) were amplified using genomic DNA as template and oligodeoxynucleotides corresponding to mtDNA at positions 3861-4560, and subsequently digested with the restriction enzyme NlaIII. In fact, the A4435G mutation creates a novel site for this enzyme. Equal amounts of various digested samples were then analyzed by electrophoresis through 7% polyacrylamide gel. The proportions of digested and undigested PCR product were determined by computer (Image-Quant; Molecular Dynamics, Sunnyvale, CA) program after ethidium bromide staining to determine whether the G11778A or A4435G mutation is homoplasmic in these subjects. 
The entire mitochondrial genome of an affected patient III-2 was PCR amplified in 24 overlapping fragments, by using sets of the light (L) strand and the heavy (H) strand oligonucleotide primers as described previously. 21 Each fragment was purified and subsequently analyzed by direct sequencing (model 3700 automated DNA sequencer using the Big Dye Terminator Cycle sequencing reaction kit; Applied Biosystems, Inc. [ABI], Foster City, CA). These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_001807). 19 DNA and protein sequence alignments were performed using the seqweb program GAP (GCG). 
Mitochondrial tRNA Analysis
Lymphoblastoid cell lines were immortalized by transformation with the Epstein-Barr virus, as described elsewhere. 22 Cell lines derived from one affected individual (III-2) carrying both the G11778A and A4435G mutations and married-in control subject (II-1) in this Chinese family and an affected individual (WZ4-IV-2) from the other Chinese family carrying only the G11778A mutation 18 were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS). Total mitochondrial RNA was obtained (Totally RNA kit; Ambion, Austin, TX) from mitochondria isolated from lymphoblastoid cell lines (∼4.0 × 108 cells), as described previously. 23 Five micrograms of total mitochondrial RNA was electrophoresed through a 10% polyacrylamide, 7-M urea gel in Tris-borate-EDTA buffer (TBE; after heating the sample at 65°C for 10 minutes), and then electroblotted onto a positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) for the hybridization analysis with oligodeoxynucleotide probes. For the detection of tRNAMet, tRNALeu(UUR), tRNALys, and tRNAGln, the following nonradioactive digoxigenin (DIG)-labeled oligodeoxynucleotides specific for each RNA were used: 5′-TAGTACGGGAAGGGTATAACC-3′ (tRNAMet); 5′-TCACTGTAAAGAGGTGTTGG-3′ (tRNALys); 5′-TGTTAAGAAGAGGAATTGAA-3′ (tRNALeu(UUR)); 5′-CTAGGACTATGAGAATCGAA-3′ (tRNAGln). 19 DIG-labeled oligodeoxynucleotides were generated by using the DIG oligonucleotide tailing kit (Roche Diagnostics). The hybridization was performed as detailed elsewhere. 24 Quantification of density in each band was made as detailed previously. 24 25  
Results
Clinical Presentation
The proband (III-2) came to the ophthalmology clinic of Wenzhou Medical College at the age of 18 years. He had begun to experience painless, progressive deterioration of bilateral visual impairment at the age of 12. His visual impairment occurred within 10 days, first in the right eye and then in the left. He saw a dark cloud in the center of vision and had problems appreciating colors that all seemed a dark gray. The ophthalmic evaluation showed that his visual acuity was 0.01 and 0.02 in his right and left eyes, respectively. Visual field testing demonstrated large cecocentral scotomata in both of his eyes. As showed in Figure 2 , a fundus examination showed that both his optic discs were abnormal, with vascular tortuosity of the central retinal vessels, a circumpapillary telangiectatic microangiopathy, and swelling of the retinal nerve fiber layer. Therefore, he exhibited a typical clinical feature of LHON. No other abnormality was found on radiologic and neurologic examination. Furthermore, he had no other significant medical history. 
The family originated from Anhui Province in Eastern China, and most of the family members lived in the same area. As shown in Figure 1 , this familial history is consistent with a maternal inheritance. None of the offspring of vision-impaired fathers has a visual impairment. Of 13 matrilineal relatives who are the offspring of subject I-2, six of seven male and two of six female matrilineal relatives exhibited the bilateral and symmetric visual impairment as the sole clinical symptom, whereas one male and four female matrilineal relatives had normal version. These affected matrilineal relatives of this family exhibited early-onset, progressive, but not congenital, visual impairment. As shown in Table 1 , visual acuity examination showed a variable severity of visual impairment in the maternal kindred, ranging from profound visual impairment (II-4, II-5, III-2), to severe visual impairment (II-6, II-10, III-3), to moderate visual impairment (II-8 and III-4), to completely normal vision (five matrilineal relatives). In addition, the age at onset of visual impairment in this family varied from 11 years to 16 years. There was no evidence that any member of this family had any other known cause to account for visual impairment. Comprehensive family medical histories of these individuals showed no other clinical abnormalities, including diabetes, muscular diseases, hearing impairment, or neurologic disorders. 
