Mutations in the mitochondrial tRNA Ser(AGY) gene are associated with deafness, retinal degeneration, myopathy and epilepsy

Although over 200 pathogenic mitochondrial DNA (mtDNA) mutations have been reported to date, determining the genetic aetiology of many cases of mitochondrial disease is still not straightforward. Here, we describe the investigations undertaken to uncover the underlying molecular defect(s) in two unrelated Caucasian patients with suspected mtDNA disease, who presented with similar symptoms of myopathy, deafness, neurodevelopmental delay, epilepsy, marked fatigue and, in one case, retinal degeneration. Histochemical and biochemical evidence of mitochondrial respiratory chain deficiency was observed in the patient muscle biopsies and both patients were discovered to harbour a novel heteroplasmic mitochondrial tRNA (mt-tRNA)(Ser(AGY)) (MTTS2) mutation (m.12264C>T and m.12261T>C, respectively). Clear segregation of the m.12261T>C mutation with the biochemical defect, as demonstrated by single-fibre radioactive RFLP, confirmed the pathogenicity of this novel variant in patient 2. However, unusually high levels of m.12264C>T mutation within both COX-positive (98.4 ± 1.5%) and COX-deficient (98.2 ± 2.1%) fibres in patient 1 necessitated further functional investigations to prove its pathogenicity. Northern blot analysis demonstrated the detrimental effect of the m.12264C>T mutation on mt-tRNA(Ser(AGY)) stability, ultimately resulting in decreased steady-state levels of fully assembled complexes I and IV, as shown by blue-native polyacrylamide gel electrophoresis. Our findings expand the spectrum of pathogenic mutations associated with the MTTS2 gene and highlight MTTS2 mutations as an important cause of retinal and syndromic auditory impairment.     

Mitochondrial myopathy associated with a novel 5522G>A mutation in the mitochondrial tRNATrp gene

We report a novel pathogenic mutation of the mitochondrial transfer RNA (tRNA) gene for tryptophan in a patient with isolated myopathy and persistently elevated creatine kinase. Muscle studies revealed ragged red fibres and decreased activity of respiratory chain complex I and cytochrome c oxidase (COX). Sequencing of the 22 mitochondrial tRNA genes revealed a mutation m.5522G>A, which alters a conserved base pairing in the D-stem of the tRNA for tryptophan. The mutation was heteroplasmic with a mutational load between 88 and 99% in COX-negative fibres. This case contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNA genes.     

A novel mutation in the mitochondrial tRNAPro gene associated with late-onset ataxia, retinitis pigmentosa, deafness, leukoencephalopathy and complex I deficiency

We present a patient with ataxia, retinitis pigmentosa, dysarthria, neurosensorial deafness, nystagmus and leukoencephalopathy. A novel heteroplasmic G to A transition at nucleotide 15 975 was found, affecting the T arm of the mitochondrial (mt) tRNA^Pro gene. A biochemical analysis of respiratory chain enzymes in muscle revealed isolated complex I deficiency. This is the fourth pathogenic tRNA^Pro point mutation to be associated with an mt disorder. The result highlights the importance of molecular dissection of mtDNA in patients with defined mt disorder and confirms the clinical and biochemical heterogeneity associated with tRNA^Pro mutations.     

Deleterious mutation in FDX1L gene is associated with a novel mitochondrial muscle myopathy

Isolated metabolic myopathies encompass a heterogeneous group of disorders, with mitochondrial myopathies being a subgroup, with depleted skeletal muscle energy production manifesting either by recurrent episodes of myoglobinuria or progressive muscle weakness. In this study, we investigated the genetic cause of a patient from a consanguineous family who presented with adolescent onset autosomal recessive mitochondrial myopathy. Analysis of enzyme activities of the five respiratory chain complexes in our patients'' skeletal muscle showed severely impaired activities of iron sulfur (Fe-S)-dependent complexes I, II and III and mitochondrial aconitase. We employed exome sequencing combined with homozygosity mapping to identify a homozygous mutation, c.1A>T, in the FDX1L gene, which encodes the mitochondrial ferredoxin 2 (Fdx2) protein. The mutation disrupts the ATG initiation translation site resulting in severe reduction of Fdx2 content in the patient muscle and fibroblasts mitochondria. Fdx2 is the second component of the Fe-S cluster biogenesis machinery, the first being IscU that is associated with isolated mitochondrial myopathy. We suggest adding genetic analysis of FDX1L in cases of mitochondrial myopathy especially when associated with reduced activity of the respiratory chain complexes I, II and III.     

