Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees.

Biochemical and molecular genetic evidence is presented that in six independent pedigrees the development of Leber hereditary optic neuropathy (LHON) is due to the same primary mutation in the mitochondrial ND1 gene. A LHON family from the Newcastle area of Great Britain was analyzed in depth to determine the mitochondrial genetic etiology of their disease. Biochemical assays of mitochondrial electron transport in organelles isolated from the platelet/white-blood-cell fraction have established that the members of this family have a substantial and specific lowering of flux through complex I (NADH-ubiquinone oxidoreductase). To determine the site of the primary mitochondrial gene mutation in this pedigree, all seven mitochondrial complex I genes were sequenced, in their entirety, from two family members. The primary mutation was identified as a homoplasmic transition at nucleotide 3460, which results in the substitution of threonine for alanine at position 52 of the ND1 protein. This residue occurs within a very highly conserved hydrophilic loop, is invariantly alanine or glycine in all ND1 proteins, and is adjacent to an invariant aspartic acid residue. This is only the second instance in which both a biochemical abnormality and a mitochondrial gene mutation have been identified in an LHON pedigree. The sequence analysis of the ND81 gene was extended to a further 11, unrelated LHON pedigrees that had been screened previously and found not to carry the mitochondrial ND4/R340H mutation. The ND1/A52T mutation at nucleotide 3460 was found in five of these 11 pedigrees. In contrast, this sequence change was not found in any of the 47 non-LHON controls. The possible role of secondary complex I mutations in the etiology of LHON is also addressed in these studies.     

The impact of mitochondrial tRNA mutations on the amount of ATP synthase differs in the brain compared to other tissues

The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNA(Lys) (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNA(LeuUUR). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits.     

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

The m.3243A>G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.     

Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia.

A rare form of Leber hereditary optic neuropathy (LHON) that is associated with hereditary spastic dystonia has been studied in a large Dutch family. Neuropathy and ophthalmological lesions were present together in some family members, whereas only one type of abnormality was found in others. mtDNA mutations previously reported in LHON were not present. Sequence analysis of the protein-coding mitochondrial genes revealed two previously unreported mtDNA mutations. A heteroplasmic A-->G transition at nucleotide position 11696 in the ND4 gene resulted in the substitution of an isoleucine for valine at amino acid position 312. A second mutation, a homoplasmic T-->A transition at nucleotide position 14596 in the ND6 gene, resulted in the substitution of a methionine for the isoleucine at amino acid residue 26. Biochemical analysis of a muscle biopsy revealed a severe complex I deficiency, providing a link between these unique mtDNA mutations and this rare, complex phenotype including Leber optic neuropathy.     

6个线粒体脑肌病伴高乳酸血症和卒中样发作综合征(MELAS)家系先证者线粒体基因全序列比较分析

线粒体脑肌病伴高乳酸血症和卒中样发作综合征(Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes, MELAS)是一种异质性很强的遗传代谢性疾病,而位于tRNA ^Leu(UUR)基因的A3243G突变是该疾病最常见的致病位点。文章对6个汉族MELAS家系的先证者进行了临床病理、分子遗传学特征分析,探讨了线粒体基因多态性对MELAS病人表型可能产生的影响。线粒体基因检测结果显示,4例先证者为A3243G阳性,其异质性比例介于29%~59%之间,临床症状的严重性和异质性程度大致呈正相关;2例MELAS/Leigh叠加综合征先证者为A3243G阴性,复发次数和严重程度重于其他4例先证者,其中1例先证者的血液和肌肉组织中发现ND5基因T13094C突变,该位点已报道与MELAS/Leigh叠加综合征、小脑共济失调相关。另外,线粒体基因全序列测序结果显示：除主要致病突变外,还存在多个与耳聋、癫痫、糖尿病、心肌病、Leigh综合征相关的线粒体基因多态位点,临床症状严重的患者其多态位点也更多。这表明MELAS综合征的复杂表型不仅受致病突变位点的直接影响,也可能受到其他与疾病相关的多态性位点的修饰作用。     