Mitochondrial DNA Analysis
The maternal transmission of visual dysfunction in this family suggested mitochondrial involvement and led us to analyze the mitochondrial genome of matrilineal relatives. First, we examined three commonly known LHON-associated mtDNA mutations (G3460A, G11778A, and T14484C) by PCR amplification and subsequent restriction enzyme digestion analysis of PCR fragments derived from four matrilineal relatives (proband III-2, his mother II-2, unaffected male III-10, and affected male II-13) and two unrelated Chinese control subjects. The results revealed the presence of the G11778A mutation, but the absence of the G3460A and T14484C mutations in those subjects. 
To determine further the presence and amount of the G11778A mutation in these matrilineal relatives, nested PCR amplification, as detailed in the Materials and Methods section, was performed to rule out the possible coamplification of nuclear pseudogenes. 20 The resultant 212-bp PCR segments corresponding to mtDNA at positions 11,654-11,865 were then digested by another restriction enzyme, Tsp45I, and separated by electrophoresis on a 7% polyacrylamide gel. There was no detectable wild-type DNA, indicating that the G11778A mutation appears to be homoplasmic in these matrilineal relatives of this Chinese family (data not shown). As expected, the G11778A mutation was absent in PCR products derived from married-in control members in the family. This strongly indicated that the levels of G11778A mutation in those matrilineal relatives did not correlate with the variability of visual dysfunction including the severity and age at onset of visual impairment. 
To determine the role of mitochondrial haplotypes in the phenotypic manifestation of the G11778A mutation, the DNA fragments spanning the entire mtDNA of an affected patient III-2 were PCR amplified. Each fragment was purified and subsequently analyzed by direct sequence. The comparison of the resultant sequences with the Cambridge consensus sequence 19 identified a number of nucleotide changes, as shown in Table 2 . All the nucleotide changes were verified in three additional matrilineal relatives of this family (proband III-2, his mother II-2, unaffected male III-10, and affected male II-13) by sequence analysis and appeared to be homoplasmic. Sequence analysis confirmed the presence of the G11778A mutation in matrilineal relatives of this family. 
Of other nucleotide changes, the novel A-to-G transition at position 4435 (A4435G) in the tRNAMet gene (Fig. 3A)is of special interest. The A4435G mutation, as shown in Figure 3C , is located immediately at the 3′ end of the anticodon, corresponding to the conventional position 37 of tRNAMet. 26 Phylogenetic analysis, as shown in Figure 4 , revealed that an adenine at this position is an extraordinarily conserved base in every sequenced methionine tRNA from bacteria to human mitochondria. 26 27 Of interest, the nucleotide at the position 37 is more prone to modification than those at other places of tRNA. 28 In fact, the nucleotide modification at this position has been shown to play a pivotal role in the stabilization of tertiary structure and the biochemical function of tRNA. 28 To determine whether the A4435G mutation is homoplasmic, the fragments spanning the tRNAMet gene were PCR amplified and subsequently digested with NlaIII. As shown in Figure 3B , there was no detectable wild-type DNA in 13 matrilineal relatives, indicating that the A4435G mutation was homoplasmic in these matrilineal relatives. In addition, this mutation was absent in 164 Chinese control subjects. 
Furthermore, nine polymorphisms in the D-loop region, 3 variants in the 12S rRNA gene, one variant in the 16S rRNA gene and 19 variants in protein-encoding genes were previously identified in the control population, 29 whereas the variants A12026G (isoleucine to valine) and A13266G (silent mutation) in ND5 gene appear to be novel polymorphisms. In addition to the G11778G mutation, as shown in Table 2 , seven amino acid substitutions caused by corresponding mtDNA variants occurred in different polypeptides in this matrilineal relative. These variants in rRNAs and polypeptides were further evaluated by phylogenetic analysis of these variants and sequences from other organisms including mouse, 30 bovine, 31 and Xenopus laevis. 32 However, none of the variants in the polypeptides was highly evolutionarily conserved or shown to have a significantly functional effect. 