The mitochondrial 13513G>A mutation is associated with Leigh disease phenotypes independent of complex I deficiency in muscle

The mitochondrial 13513G>A (D393N) mutation in the ND5 subunit of the respiratory chain complex I was initially described in association with MELAS syndrome. Recent observations have linked this mutation to Leigh disease. We screened for the 13513G>A mutation in a cohort of 265 patients with Leigh and Leigh-like disease. The mutation was found in a total of 5 patients. An additional patient who had clinical presentation consistent with a Leigh-like phenotype but with a normal brain MRI was added to the cohort. None of an additional 88 patients meeting MELAS disease criteria, nor 56 patients with respiratory chain deficiency screened for the 13513G>A were found positive for the mutation. The most frequent clinical manifestations in our patients were hypotonia, ocular and cerebellar involvement. Low mutation heteroplasmy in the range of 20–40% was observed in all 6 patients. This observation is consistent with the previously reported low heteroplasmy of this mutation in some patients with the 13513G>A mutation and complex I deficiency. However, normal complex I activity was observed in two patients in our cohort. As most patients with Leigh-like disease and the 13513G>A mutation have been described with complex I deficiency, this report adds to the previously reported subset of patients with normal respiratory complex function. We conclude that in any patient with Leigh or Leigh-like disease, testing for the 13513G>A mutation is clinically relevant and low mutant loads in blood or muscle may be considered pathogenic, in the presence of normal respiratory chain enzyme activities.     

A novel mutation in the mitochondrial tRNA(Ser(UCN)) gene in a family with non-syndromic sensorineural hearing impairment

We describe a family with non-syndromic sensorineural hearing impairment inherited in a manner consistent with maternal transmission. Affected members were found to have a novel heteroplasmic mtDNA mutation, T7510C, in the tRNA^Ser(UCN) gene. This mutation was not found in 661 controls, is well conserved between species, and disrupts base pairing in the acceptor stem of the tRNA, making it the probable cause of hearing impairment in this family. Sequencing of the other mitochondrial tRNA genes did not show any other pathogenic mutations. Four other mutations causing hearing impairment have been reported in the tRNA^Ser(UCN) gene, two having been shown to affect tRNA^Ser(UCN) levels. With increasing numbers of reports of mtDNA mutations causing hearing impairment, screening for such mutations should be considered in all cases unless mitochondrial inheritance can be excluded for certain.


Keywords: hearing impairment; mtDNA mutation; tRNA^Ser(UCN)     

A novel mitochondrial ATP6 frameshift mutation causing isolated complex V deficiency,ataxia and encephalomyopathy

We describe a novel frameshift mutation in the mitochondrial ATP6 gene in a 4-year-old girl associated with ataxia, microcephaly, developmental delay and intellectual disability.A heteroplasmic frameshift mutation in the MT-ATP6 gene was confirmed in the patient's skeletal muscle and blood. The mutation was not detectable in the mother's DNA extracted from blood or buccal cells. Enzymatic and oxymetric analysis of the mitochondrial respiratory system in the patients' skeletal muscle and skin fibroblasts demonstrated an isolated complex V deficiency. Native PAGE with subsequent immunoblotting for complex V revealed impaired complex V assembly and accumulation of ATPase subcomplexes. Whilst northern blotting confirmed equal presence of ATP8/6 mRNA, metabolic ³⁵S-labelling of mitochondrial translation products showed a severe depletion of the ATP6 protein together with aberrant translation product accumulation. In conclusion, this novel isolated complex V defect expands the clinical and genetic spectrum of mitochondrial defects of complex V deficiency. Furthermore, this work confirms the benefit of native PAGE as an additional diagnostic method for the identification of OXPHOS defects, as the presence of complex V subcomplexes is associated with pathogenic mutations of mtDNA.     