A family with 3460G>A and 11778G>A mutations and haplogroup analysis of Polish Leber hereditary optic neuropathy patients

Three mutations in mitochondrial DNA complex I genes are responsible for over 90% of Leber hereditary optic neuropathy (LHON) cases in Europe. A family with two LHON mutations--practically homoplasmic 11778G>A and varying levels of 3460G>A--was found during analysis of Polish patients. DNA and visual acuity was analyzed in four affected brothers and their unaffected sister and mother as well as in their step brother. Four male patients experienced vision loss around the age of 20 while for their step brother the onset was late--at the age of 33. No additional neurological symptoms were observed and both women were completely asymptomatic. The mutation occurred in a haplogroup H background, the most common one in both the Polish population and among patients. Double LHON mutations are extremely rare, and this particular combination has not been previously described in the literature.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease

Mutations of the genes encoding aminoacyl-t RNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5 AG, p.D2 G; c.1367 CT, p.S456 L; c.1535 GA, p.R512 Q and c.1846_1847 del, p.Y616 Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-t RNA synthetase(h Arg RS), are linked to Pelizaeus-Merzbacher-like disease(PMLD) with unclear pathogenesis. Among these mutations, c.5 AG is the most extensively reported mutation, leading to a p.D2 G mutation in the N-terminal extension of the long-form h Arg RS. Here, we showed the detrimental effects of R512 Q substitution and ΔC mutations on the structure and function of h Arg RS, while the most frequent mutation c.5 AG, p.D2 G acted in a different manner without impairing h Arg RS activity. The nucleotide substitution c.5 AG reduced translation of h Arg RS m RNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778, or 14484 Leber's hereditary optic neuropathy mitochondrial DNA mutation

Leber's hereditary optic neuropathy (LHON) is a form of blindness caused by mitochondrial DNA (mtDNA) mutations in complex I genes. We report an extensive biochemical analysis of the mitochondrial defects in lymphoblasts and transmitochondrial cybrids harboring the three most common LHON mutations: 3460A, 11778A, and 14484C. Respiration studies revealed that the 3460A mutation reduced the maximal respiration rate 20-28%, the 11778A mutation 30-36%, and the 14484C mutation 10-15%. The respiration defects of the 3460A and 11778A mutations transferred in cybrid experiments linking these defects to the mtDNA. Complex I enzymatic assays revealed that the 3460A mutation resulted in a 79% reduction in specific activity and the 11778A mutation resulted in a 20% reduction, while the 14484C mutation did not affect the complex I activity. The enzyme defect of the 3460A mutation transferred with the mtDNA in cybrids. Overall, these data support the conclusion that the 3460A and 11778A mutants result in complex I defects and that the 14484C mutation causes a much milder biochemical defect. These studies represent the first direct comparison of oxidative phosphorylation defects among all of the primary LHON mtDNA mutations, thus permitting insight into the underlying pathophysiological mechanism of the disease.     

A point mutation in the mitochondrial tRNA(Leu)(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes)

Mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episode (MELAS) is a major group of heterogeneous mitochondrial disorders. To identify the defective gene, mitochondrial DNA from a patient with MELAS was sequenced by using amplified DNA fragments as sequencing templates. In 14.1 kbp determined out of 16.6 kbp of the whole mitochondrial gene, at least 21 nucleotides were different from those of a control human mitochondrial DNA. One of the substitutions was a transition of A to G in the tRNA(Leu) (UUR) gene at Cambridge nucleotide number 3,243. This nucleotide is conserved not only in many mitochondrial tRNAs but in most cytosolic tRNA molecules. An Apa I restriction site was gained by the substitution of this nucleotide. The Apa I digestion of the amplified DNA fragment revealed that all independent 6 patients had G at nucleotide number 3,243 in their mitochondrial DNAs, but none of 11 control individuals had G at this position. This result strongly suggests that the mutation in the mitochondrial tRNALeu gene causes MELAS.     