Mitochondrial tRNA Analysis
To examine whether the A4435G mutation affects the stability of the tRNAMet, we determined the steady state level of the tRNAMet determined by isolating total mitochondrial RNA from lymphoblastoid cell lines, separating them on a 10% polyacrylamide, 7–M urea gel, electroblotting and hybridizing with a nonradioactive DIG-labeled oligodeoxynucleotide probe specific for tRNAMet. After the blots were stripped, the DIG-labeled probes, including tRNALeu(UUR) and tRNALys as representatives of the whole H-strand transcription unit and tRNAGln derived from the L-strand transcription unit, 25 were hybridized with the same blots for normalization purposes. 
As shown in Figure 5A , the amount of tRNAMet in the mutant cell line derived from proband III-2, who carried both the G11778A and A4435G mutations were markedly decreased, compared with those in the cell line derived from the married-in control (II-1) subject, who lacked those mtDNA mutations. For comparison, the average levels of tRNAMet, in various control or mutant cell lines were normalized to the average levels in the same cell line for the tRNALeu(UUR), tRNALys, and tRNAGln, respectively. As shown in Figure 5B , the average levels of tRNAmet in the mutant cell line derived from III-2 ranged between ∼43% of the control after normaliza-tion to tRNALys, ∼51% of the control after normalization to tRNALeu(UUR), and ∼68% of the control after normalization to tRNAGln. However, the levels of tRNAMet in one cell line derived from an affected Chinese individual (WZ4-IV-2) carrying the G11778A mutation 18 were comparable with those in the cell line derived from the married-in control subject (II-1). 
Discussion
In the present study, we undertook the clinical, genetic, and molecular characterization of a Chinese family with LHON. The visual impairment as a sole clinical phenotype was present only in the maternal lineage of this three-generation pedigree, suggesting that the mtDNA mutation(s) is the molecular basis for this disorder. In fact, the molecular analysis identified the homoplasmic G11778A mutation in the ND4 gene in matrilineal relatives of this Chinese family. 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: being the rapid, painless, bilateral loss of central vision. Strikingly, this Chinese family carrying the G11778A mutation exhibited high penetrance and occurrence of visual impairment. In particular, the average age at onset was 13.9 years in this family. However, as shown in Table 3 , matrilineal relatives in another four Chinese families experienced onset of optic neuropathy at the average of 17, 18, 20, and 22 years of age, 12 18 whereas the average age at onset for visual loss were 24 and 28 years old from matrilineal relatives of 66 and 49 white pedigrees carrying the G11778A mutation, respectively. 33 34 Thus, matrilineal relatives in this family had much earlier age at onset than those in other families carrying the G11778A mutation. The ratios between affected male and female matrilineal relatives were 3:0, 1:1, 1.2:1, and 6:0 in the other four Chinese families, 12 18 whereas the ratio (affected male to female) was 3:1 in the Chinese family in the present study. Indeed, this ratio is comparable with the ratios of 4.5:1 and 3.7:1 in two large cohorts of white pedigrees carrying the G11778A mutation. 33 34 In addition, 86% of the male and 29% of the female matrilineal relatives in this Chinese family exhibited development of visual loss, in contrast with the fact that only 33%, 36%, 36%, and 18% of matrilineal relatives showed visual loss in the other four Chinese families. 12 18 However, ∼50% of white males and ∼10% of white females carrying one LHON–associated mutation (G3460A, G11778A, or T14484C) had the optic neuropathy. 2 35  