Identification of 45 novel mutations in the nebulin gene associated with autosomal recessive nemaline myopathy

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder of skeletal muscle caused by mutations in at least five different genes encoding thin filament proteins of the striated muscle sarcomere. We have previously described 18 different mutations in the last 42 exons of the nebulin gene (NEB) in 18 families with NM. Here we report 45 novel NEB mutations detected by denaturing high-performance liquid chromatography (dHPLC) and sequence analysis of all 183 NEB exons in NM patients from 44 families. Altogether we have identified, including the deletion of exon 55 identified in the Ashkenazi Jewish population, 64 different mutations in NEB segregating with autosomal recessive NM in 55 families. The majority (55%) of the mutations in NEB are frameshift or nonsense mutations predicted to cause premature truncation of nebulin. Point mutations (25%) or deletions (3%) affecting conserved splice signals are predicted in the majority of cases to cause in-frame exon skipping, possibly leading to impaired nebulin-tropomyosin interaction along the thin filament. Patients in 18 families had one of nine missense mutations (14%) affecting conserved amino acids at or in the vicinity of actin or tropomyosin binding sites. In addition, we found the exon 55 deletion in four families. The majority of the patients (in 49/55 families) were shown to be compound heterozygous for two different mutations. The mutations were found in both constitutively and alternatively expressed exons throughout the NEB gene, and there were no obvious mutational hotspots. Patients with more severe clinical pictures tended to have mutations predicted to be more disruptive than patients with milder forms.     

线粒体tRNAThr A15951G可能是与Leber遗传性视神经病变相关的基因突变

目的 进一步分析中国汉族Leber遗传性视神经病变(Leber's hereditary optic neuropathy,LHON)家系的临床和分子遗传学特征,阐明LHON的分子致病机制.方法 对2例具有典型LHON临床特征的先证者和家系其他成员进行眼科学及其临床检查.对这2个家系先证者使用24对有部分重叠的引物进行线粒体DNA(mitochondrial DNA,mtDNA)全序列扩增分析.结果 检查发现这些家系成员中视力损害的外显率分别为5.3％(1/19)、18.2％(4/22).经mtDNA测序分析,并没有发现mtDNA G11778A、G3460A和T14484C 3个常见的突变,在tRNAThr上发现了A15951G同质性突变位点.线粒体DNA全序列分析显示2个家系呈现mtDNA多态性,都属于东亚单倍型D4b1.A15951G突变位于线粒体tRNAThr高度保守区(通用位点为71位),可能导致tRNA空间结构和稳定性发生改变,线粒体蛋白合成功能受损,最终发生视力损害.结论 线粒体tRNAThr A15951G可能是与Leber遗传性视神经病变相关的致病性线粒体基因突变.     

Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNA Ser(UCN) gene

We report on a new maternally-inherited syndrome characterizedby a combination of sensorineural hearing loss, ataxia and myoclonusin a large kindred from Sicily. Hearing loss was the most widespreadand sometimes the only symptom found in family members. Sequenceanalysis of the mitochondrial DNA regions encompassing the tRNAgenes revealed the presence of a heteroplasmic insertion atnucleotide position 7472. The insertion adds a seventh cytosineto a six-cytosine run that is part of the mitochondrial tRNA^Ser(UCN) gene. Conformational analysis showed that this mutationis likely to alter the structure of the T     

Expression pattern of mitochondrial respiratory chain enzymes in skeletal muscle of patients with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant

Two homoplasmic variants in tRNA^Glu (m.14674T>C/G) are associated with reversible infantile respiratory chain deficiency. This study sought to further characterize the expression of the individual mitochondrial respiratory chain complexes and to describe the natural history of the disease. Seven patients from four families with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant were investigated. All patients underwent skeletal muscle biopsy and mtDNA sequencing. Whole‐genome sequencing was performed in one family. Western blot and immunohistochemical analyses were used to characterize the expression of the individual respiratory chain complexes. Patients presented with hypotonia and feeding difficulties within the first weeks or months of life, except for one patient who first showed symptoms at 4 years of age. Histopathological findings in muscle included lipid accumulation, numerous COX‐deficient fibers, and mitochondrial proliferation. Ultrastructural abnormalities included enlarged mitochondria with concentric cristae and dense mitochondrial matrix. The m.14674T>C variant in MT‐TE was identified in all patients. Immunohistochemistry and immunoblotting demonstrated pronounced deficiency of the complex I subunit NDUFB8. The expression of MTCO1, a complex IV subunit, was also decreased, but not to the same extent as NDUFB8. Longitudinal follow‐up data demonstrated that not all features of the disorder are entirely transient, that the disease may be progressive, and that signs and symptoms of myopathy may develop during childhood. This study sheds new light on the involvement of complex I in reversible infantile respiratory chain deficiency, it shows that the disorder may be progressive, and that myopathy can develop without an infantile episode.     

