De novo frameshift variant in MT-ND1 causes a mitochondrial complex I deficiency associated with MELAS syndrome.

BACKGROUND: The variant m.3571_3572insC/MT-ND1 thus far only reported in oncocytic tumors of different tissues. However, the role of m.3571_3572insC in inherited mitochondrial diseases has yet to be elucidated. METHODS: A patient diagnosed with MELAS syndrome was recruited, and detailed medical records were collected and reviewed. The muscle was biopsied for mitochondrial respiratory chain enzyme activity. Series of fibroblast clones bearing different m.3571_3572insC variant loads were generated from patient-derived fibroblasts and subjected to functional assays. RESULTS: Complex I deficiency was confirmed in the patient's muscle via mitochondrial respiratory chain enzyme activity assay. The m.3571_3572insC was filtered for the candidate variant of the patient according to the guidelines for mitochondrial mRNA variants interpretation. Three cell clones with different m.3571_3572insC variant loads were generated to evaluate mitochondrial function. Blue native PAGE analysis revealed that m.3571_3572insC caused a deficiency in the mitochondrial complex I. Oxygen consumption rate, ATP production, and lactate assays found an impairment of cellular bioenergetic capacity due to m.3571_3572insC. Mitochondrial membrane potential was decreased, and mitochondrial reactive oxygen species production was increased with the variant of m.3571_3572insC. According to the competitive cell growth assay, the mutant cells had impaired cell growth capacity compared to wild type. CONCLUSIONS: A novel variant m.3571_3572insC was identified in a patient diagnosed with MELAS syndrome, and the variant impaired mitochondrial respiration by decreasing the activity of complex I. In conclusion, the genetic spectrum of mitochondrial diseases was expanded by including m.3571_3572insC/MT-ND1.

Novel biallelic mutations in TMEM126B cause splicing defects and lead to Leigh-like syndrome with severe complex I deficiency.

Leigh syndrome (LS)/Leigh-like syndrome (LLS) is one of the most common mitochondrial disease subtypes, caused by mutations in either the nuclear or mitochondrial genomes. Here, we identified a novel intronic mutation (c.82-2 A > G) and a novel exonic insertion mutation (c.290dupT) in TMEM126B from a Chinese patient with clinical manifestations of LLS. In silico predictions, minigene splicing assays and patients' RNA analyses determined that the c.82-2 A > G mutation resulted in complete exon 2 skipping, and the c.290dupT mutation provoked partial and complete exon 3 skipping, leading to translational frameshifts and premature termination. Functional analysis revealed the impaired mitochondrial function in patient-derived lymphocytes due to severe complex I content and assembly defect. Altogether, this is the first report of LLS in a patient carrying mutations in TMEM126B. Our data uncovers the functional effect and the molecular mechanism of the pathogenic variants c.82-2 A > G and c.290dupT, which expands the gene mutation spectrum of LLS and clinical spectrum caused by TMEM126B mutations, and thus help to clinical diagnosis of TMEM126B mutation-related mitochondrial diseases.

Biallelic Mutations in ACACA Cause a Disruption in Lipid Homeostasis That Is Associated With Global Developmental Delay, Microcephaly, and Dysmorphic Facial Features.

OBJECTIVE: We proposed that the deficit of ACC1 is the cause of patient symptoms including global developmental delay, microcephaly, hypotonia, and dysmorphic facial features. We evaluated the possible disease-causing role of the ACACA gene in developmental delay and investigated the pathogenesis of ACC1 deficiency. METHODS: A patient who presented with global developmental delay with unknown cause was recruited. Detailed medical records were collected and reviewed. Whole exome sequencing found two variants of ACACA with unknown significance. ACC1 mRNA expression level, protein expression level, and enzyme activity level were detected in patient-derived cells. Lipidomic analysis, and in vitro functional studies including cell proliferation, apoptosis, and the migratory ability of patient-derived cells were evaluated to investigate the possible pathogenic mechanism of ACC1 deficiency. RNAi-induced ACC1 deficiency fibroblasts were established to assess the causative role of ACC1 deficit in cell migratory disability in patient-derived cells. Palmitate supplementation assays were performed to assess the effect of palmitic acid on ACC1 deficiency-induced cell motility deficit. RESULTS: The patient presented with global developmental delay, microcephaly, hypotonia, and dysmorphic facial features. A decreased level of ACC1 and ACC1 enzyme activity were detected in patient-derived lymphocytes. Lipidomic profiles revealed a disruption in the lipid homeostasis of the patient-derived cell lines. In vitro functional studies revealed a deficit of cell motility in patient-derived cells and the phenotype was further recapitulated in ACC1-knockdown (KD) fibroblasts. The cell motility deficit in both patient-derived cells and ACC1-KD were attenuated by palmitate. CONCLUSION: We report an individual with biallelic mutations in ACACA, presenting global development delay. In vitro studies revealed a disruption of lipid homeostasis in patient-derived lymphocytes, further inducing the deficit of cell motility capacity and that the deficiency could be partly attenuated by palmitate.

