Analysis of Three Primary mtDNA Mutations in 155 Patients with Leber’s Hereditary Optic Neuropathy

Background: Leber’s Hereditary Optic Neuropathy (LHON) is a maternal inherited disease caused by mitochondrial DNA (mtDNA) mutations. The aim of the current study is to analysis the frequencies of mitochondrial ND1 G3460A, ND4 G11778A and ND6 T14484C mutations in patients with LHON. Methods: Our study enrolled 155 patients with LHON and 83 controls, PCR-Sanger sequencing was performed to screen the presence of these primary mutations. Moreover, we performed clinical, genetic and molecular characterizations of ve Chinese families carrying LHON-related three primary mutations. Results: 28 patients with G3460A (18.1%), 86 patients with G11778A (55.5%) and 32 patients carrying T14484C mutation (20.6%) were identied. However, none of these primary mutations were identied in controls. Among them, one patient carrying G3460A, two patients with G11778A and two patients with T14484C mutation had an obvious family history of LHON. Clinical evaluation of these pedigrees showed the variable clinical phenotypes with different age at onset of LHON. Sequence analysis of the complete mtDNA genes from the matrilineal relatives suggested the presence of these primary mutations. However, the lack of any functional variants in mtDNA genes revealed that mitochondrial haplogroups or haplotypes may not play important roles in the clinical phenotypic manifestation of LHON-associated primary mutations. Conclusions: Our data indicated that screening for the mtDNA primary mutations was necessary for early detection, prevention and diagnosis of LHON.


Background
LHON was a common maternally inherited disease caused by mtDNA mutations, which led to subacute optic atrophy resulting in severe visual impairment in most affected persons [1]. Several documented LHON cases had involved abnormal symptoms, including movement disorders, dystonia or multiple-sclerosis-like symptoms, which remains a big challenge for clinicians [2,3].
To date, the molecular mechanism underlying LHON was still not fully understood. Since the landmark discovery of the LHONassociated mtDNA ND4 G11778A mutation [4], more than 30 LHON-related mtDNA mutations had been reported among various ethnic population [5]. Notably, three primary mtDNA mutations: ND1 G3460A, ND4 G11778A and ND6 T14484C, which involved genes encoding the subunits of respiratory chain complex I, accounted for > 90% of LHON pedigrees in some countries [6,7]. Moreover, LHON mutations were predominantly homoplasmic with incomplete penetrance and marked male bias concerning clinical affection [8], suggesting that additional factors may in uence the conversion to symptomatic LHON, like mtDNA genetic background, nuclear genes and environmental factors.
With the aim of exploring the molecular basis for LHON, we recently initiated a mutational screening for the three primary mtDNA mutations in a cohort of 155 LHON patients and 83 healthy subjects. Moreover, we performed clinical, genetic and molecular analysis of ve Han Chinese families with LHON-associated mtDNA primary mutations.

Materials And Methods
Subjects and ethics statement 155 patients (99 males and 56 females, aged from 4 to 33 years, with an average of 18 years) received ophthalmological examinations at Fuzhou Second Hospital, a liated to Xiamen University, were categorized according to the expression of LHON clinical features, such as painless, acute or subacute vision loss. Moreover, a total of 83 control subjects (41 males and 42 females, aged from 8 to 24 years, with an average of 13 years) were obtained from the Physical Examination Center of our hospital. The written informed consents conforming to the tenets of the Declaration of Helsinki were obtained from each participant prior to the study. The institutional review boards of Fuzhou Second Hospital, Xiamen University approved this study.
Clinical and molecular features of ve LHON pedigrees with three primarymutations Five Chinese pedigrees, as shown in Figure 2, were ascertained in the Department of Ophthalmology, Fuzhou Second Hospital, Xiamen University. Members of these pedigrees were interviewed at length to identify both personal or family medical histories of visual impairments, and other clinical abnormalities. The ophthalmic examinations of the probands and other matrilineal relatives, including visual eld test, visual acuity examination, fundus photography, visual evoked potentials and determination of the degree of visual impairment, as well as other clinical evaluations, were conducted as described previously [14]. The degree of visual loss was classi ed based on the following criteria: healthy, ≥0.3, mild, 0.1 to 0.299; moderate, 0.05 to 0.099; severe, 0.02 to 0.049; and profound, <0.02.
The complete mitochondrial genomes of the affected LHON individuals (FZ010: II-1 and III-8; FZ011: II-6 and III-2; FZ012: III-1 and III-4; FZ013: III-14 and IV-6; FZ014: III-5) were PCR ampli ed by using 24 primers, as described in a previous study [15]. Each fragment was puri ed and subsequently was analyzed by direct sequencing in an ABI 3700 automated DNA sequencer using the Big Dye Terminator Cycle sequencing reaction kit (Applied Biosystems). These sequence results were compared with the rCRS (GenBank accession number: NC_012920.1) to detect the mutations/variants [13].