The various degrees of penetrance and severity and age at onset of visual impairment between this Chinese family and other pedigrees probably reflects different modifying factors including nuclear backgrounds, other environmental factors, and mitochondrial haplotypes. The phenotypic variability of members including a wide range of severity and age at onset of visual loss in this Chinese pedigree suggests the possible involvement of nuclear modifier gene(s) in the development of the visual impairment as described in the other families. 3 18 33 Furthermore, it is possible that environmental factors contributed to the phenotypic variability of visual loss in matrilineal relatives of this family. In fact, the background sequences (haplotype) of the mtDNA have been shown to influence the penetrance and expressivity of visional loss associated with primary mtDNA mutations. In particular, secondary LHON mutations at positions 4,216 and 13,708 may increase the penetrance and expressivity of LHON associated with the primary LHON mutations, including 11778A and T14484C, 15 16 36 or deafness associated with the primary mtDNA A7445G mutation. 25 Furthermore, the G7444A mutation in the CO1 and tRNASer(UCN) genes has also been implicated as influencing the penetrance and phenotypic expression of visual loss associated with the primary LHON mutations 6 and of hearing loss associated with the 12S rRNA A1555G mutation. 37 The G7444A mutation results in a read-through of the stop codon AGA of the COI message, thereby adding three amino acids (Lys, Gln, and Lys) to the C-terminal of the polypeptide. Alternatively, the G7444A mutation, which is adjacent to the site of 3′ end endonucleoly-tic processing of the L-strand RNA precursor, spanning tRNASer(UCN) and ND6 mRNA, 25 may lead to a defect in the processing of the L-strand RNA precursor, thus influencing the phenotypic expression of the A1555G or G11778A mutation. In this Chinese pedigree, apart from the G11778A and A4435G mutations, another 34 variants in this mitochondrial genome belonging to the Eastern Asian haplogroup D5 38 showed no evolutionary conservation. In contrast, the mitochondrial genomes in four other Chinese families carrying the G11778A mutation belong to the Eastern Asian haplogoups B5, 12 F1, D4, and M10. 18 This suggests that the G11778A mutations, similar to white families, 15 occurred sporadically and multiplied through evolution of the mtDNA in China. Here, the presence of the homoplasmic A4435G mutation in the tRNAMet in this Chinese pedigree, but its absence in the previous identified Chinese pedigrees with lower penetrance and less expressivity, 12 18 seems to account for the different penetrance and expressivity between this Chinese pedigree and other Chinese families. 
The A4435G mutation is localized at 3′ end adjacent to the anticodon (position 37) of tRNAMet. 26 In fact, the adenine at this position of tRNAMet is extraordinarily conserved from bacteria to human mitochondria. 27 Almost all A37 in tRNAs are modified, by such processes as thiolation and methylation. 28 Indeed, this modified nucleotide contributes to the high fidelity of codon recognition, the structural formation and stabilization of functional tRNAs. 39 In Escherichia coli, nucleotide modifications at positions 37 and 34 are responsible for the stabilization of the canonical loop structure in the anticodon domain of tRNALys. 40 Also, it has been shown that the modification of A37 stabilizes the 3′ stacking features of the anticodon, thereby improving its interaction with the codon. 41 Thedeficient modification of A37 decreases the activity of the corresponding tRNA 42 and increases +1 frameshifts for tRNAPhe, 43 whereas the A-to-G substitution at position 37 leads to a 10-fold reduction in the section of tRNAs at the A-site. 44 Most recently, the T4291C mutation at the anticodon region of mitochondrial tRNAIle has been associated with hypertension, hypercholesterolemia, and hypomagnesemia. 45 The lack of modification at U34 of anticodon in the tRNALys has been observed in cells carrying the MERRF-associated A8344G mutation in this gene. 46 In the current study, compared with control cells lacking those mutations, a ∼50% reduction in the level of tRNAMet was observed in cells carrying both the G11778A and A4435G mutations, whereas there was no significant reduction in the level of tRNAMet in cells carrying the G11778A mutation. The lower level of tRNAMet in cells carrying the A4435G mutation most probably results from a defect in nucleotide modification at position 37 of tRNAMet. As a result, a failure in mitochondrial tRNA metabolism, caused by the A4435G mutation, may lead to impairment of mitochondrial translation, especially for seven subunits, including ND4 of NADH (complex I) encoded by mtDNA. Thus, the mitochondrial dysfunction, particularly in deficient activities of complex I, caused by ND4 G11778 mutation, would be worsened by the A4435G mutation, similar to other secondary LHON missense mutations or G7444A mutation. Therefore, the A4435G mutation may have a modifying role in increasing the penetrance and expressivity of the primary LHON-associated G11778A mutation in this Chinese family. 
 