Nanoscopic quantification of sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells derived from patients with mitochondrial diseases.

SLC25A46 mutations have been found to lead to mitochondrial hyper-fusion and reduced mitochondrial respiratory function, which results in optic atrophy, cerebellar atrophy, and other clinical symptoms of mitochondrial disease. However, it is generally believed that mitochondrial fusion is attributable to increased mitochondrial oxidative phosphorylation (OXPHOS), which is inconsistent with the decreased OXPHOS of highly-fused mitochondria observed in previous studies. In this paper, we have used the live-cell nanoscope to observe and quantify the structure of mitochondrial cristae, and the behavior of mitochondria and lysosomes in patient-derived SLC25A46 mutant fibroblasts. The results show that the cristae have been markedly damaged in the mutant fibroblasts, but there is no corresponding increase in mitophagy. This study suggests that severely damaged mitochondrial cristae might be the predominant cause of reduced OXPHOS in SLC25A46 mutant fibroblasts. This study demonstrates the utility of nanoscope-based imaging for realizing the sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells, which may be particularly valuable for the quick evaluation of pathogenesis of mitochondrial morphological abnormalities.

Association of macrophage inhibitory factor -1 polymorphisms with antiviral efficacy of type 1b chronic hepatitis C.

The expression of macrophage inhibitory factor-1 (MIC-1) increases in patients with chronic hepatitis C (CHC), but whether MIC-1 level and its polymorphism affect the antiviral efficacy of CHC has not yet been reported. The present study aimed to investigate the association between MIC-1 polymorphism and antiviral efficacy in patients with CHC genotype 1b (CHC 1b). A total of 171 patients with CHC1b were recruited. The polymorphisms of rs1059369 and rs1059519 in MIC-1 were detected by DNA sequencing. All patients received a standard dose of polyethylene glycol interferon + ribavirin (PR regimen), and divided into response, nonresponse, sustained virological response (SVR), and non-sustained virological response (NSVR) groups based on HCV RNA levels. The genotype distribution of the two single nucleotide polymorphisms (SNPs) did not differ between the response and nonresponse groups, SVR and non-SVR groups. However, the level of MIC-1 was positively correlated with ALT, AST, PIIINP, CIV, and HCV RNA (P < 0.05). Compared to before treatment, the level of MIC-1 in plasma was significantly decrease in the response group but not in the non-responsive group. Our results suggest that the level of MIC-1 in CHC1b is correlated with liver cell injury, liver fibrosis index, and viral load. However, the polymorphism of rs1059369 and rs1059519 may have negligible impact in expression of MIC-1 and efficacy of antiviral therapy in CHC patient.

Mitochondrial genome variant m.3250T>C as a possible risk factor for mitochondrial cardiomyopathy.