Two novel mutations in the BCKDHB gene (R170H, Q346R) cause the classic form of maple syrup urine disease (MSUD)

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509G>A) in exon 5 and Q346R (c.1037 A>G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β' and S206-α, which results in unstable β-β' assembly and an unstable K(+) ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β', which reduces the affinity of the β-β' subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD.     

Screening of common mitochondrial mutations in Chinese patients with mitochondrial encephalomyopathies

To investigate the spectrum of common mitochondrial mutations in Northern China during the years of 2000-2005, 552 patients of mitochondrial encephalomyopathies clinically diagnosed as MELAS, MERRF or Leigh's syndrome, 14 cases of LHON and 46 cases of aminoglycoside induced deafness along with their family members, accepted routine point mutation tests at nucleotide positions 3243, 8344, 8993, 11778 or 1555 in mitochondrial genome. PCR-RFLP analysis, site-specific PCR and PCR-sequencing methods were used to identify the mutations. Fifty-seven cases with A3243G mutation, 4 cases with A8344G, 2 cases with T8993C and 1 case with T8993G were identified from the 552 encephalomyopathy patients. In addition, one case with G11778A was found from the 14 cases of LHON, and 5 cases with A1555G from the 46 cases of aminoglycoside ototoxicity patients. Additional screening for T8356G and T3271C merely had limited significance for the diagnosis of MERRF and MELAS. Differential diagnosis among mitochondrial encephalomyopathies was often complicated due to many similar clinical manifestations. For A3243G mutation, the proportion of mutant mtDNA was not related to severity of the disease but to the age of onset.     

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.     

Leber遗传性视神经病变研究进展和挑战

Leber遗传性视神经病变(Leber hereditary optic neuropathy, LHON; MIM535000)是最典型的线粒体遗传病之一, 主要由线粒体DNA (Mitochondrial DNA, mtDNA)3个原发突变(Primary mutation, m.11778G>A、m.3460G> A 和m.14484T>C)引起。患者表现为无痛性双侧视力下降或丧失, 主要易感人群为青壮年男性。不完全外显(Incomplete penetrance)和性别偏好(Gender bias)是该病亟待解决的两大难题, 目前尚无有效的预防及治疗措施。文章对近年来LHON 的分子发病机制、临床症状及特点、体外实验和动物模型研究、预防及治疗等方面的研究进展进行综述, 并集中介绍了我们近期对于我国LHON患者的研究结果。     

The altered activity of complex III may contribute to the high penetrance of Leber's hereditary optic neuropathy in a Chinese family carrying the ND4 G11778A mutation

The ND4 G11778A mutation is the most common mitochondrial DNA mutation leading to Leber's hereditary optic neuropathy (LHON). Despite considerable clinical evidences, the modifier role of nuclear background and mitochondrial haplotypes in phenotypic manifestation of LHON remains poorly understood. We investigated the effect of these modifiers on bioenergetics in lymphoblastoid cell lines derived from five affected subjects of one Chinese family carrying the G11778A mutation and five Chinese controls. Significant reductions in the activities of complexes I and III were observed in mutant cell lines from the Chinese family, whereas the mutant cell lines from other families carrying the same mutation exhibited only reduced activity of complex I. The reduced activities of complexes I and III caused remarkably higher reductions of ATP synthesis in mutant cell lines from the Chinese family than those from other families. The deficient respiration increased generation of reactive oxygen species. The defect in complex III activity, likely resulting from the mitochondrial haplotype or nuclear gene alteration, worsens mitochondrial dysfunction caused by the G11778A mutation, thereby causing extremely high penetrance and expressivity of optic neuropathy in this Chinese family. Our data provide the first experimental evidence that altered activity of complex III modulates the phenotypic manifestation of LHON-associated G11778A mutation. Thus, our findings may provide new insights into the pathophysiology of LHON.     

GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene

A maternally inherited and practically homoplasmic mitochondrial (mtDNA) mutation, 8527A>G, changing the initiation codon AUG into GUG, normally coding for a valine, was observed in the ATP6 gene encoding the ATPase subunit a. No alternate Met codon could replace the normal translational initiator. The patient harboring this mutation exhibited clinical symptoms suggesting a mitochondrial disease but his mother who carried the same mtDNA mutation was healthy. The mutation was absent from 100 controls and occurred once amongst 44 patients suspected of Leber Hereditary Optic Neuropathy (LHON) but devoid of typical LHON mutations. In patient fibroblasts, no effect of 8527A>G mutation could be demonstrated on the biosynthesis of mtDNA-encoded proteins, on size and the content of ATPase subunit a, on ATP hydrolysis and on mitochondrial membrane potential. In addition, ATP synthesis was barely decreased. Therefore, GUG is a functional initiation codon for the human ATP6 gene.     

Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas

Defects in complex I (NADH:ubiquinone oxidoreductase (EC 1.6.5.3)) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which may hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (i) respiratory complex-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (ii) transformation of the mitochondrial genome is feasible, and (iii) Chlamydomonas complex I is similar to that of humans. To illustrate this proposal, we introduced a Leu157Pro substitution into the Chlamydomonas ND4 subunit of complex I in two recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defects when present in the heteroplasmic state in a patient with chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent assembly of whole complex I (950 kDa) and the NADH dehydrogenase activity of the peripheral arm of the complex is mildly affected. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect binding of ubiquinone to the membrane domain. The in vitro defects correlate with a decrease in dark respiration and growth rate in vivo.     

线粒体ND1基因T3866C突变可能是Leber’s遗传性视神经病和四肢畸形跛行相关的突变

线粒体DNA(Mitochondrial DNA,mtDNA)突变与人类许多疾病的发病机制相关。现报道1个具有典型母系遗传特征的中国人Leber’s遗传性视神经病和四肢畸形跛行的家系。该家系共5代60人,共27名母系成员,其中4人只有Leber’s遗传性视神经病症状,1人呈现四肢畸形跛行症状,4人同时具有上述两种临床症状,而其他成员无临床症状。对先证者的mtDNA全序列进行分析,发现ND1基因T3866C突变位点和43个多态位点,经系统进化树分析属于东亚单体型D4a3。MtDNAND13866位点T-C碱基的改变使ND1亚基第187位进化高度保守的异亮氨酸转变为苏氨酸,从而改变该蛋白的结构,进而影响其功能。在135名正常对照中未发现该突变。因此,线粒体ND1T3866C可能是与Leber’s遗传性视神经病和四肢畸形跛行相关的线粒体基因突变。     

Mitochondrial DNA Haplogroups M7b1′2 and M8a Affect Clinical Expression of Leber Hereditary Optic Neuropathy in Chinese Families with the m.11778G→A Mutation

Leber hereditary optic neuropathy (LHON) is the most extensively studied mitochondrial disease, with the majority of the cases being caused by one of three primary mitochondrial DNA (mtDNA) mutations. Incomplete disease penetrance and gender bias are two features of LHON and indicate involvement of additional genetic or environmental factors in the pathogenesis of the disorder. Haplogroups J, K, and H have been shown to influence the clinical expression of LHON in subjects harboring primary mutations in European families. However, whether mtDNA haplogroups would affect the penetrance of LHON in East Asian families has not been evaluated yet. By studying the penetrance of LHON in 1859 individuals from 182 Chinese families (including one from Cambodia) with the m.11778G→A mutation, we found that haplogroup M7b1′2 significantly increases the risk of visual loss, whereas M8a has a protective effect. Analyses of the complete mtDNA sequences from LHON families with m.11778G→A narrow the association of disease expression to m.12811T→C (Y159H) in the NADH dehydrogenase 5 gene (MT-ND5) in haplogroup M7b1′2 and suggest that the specific combination of amino acid changes (A20T-T53I) in the ATP synthase 6 protein (MT-ATP6) caused by m.8584G→A and m.8684C→T might account for the beneficial background effect of M8a. Protein secondary-structure prediction for the MT-ATP6 with the two M8a-specific amino acid changes further supported our inferences. These findings will assist in further understanding the pathogenesis of LHON and guide future genetic counseling in East Asian patients with m.11778G→A.