Figure 1.
 
The Chinese pedigree with LHON. Filled symbols: vision-impaired individuals.
Figure 1.
 
The Chinese pedigree with LHON. Filled symbols: vision-impaired individuals.
Figure 2.
 
Fundus photograph of an affected subject (III-2) and a married-in control (II-1)
Figure 2.
 
Fundus photograph of an affected subject (III-2) and a married-in control (II-1)
Table 1.
 
Summary of Clinical Data for Some Members in the Chinese Pedigree
Table 1.
 
Summary of Clinical Data for Some Members in the Chinese Pedigree
Patient Sex Age of Test (y) Age of Onset (y) Visual Acuity Level of Visual Impairment
Right Left
II-2 F 46 1.0 1.0 Normal
II-4 F 51 13 0.02 0.01 Profound
II-5 M 22 16 <0.01 <0.01 Profound
II-6 M 48 16 0.05 0.04 Severe
II-8 M 41 14 0.12 0.08 Moderate
II-10 M 38 15 0.08 0.02 Severe
II-13 F 35 1.2 1.2 Normal
III-1 F 21 1.2 1.2 Normal
III-2 M 18 12 0.01 0.02 Profound
III-3 F 23 14 0.05 0.04 Severe
III-4 M 19 11 0.07 0.09 Moderate
III-9 F 11 1.2 1.2 Normal
III-10 M 7 1.2 1.2 Normal
Table 2.
 
mtDNA Mutations in the Chinese Family with Leber Hereditary Optic Neuropathy
Table 2.
 
mtDNA Mutations in the Chinese Family with Leber Hereditary Optic Neuropathy
Gene Position Replacement Conservation* (H/B/M/X) Previously Reported, †
D-Loop 73 A to G Yes
150 C to T Yes
263 A to G Yes
310 T to CTC Yes
489 T to C Yes
16189 T to C Yes
16244 C to T Yes
16362 C to T Yes
16521 A to G Yes
12S rRNA 750 A to G A/A/G/– Yes
752 C to T C/C/A/– Yes
1107 T to C A/G/A/A Yes
16S rRNA 2706 A to G A/G/A/A Yes
ND1 3348 A to G Yes
tRNAMet , ‡ 4435 A to G A/A/A/A No
ND2 4769 A to G Yes
4883 C to T Yes
5178 C to A (Leu to Met) L/T/T/T Yes
CO1 7028 C to T Yes
A6 8701 A to G (Thr to Ala) T/S/L/Q Yes
8860 A to G (Thr to Ala) T/A/A/T Yes
9180 Yes
CO3 9540 T to C Yes
ND3 10397 Yes
10398 A to G (Thr to Ala) T/T/T/A Yes
10400 C to T Yes
ND4 10873 T to C Yes
11719 G to A Yes
11778 G to A (Arg to His) R/R/R/R Yes
11944 T to C Yes
12026 A to G (Ile to Val) I/I/M/I Yes
12131 T to C (Ser to Pro) S/L/T/F No
ND5 12705 C to T Yes
13266 A to G No
Cytochrome b 15301 G to A Yes
15326 A to G (Thr to Ala) T/M/I/I Yes
Figure 3.
 