Haplogroup analysis supports a pathogenic role for the 7510T>C mutation of mitochondrial tRNA(Ser(UCN)) in sensorineural hearing loss

We ascertained a large North American family, LMG309, with matrilineal transmission of non-syndromic, progressive sensorineural hearing loss (SNHL). There was no history of aminoglycoside exposure, and penetrance was complete. We sequenced the entire mitochondrial genome and identified the previously reported 7510T>C transition in the tRNA^Ser(UCN) gene. The 7510T>C was homoplasmic in all affected members. The LMG309 mitochondrial sequence belongs to an unnamed subgroup of mitochondrial haplogroup H. We demonstrate that the previously reported Spanish family S258 carries 7510T>C on a different mitochondrial sub-haplogroup, H1. We did not detect 7510T>C among 79 Caucasian haplogroup H control samples, including 11 from sub-haplogroup H1 and one from the same sub-haplogroup as LMG309. Our results provide strong genetic evidence that 7510T>C is a pathogenic mutation that causes non-syndromic SNHL.     

糖尿病家系的线粒体基因tRNALeu(UUR)A3243G突变分析

目的 分析线粒体基因tRNALeu(UUR)A3243G突变的糖尿病家系中发病规律.方法 筛选临床疑似线粒体糖尿病家系,采用PCR、DNA直接测序技术对3个家系19例临床疑似线粒体基因突变糖尿病家系进行线粒体基因突变高发区域tRNALeu(UUR)基因的检测.结果 3个家系发现与糖尿病发病有关的突变位点均位于nt3243A→G突变,且家系中大部分患者伴有消瘦、耳聋、β细胞功能低下、发病年龄低的特点.结论 线粒体tRNALeu(UUR)基因3243位点A→G突变可导致糖尿病和耳聋.     

A distinct mitochondrial myopathy,lactic acidosis and sideroblastic anemia (MLASA) phenotype associates with YARS2 mutations

Nuclear‐encoded disorders of mitochondrial translation are clinically and genetically heterogeneous. Genetic causes include defects of mitochondrial aminoacyl‐tRNA synthetases, and factors required for initiation, elongation and termination of protein synthesis as well as ribosome recycling. We report on a new case of myopathy, lactic acidosis and sideroblastic anemia (MLASA) syndrome caused by defective mitochondrial tyrosyl aminoacylation. The patient presented at 1 year with anemia initially attributed to iron deficiency. Bone marrow aspirate at 5 years revealed ringed sideroblasts but transfusion dependency did not occur until 11 years. Other clinical features included lactic acidosis, poor weight gain, hypertrophic cardiomyopathy and severe myopathy leading to respiratory failure necessitating ventilatory support. Long‐range PCR excluded mitochondrial DNA rearrangements. Clinical diagnosis of MLASA prompted direct sequence analysis of the YARS2 gene encoding the mitochondrial tyrosyl‐tRNA synthetase, which revealed homozygosity for a known pathogenic mutation, c.156C>G;p.F52L. Comparison with four previously reported cases demonstrated remarkable clinical homogeneity. First line investigation of MLASA should include direct sequence analysis of YARS2 and PUS1 (encoding a tRNA modification factor) rather than muscle biopsy. Early genetic diagnosis is essential for counseling and to facilitate appropriate supportive therapy. Reasons for segregation of specific clinical phenotypes with particular mitochondrial aminoacyl tRNA‐synthetase defects remain unknown. © 2013 Wiley Periodicals, Inc.     