Phylogenetic analysis
The mtDNA sequences of 15 different vertebrates were used to conduct an inter-speci c analysis as reported previously [16].
The conservation index (CI) of each mtDNA mutation was calculated, we regarded the CI≥75% as having functional potential [17].

Haplogroup analysis
The entire mtDNA sequences of the ve Chinese pedigrees carrying the mtDNA primary mutations were assigned to the Asian mitochondrial haplogroups by using the nomenclature of mitochondrial haplogroups [18].

Mutational screening of LHON-associated three primary mutations
The study samples with LHON consisted of 99 males and 56 females. All participants were Han Chinese subjects recruited from Department of Ophthalmology, Fuzhou Second Hospital, Xiamen University in China. After PCR-Sanger sequencing, we identi ed 28 patients with G3460A mutation (18.1%), 86 patients with G11778A mutation (55.5%) and 32 patients carrying T14484C mutation (20.6%) (Fig. 1). But we failed to detect these primary mutations in 83 healthy controls.
Clinical characterization of ve Han Chinese families with mtDNA primary mutations In our case-control study for genetic screening of LHON-related three primary mtDNA mutations, ve Han Chinese families, as shown in Fig. 2, were ascertained in Fuzhou Second Hospital, Xiamen University. In family FZ010, the proband (II-1) was 31year-old man who lived in Fuzhou City of Fujian Province. The comprehensive history interview indicated that he did not have any other clinical abnormalities such as diabetes, cardiovascular disease, deafness, cancer or neurological disorders. In addition, he began to suffer from painless and progressive bilateral vision loss (right eye: 0.01 and left eye: 0.03) at the age of 18, he observed a dark cloud in the center of his vision and had di culty discerning colors, which all appeared dark grey.
In family FZ011, the proband (III-2) complained of painless, progressive deterioration of bilateral vision impairment at the age of 4 and went to the Eye Center of Fuzhou Second Hospital, Xiamen University. His visual acuity was 0.08 and 0.04 at the right and left. Ophthalmological and other clinical evaluations showed that he had a typical clinical feature of LHON. In addition, the matrilineal relative (II-6) was also LHON carrier.
In family FZ012, the proband (III-1) began suffering from painless, progressive deterioration of bilateral vision impairment at the age of 11 years old and came to Ophthalmology Clinic at the Fuzhou Second Hospital, Xiamen University. She saw a dark cloud in the center of vision and had problems appreciating colors that all seemed dark gray. Her visual acuity was 0.01 and 0.03 at the right and left. Among other matrilineal relatives, the subject (III-4) experienced loss of vision at the age of 12, visual acuity of III-4 was 0.1 in both eyes.
In family FZ013, the proband (III-14) was diagnosed with LHON at the age of 25 by the Ophthalmology Clinic at Fuzhou Second Hospital, Xiamen University. His visual acuity was 0.2 in both eyes. Among other members in this pedigree, the subject (IV-6) was LHON carrier, and her visual acuity was 0.03 in both eyes. However, other members in this family were normal vision.
In family FZ014, the proband (III-5) came to the Eye Center of Fuzhou Second Hospital, Xiamen University at the age of 12, he suffered painless, progressive deterioration of visual impairment at the age of 12. He saw a dark cloud in the center of vision and had problems appreciating colors that all seemed a dark gray, and his visual acuity was 0.1 in both eyes. However, none of other members in this family had any vision de cit. The clinical features of several members from these pedigrees were listed in Table 1. To explore the molecular basis for LHON, we performed the mutational screening for the mtDNA variants in these matrilineal relatives (FZ010: II-1 and III-8; FZ011: II-6 and III-2; FZ012: III-1 and III-4; FZ013: III-14 and IV-6; FZ014: III-5), as well as the healthy subjects. We rst ampli ed the complete mitochondrial genomes of these LHON patients by using 24 primers as previously described [14], the PCR products were puri ed and subsequently analyzed in an ABI 3700 automated DNA sequencer using the Big Dye Terminator Cycle sequencing reaction kit. Sequences were handled by the DNASTAR program (DNAS Inc, Madison, USA). Compared with the rCRS, matrilineal relatives of these families revealed sets of genetic variations belonging to different mitochondrial haplogroup F1a, D5b1, F1, M7b1, M10a, respectively [18] ( Table 2). Among them, there were 22 variants in D-loop, 4 variants in 12S rRNA and 3 variants in 16S rRNA, one common 9-bp deletion in the conjunction between tRNA Lys and CO2 gene, while other variants were found in mitochondrial protein-coding genes. Furthermore, 20 missense mutations were found, including the ND1 G3316A (Ala to Thr), G3460A (Ala to Thr) and G4048A (Asp to Asn), ND2 C5178A (Leu to Met), ATP8 C8414T (Leu to Phe) and A8498G (Lys to Glu), ATP6 G8584A (Ala to Thr), A8701G (Thr to Ala), A8860G (Thr to Ala) and C9071T (Ser to Leu), ND3 A10398G (Thr to Ala), ND4 G11778A (Arg to His), ND5 A12361G (Thr to Ala), A13681G (Thr to Ala) and G13708A (Ala to Thr), ND6 T14484C (Met to Val), CytB C14766T (Thr to Ile), C14883T (Thr to Ile), A15236G (Ile to Val) and A15326G (Thr to Ala). In addition, evolutionary conservation analysis was performed for these variants between 15 species, including mouse [19], bovine [20] and Xenopus laevis [21]. However, besides the ND4 G11778A mutation, other mutations/variants were well conserved.
A to G (Thr to Ala) A to G (Thr to Ala)   Chinese families ranged from 16.7 to 50%, with the average of 26.12%. Furthermore, the average age-of-onset for optic neuropathy ranged from 6 to 28.7 years, with the average of 18.7 years in other 14 Chinese families [9,12,[27][28] ( Table 3).
The relative low penetrances of LHON in these ve families strongly indicated that the ND1 G3460A, ND4 G11778A and ND6 T14484C mutations were not su cient to produce the clinical phenotypes, hence, other modi ed factors such as environmental factors, nuclear genes, mtDNA haplogroups and epigenetic modi cation may contribute to the clinical manifestation of LHON-associated mtDNA primary mutations [29,30].
Recently, many studies reported that mtDNA background affected the expression of LHON. For example, in the European families, the risk of optic neuropathy increased when the ND4 G11778A and ND6 T14484C mutations were present in the haplogroup J [31,32], and when the ND1 G3460A mutation occurred in the haplogroup K [32]. Furthermore, secondary LHON mutations such as ND1 T4216C and ND5 G13708A may increase the penetrance and expressivity of LHON associated with the primary ND4 G11778A and ND6 T14484C mutations [32]. In the Chinese families carrying the G11778A mutation, the haplogroup M7b1'2 signi cantly increased the risk of optic neuropathy, whereas the haplogroup M8a had a protective effect [33].  29]. These data suggested that these mtDNA haplogroup-speci c variants may not play an important role in the phenotypic expression of the LHON-related mtDNA primary mutations in these pedigrees.

Conclusions
In summary, our study indicated that mitochondrial ND1 G3460A, ND4 G11778A and ND6 T14484C mutations were the molecular basis for LHON, screening for these primary mtDNA mutations were recommended for early detection and diagnosis of inherited optic neuropathy. The main limitation of this study was the relatively small sample size, further studies including more LHON patients were needed to be performed.  Five Han Chinese pedigrees with LHON, arrows indicate the proband, the affected subjects are indicated by lled symbols.