Identification of the A4435G mutation in the mitochondrial tRNAMet gene. (A) Partial sequence chromatograms of the tRNAMet gene from affected individual III-2 and married-in control subject II-3. Arrow: location of the base changes at position 4435. (B) Quantification of A4435G mutation in the tRNAMet gene of 13 mutants and 1 control subject derived from the Chinese family. PCR products around the A4435G mutation were digested with NlaIII and analyzed by electrophoresis in a 7% polyacrylamide gel stained with ethidium bromide. Patients and control individuals are indicated. (C) The location of the A4435G mutation in the mitochondrial tRNAMet. Cloverleaf structure of human mitochondrial is derived from Florentz et al. 26 Arrow: position of the A4435G mutation.
Figure 3.
 
Identification of the A4435G mutation in the mitochondrial tRNAMet gene. (A) Partial sequence chromatograms of the tRNAMet gene from affected individual III-2 and married-in control subject II-3. Arrow: location of the base changes at position 4435. (B) Quantification of A4435G mutation in the tRNAMet gene of 13 mutants and 1 control subject derived from the Chinese family. PCR products around the A4435G mutation were digested with NlaIII and analyzed by electrophoresis in a 7% polyacrylamide gel stained with ethidium bromide. Patients and control individuals are indicated. (C) The location of the A4435G mutation in the mitochondrial tRNAMet. Cloverleaf structure of human mitochondrial is derived from Florentz et al. 26 Arrow: position of the A4435G mutation.
Figure 4.
 
Alignment of tRNAMet genes from different species. Arrow: position of the A37 at the anticodon loop of tRNA, corresponding to the A4435G mutation.
Figure 4.
 
Alignment of tRNAMet genes from different species. Arrow: position of the A37 at the anticodon loop of tRNA, corresponding to the A4435G mutation.
Figure 5.
 
Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (5 μg) of total mitochondrial RNA from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with DIG-labeled oligonucleotide probes specific for the tRNAMet. The blots were then stripped and rehybridized with DIG-labeled tRNALeu(UUR), tRNALys and tRNAGln, respectively. (B) Quantification of mitochondrial tRNA levels. Average relative tRNAMet, content per cell, normalized to the average content per cell of tRNALeu(UUR), tRNALys, or tRNAGln in one control cell line (II-1) and in one mutant cell line (WZ4-IV-2) carrying the G11778A mutation and one cell line (III-2) carrying both G11778A and A4435G mutations. The values for the latter are expressed as percentages of the average values in the control cell line. The calculations are based on three independent determinations of tRNAMet content in each cell line and three determinations of the content of each reference RNA marker in each cell line.
Figure 5.
 
Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (5 μg) of total mitochondrial RNA from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with DIG-labeled oligonucleotide probes specific for the tRNAMet. The blots were then stripped and rehybridized with DIG-labeled tRNALeu(UUR), tRNALys and tRNAGln, respectively. (B) Quantification of mitochondrial tRNA levels. Average relative tRNAMet, content per cell, normalized to the average content per cell of tRNALeu(UUR), tRNALys, or tRNAGln in one control cell line (II-1) and in one mutant cell line (WZ4-IV-2) carrying the G11778A mutation and one cell line (III-2) carrying both G11778A and A4435G mutations. The values for the latter are expressed as percentages of the average values in the control cell line. The calculations are based on three independent determinations of tRNAMet content in each cell line and three determinations of the content of each reference RNA marker in each cell line.
Table 3.
 