A novel recurrent mitochondrial DNA mutation in ND3 gene is associated with isolated complex I deficiency causing Leigh syndrome and dystonia

Defects in NADH:ubiquinone oxidoreductase (complex I), the largest complex of the mitochondrial respiratory chain, account for most cases of respiratory chain deficiency in human. Complex I contains at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). Here we report a novel 10197G>A mutation of the ND3 gene in three unrelated families with Leigh syndrome (LS) or dystonia. Variable degrees of heteroplasmy were found in all tissues tested and a high percentage of mutant mtDNA was observed in muscle. The 10197G>A mutation modifies a hydrophobic alanine residue into a hydrophilic threonine (A47T) in a highly conserved domain of ND3 subunit. Furthermore, this defect could be transferred along with the mutant mtDNAs to rho degrees lymphoblastoid cells in cybrid experiments. However, nuclear modifier genes may also play a role in the phenotypic expression and severity of the 10197G>A mutation. The association of the 10197G>A ND3 mutation with an isolated biochemical defect involving complex I and the discovery of the 10197G>A mutation with a similar phenotype in three unrelated families establish its pathogenicity and demonstrate that the amino acid position A47 is important for the function of complex I. These results show that the 10197G>A mutation in the mitochondrial ND3 gene should be considered as a common mtDNA mutation responsible for LS and dystonia.     

Nephropathy and growth hormone deficiency in a patient with mitochondrial tRNA(Leu(UUR)) mutation.

A mitochondrial A 3243 G mutation in the tRNA(Leu(UUR)) gene was first described as a common cause of MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like syndrome). This same mutation is also the cause of a totally different disorder, a subtype of diabetes mellitus which is inherited maternally and often associated with sensorineural hearing loss. In this paper, we report on a Japanese boy with A 3243 G who developed a previously undescribed combination of symptoms, nephropathy and growth hormone deficiency. The patient first presented with short stature and moderate mental retardation. Growth hormone (GH) provocation tests showed deficient growth hormone secretion. During the course of follow up, he presented with progressive nephropathy followed by the development of diabetes mellitus. The results of laboratory tests and renal biopsy were against incidental association of known types of nephropathy. On PCR-RFLP analysis, the percentage of mutated mtDNA was higher in the renal biopsy specimen than 12 peripheral blood leucocytes. Our case suggests that mitochondrial diseases should be taken into account when there is nephropathy of unknown cause. In addition, the presence of growth hormone deficiency may account for part of the mechanism leading to short stature commonly seen in these patients.     

A homozygous variant in NDUFA8 is associated with developmental delay,microcephaly, and epilepsy due to mitochondrial complex I deficiency

Mitochondrial complex I deficiency is caused by pathogenic variants in mitochondrial and nuclear genes associated with complex I structure and assembly. We report the case of a patient with NDUFA8-related mitochondrial disease. The patient presented with developmental delay, microcephaly, and epilepsy. His fibroblasts showed apparent biochemical defects in mitochondrial complex I. Whole-exome sequencing revealed that the patient carried a homozygous variant in NDUFA8. His fibroblasts showed a reduction in the protein expression level of not only NDUFA8, but also the other complex I subunits, consistent with assembly defects. The enzyme activity of complex I and oxygen consumption rate were restored by reintroducing wild-typeNDUFA8 cDNA into patient fibroblasts. The functional properties of the variant in NDUFA8 were also investigated using NDUFA8 knockout cells expressing wild-type or mutated NDUFA8 cDNA. These experiments further supported the pathogenicity of the variant in complex I assembly. This is the first report describing that the loss of NDUFA8, which has not previously been associated with mitochondrial disease, causes severe defect in the assembly of mitochondrial complex I, leading to progressive neurological and developmental abnormalities.     

线粒体tRNALeu基因突变导致糖尿病分子机制进展

尽管线粒体糖尿病被发现有多个致病点突变,但tRNALeu(UUR)3243A>G突变仍是目前国际上惟一公认的位点.胰岛β细胞过早的功能衰退和凋亡,胰岛素合成减少以及ATP/ADP介导的胰岛分泌功能障碍可能是该位点突变致糖尿病发生发展的主要原因.本文就该突变类型所致糖尿病的分子发病机制作一综述.