The MT-TL1 gene codes for the mitochondrial leucine transfer RNA (tRNALeu(UUR) ) necessary for mitochondrial translation. Pathogenic variants in the MT-TL1 gene result in mitochondriopathy in humans. The m.3250T>C variant in the MT-TL1 gene has been previously associated with exercise intolerance and mitochondrial myopathy, yet disease classification for this variant has not been consistently reported. Molecular studies suggest the m.3250T>C variant does not alter tRNALeu(UUR) structure but may have a modest impact on aminoacylation capacity. However, functional studies are limited. Our study aimed to further define the clinical presentation, inheritance pattern, and molecular pathology of the m.3250T>C variant. Families with the m.3250T>C variant were recruited from the Mitochondrial Disease Clinic at Cincinnati Children's Hospital Medical Center and GeneDx laboratory database. Affected individuals most frequently presented with cardiac findings, exercise intolerance, and muscle weakness. Hypertrophic cardiomyopathy was the most frequent cardiac finding. Many asymptomatic individuals had homoplasmic or near homoplasmic levels of the m.3250T>C variant, suggesting the penetrance is incomplete. Patient-derived fibroblasts demonstrated lowered ATP production and increased levels of reactive oxygen species. Our results demonstrate that the m.3250T>C variant exhibits incomplete penetrance and may be a possible cause of cardiomyopathy by impacting cellular respiration in mitochondria.

Systematic analysis of a mitochondrial disease-causing ND6 mutation in mitochondrial deficiency.

BACKGROUND: The m.14487T>C mutation is recognized as a diagnostic mutation of mitochondrial disease during the past 16 years, emerging evidence suggests that mutant loads of m.14487T>C and disease phenotype are not closely correlated. METHODS: Immortalized lymphocytes were generated by coculturing the Epstein-Barr virus and lymphocytes from m.14487T>C carrier Chinese patient with Leigh syndrome. Fifteen cytoplasmic hybrid (cybrid) cell lines were generated by fusing mtDNA lacking 143B cells with platelets donated by patients. Mitochondrial function was systematically analyzed at transcriptomic, metabolomic, and biochemical levels. RESULTS: Unlike previous reports, we found that the assembly of mitochondrial respiratory chain complexes, mitochondrial respiration, and mitochondrial OXPHOS function was barely affected in cybrid cells carrying homoplastic m.14487T>C mutation. Mitochondrial dysfunction associated transcriptomic and metabolomic reprogramming were not detected in cybrid carrying homoplastic m.14487T>C. However, we found that mitochondrial function was impaired in patient-derived immortalized lymphocytes. CONCLUSION: Our data revealed that m.14487T>C mutation is insufficient to cause mitochondrial deficiency; additional modifier genes may be involved in m.14487T>C-associated mitochondrial disease. Our results further demonstrated that a caution should be taken by solely use of m.14487T>C mutation for molecular diagnosis of mitochondrial disease.

Systemic administration of AAV-Slc25a46 mitigates mitochondrial neuropathy in Slc25a46-/- mice.

Mitochondrial disorders are the result of nuclear and mitochondrial DNA mutations that affect multiple organs, with the central and peripheral nervous system often affected. Currently, there is no cure for mitochondrial disorders. Currently, gene therapy offers a novel approach for treating monogenetic disorders, including nuclear genes associated with mitochondrial disorders. We utilized a mouse model carrying a knockout of the mitochondrial fusion-fission-related gene solute carrier family 25 member 46 (Slc25a46) and treated them with neurotrophic AAV-PHP.B vector carrying the mouse Slc25a46 coding sequence. Thereafter, we used immunofluorescence staining and western blot to test the transduction efficiency of this vector. Toluidine blue staining and electronic microscopy were utilized to assess the morphology of optic and sciatic nerves following treatment, and the morphology and respiratory chain activity of mitochondria within these tissues were determined as well. The adeno-associated virus (AAV) vector effectively transduced in the cerebrum, cerebellum, heart, liver and sciatic nerves. AAV-Slc25a46 treatment was able to rescue the premature death in the mutant mice (Slc25a46-/-). The treatment-improved electronic conductivity of the peripheral nerves increased mobility and restored mitochondrial complex activities. Most notably, mitochondrial morphology inside the tissues of both the central and peripheral nervous systems was normalized, and the neurodegeneration, chronic neuroinflammation and loss of Purkinje cell dendrites observed within the mutant mice were alleviated. Overall, our study shows that AAV-PHP.B's neurotrophic properties are plausible for treating conditions where the central nervous system is affected, such as many mitochondrial diseases, and that AAV-Slc25a46 could be a novel approach for treating SLC25A46-related mitochondrial disorders.

Erxian decoction, a Chinese herbal formula, for menopausal syndrome: An updated systematic review.