Summary of Clinical and Molecular Data for Five Chinese Families Carrying the G11778A Mutation
Table 3.
 
Summary of Clinical and Molecular Data for Five Chinese Families Carrying the G11778A Mutation
Pedigree Ratio (Affected Male/Female) Average Age of Onset (y) Matrilineal Relatives (n) Penetrance (%)* mtDNA Haplogroup
WZ2 3:1 14 14 57.1 D5
WZ4, † 3:0 17 9 33.3 F1
WZ5 1:1 20 14 35.7 D4b2b
WZ6 1.2:1 18 31 35.5 M10a
WZ1, ‡ 6:0 22 38 15.8 B5
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Figure 1.
 
The Chinese pedigree with LHON. Filled symbols: vision-impaired individuals.
Figure 1.
 
The Chinese pedigree with LHON. Filled symbols: vision-impaired individuals.
Figure 2.
 
Fundus photograph of an affected subject (III-2) and a married-in control (II-1)
Figure 2.
 
Fundus photograph of an affected subject (III-2) and a married-in control (II-1)
Figure 3.
 
Identification of the A4435G mutation in the mitochondrial tRNAMet gene. (A) Partial sequence chromatograms of the tRNAMet gene from affected individual III-2 and married-in control subject II-3. Arrow: location of the base changes at position 4435. (B) Quantification of A4435G mutation in the tRNAMet gene of 13 mutants and 1 control subject derived from the Chinese family. PCR products around the A4435G mutation were digested with NlaIII and analyzed by electrophoresis in a 7% polyacrylamide gel stained with ethidium bromide. Patients and control individuals are indicated. (C) The location of the A4435G mutation in the mitochondrial tRNAMet. Cloverleaf structure of human mitochondrial is derived from Florentz et al. 26 Arrow: position of the A4435G mutation.
Figure 3.
 
Identification of the A4435G mutation in the mitochondrial tRNAMet gene. (A) Partial sequence chromatograms of the tRNAMet gene from affected individual III-2 and married-in control subject II-3. Arrow: location of the base changes at position 4435. (B) Quantification of A4435G mutation in the tRNAMet gene of 13 mutants and 1 control subject derived from the Chinese family. PCR products around the A4435G mutation were digested with NlaIII and analyzed by electrophoresis in a 7% polyacrylamide gel stained with ethidium bromide. Patients and control individuals are indicated. (C) The location of the A4435G mutation in the mitochondrial tRNAMet. Cloverleaf structure of human mitochondrial is derived from Florentz et al. 26 Arrow: position of the A4435G mutation.
Figure 4.
 
Alignment of tRNAMet genes from different species. Arrow: position of the A37 at the anticodon loop of tRNA, corresponding to the A4435G mutation.
Figure 4.
 
Alignment of tRNAMet genes from different species. Arrow: position of the A37 at the anticodon loop of tRNA, corresponding to the A4435G mutation.
Figure 5.
 
Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (5 μg) of total mitochondrial RNA from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with DIG-labeled oligonucleotide probes specific for the tRNAMet. The blots were then stripped and rehybridized with DIG-labeled tRNALeu(UUR), tRNALys and tRNAGln, respectively. (B) Quantification of mitochondrial tRNA levels. Average relative tRNAMet, content per cell, normalized to the average content per cell of tRNALeu(UUR), tRNALys, or tRNAGln in one control cell line (II-1) and in one mutant cell line (WZ4-IV-2) carrying the G11778A mutation and one cell line (III-2) carrying both G11778A and A4435G mutations. The values for the latter are expressed as percentages of the average values in the control cell line. The calculations are based on three independent determinations of tRNAMet content in each cell line and three determinations of the content of each reference RNA marker in each cell line.
Figure 5.
 
Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (5 μg) of total mitochondrial RNA from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with DIG-labeled oligonucleotide probes specific for the tRNAMet. The blots were then stripped and rehybridized with DIG-labeled tRNALeu(UUR), tRNALys and tRNAGln, respectively. (B) Quantification of mitochondrial tRNA levels. Average relative tRNAMet, content per cell, normalized to the average content per cell of tRNALeu(UUR), tRNALys, or tRNAGln in one control cell line (II-1) and in one mutant cell line (WZ4-IV-2) carrying the G11778A mutation and one cell line (III-2) carrying both G11778A and A4435G mutations. The values for the latter are expressed as percentages of the average values in the control cell line. The calculations are based on three independent determinations of tRNAMet content in each cell line and three determinations of the content of each reference RNA marker in each cell line.
Table 1.
 
Summary of Clinical Data for Some Members in the Chinese Pedigree
Table 1.
 
Summary of Clinical Data for Some Members in the Chinese Pedigree
Patient Sex Age of Test (y) Age of Onset (y) Visual Acuity Level of Visual Impairment
Right Left
II-2 F 46 1.0 1.0 Normal
II-4 F 51 13 0.02 0.01 Profound
II-5 M 22 16 <0.01 <0.01 Profound
II-6 M 48 16 0.05 0.04 Severe
II-8 M 41 14 0.12 0.08 Moderate
II-10 M 38 15 0.08 0.02 Severe
II-13 F 35 1.2 1.2 Normal
III-1 F 21 1.2 1.2 Normal
III-2 M 18 12 0.01 0.02 Profound
III-3 F 23 14 0.05 0.04 Severe
III-4 M 19 11 0.07 0.09 Moderate
III-9 F 11 1.2 1.2 Normal
III-10 M 7 1.2 1.2 Normal
Table 2.
 
mtDNA Mutations in the Chinese Family with Leber Hereditary Optic Neuropathy
Table 2.
 
mtDNA Mutations in the Chinese Family with Leber Hereditary Optic Neuropathy
Gene Position Replacement Conservation* (H/B/M/X) Previously Reported, †
D-Loop 73 A to G Yes
150 C to T Yes
263 A to G Yes
310 T to CTC Yes
489 T to C Yes
16189 T to C Yes
16244 C to T Yes
16362 C to T Yes
16521 A to G Yes
12S rRNA 750 A to G A/A/G/– Yes
752 C to T C/C/A/– Yes
1107 T to C A/G/A/A Yes
16S rRNA 2706 A to G A/G/A/A Yes
ND1 3348 A to G Yes
tRNAMet , ‡ 4435 A to G A/A/A/A No
ND2 4769 A to G Yes
4883 C to T Yes
5178 C to A (Leu to Met) L/T/T/T Yes
CO1 7028 C to T Yes
A6 8701 A to G (Thr to Ala) T/S/L/Q Yes
8860 A to G (Thr to Ala) T/A/A/T Yes
9180 Yes
CO3 9540 T to C Yes
ND3 10397 Yes
10398 A to G (Thr to Ala) T/T/T/A Yes
10400 C to T Yes
ND4 10873 T to C Yes
11719 G to A Yes
11778 G to A (Arg to His) R/R/R/R Yes
11944 T to C Yes
12026 A to G (Ile to Val) I/I/M/I Yes
12131 T to C (Ser to Pro) S/L/T/F No
ND5 12705 C to T Yes
13266 A to G No
Cytochrome b 15301 G to A Yes
15326 A to G (Thr to Ala) T/M/I/I Yes
Table 3.
 
Summary of Clinical and Molecular Data for Five Chinese Families Carrying the G11778A Mutation
Table 3.
 
Summary of Clinical and Molecular Data for Five Chinese Families Carrying the G11778A Mutation
Pedigree Ratio (Affected Male/Female) Average Age of Onset (y) Matrilineal Relatives (n) Penetrance (%)* mtDNA Haplogroup
WZ2 3:1 14 14 57.1 D5
WZ4, † 3:0 17 9 33.3 F1
WZ5 1:1 20 14 35.7 D4b2b
WZ6 1.2:1 18 31 35.5 M10a
WZ1, ‡ 6:0 22 38 15.8 B5
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