ETHNOPHARMACOLOGICAL RELEVANCE: Erxian decoction (EXD), a famous Chinese herbal prescription, consists of Rhizoma Curculiginis, Herba Epimedii, Radix Morindae Officinalis, Radix Angelicae Sinensis, Cortex Phellodendri, Rhizoma Anemarrhenae, all of which are recorded in the Chinese Pharmacopoeia. OBJECTIVE: To conduct an updated systematic and meta-analysis investigating efficacy and safety of EXD for menopausal syndrome. METHODS: An electronic search was conducted in eight databases from inception until July 2018. Randomized controlled trials with risk-of-bias score ≥ 7 according to the Cochrane Back Review Group were included for analyses. All participants with a diagnosis of menopausal syndrome met the established criteria. The treatment group was EXD monotherapy or adjunct therapy. Comparators were placebo, hormone replace therapy, hormone plus nonhormonal agents, nonhormonal agents and no treatment. The primary outcome measurements were the Kupperman index, total hot flush scores, total menopause rating scale (MRS) scores and total menopause-specific quality of life (MENQOL) scores. The secondary outcomes were total clinical effective rate, traditional Chinese medicine (TCM) syndrome scores, Hamilton depression (HAMD) scale scores, self-rating depression scale (SDS) scores, self-Rating Anxiety Scale (SAS) scores, athens insomnia scale (AIS) scores, serological indicators, blood pressure, and adverse events. RevMan 5.3 Software was used for data analyses. GRADE system was used to assess the level of evidence. RESULTS: Sixteen eligible studies with 1594 subjects were identified. Five studies showed EXD was contradictory results according to Kupperman index of menopausal syndrome compared with hormone. One study showed EXD significantly improved total hot flush scores, total MRS scores and total MENQOL scores compared with placebo (P < 0.05). Meta-analysis of 10 EXD monotherapy or 2 paratherapy studies showed that both can significantly improve total effective rate compared with hormone (P < 0.05); 3 studies showed that EXD plus hormone significantly reduces the TCM syndrome scores, HAMD scale scores, SDS scores and SAS scores compared with hormone (P < 0.05). One study showed a significant effect of EXD for reducing AIS scores compared with hormone (P < 0.05); 7 studies showed contradictory effects for improving serological indicators compared with hormone. Two studies reported adverse effects, whereas the other studies did not mention. The quality of the evidence of primary outcomes was moderate to high according to the GRADE profiler. CONCLUSIONS: The present findings do not allow an assessment of the evidence because the low-quality studies included cannot be reproduced. However, we identified an area, which is worthy of further research. Rigorous RCTs are still needed in the future.

Biomechanical properties of novel transpedicular transdiscal screw fixation with interbody arthrodesis technique in lumbar spine: A finite element study.

PURPOSE: The purpose of this study was to investigate finite element biomechanical properties of the novel transpedicular transdiscal (TPTD) screw fixation with interbody arthrodesis technique in lumbar spine. METHODS: An L4-L5 finite element model was established and validated. Then, two fixation models, TPTD screw system and bilateral pedicle screw system (BPSS), were established on the validated L4-L5 finite element model. The inferior surface of the L5 vertebra was set immobilised, and moment of 7.5 Nm was applied on the L4 vertebra to test the range of motion (ROM) and stress at flexion, extension, lateral bending and axial rotation. RESULTS: The intact model was validated for prediction accuracy by comparing two previously published studies. Both of TPTD and BPSS fixation models displayed decreased motion at L4-L5. The ROMs of six moments of flexion, extension, left lateral bending, right lateral bending, left axial rotation and right axial rotation in TPTD model were 1.92, 2.12, 1.10, 1.11, 0.90 and 0.87°, respectively; in BPSS model, they were 1.48, 0.42, 0.35, 0.38, 0.74 and 0.75°, respectively. The screws' peak stress of above six moments in TPTD model was 182.58, 272.75, 133.01, 137.36, 155.48 and 150.50 MPa, respectively; and in BPSS model, it was 103.16, 129.74, 120.28, 134.62, 180.84 and 169.76 MPa, respectively. CONCLUSION: Both BPSS and TPTD can provide stable biomechanical properties for lumbar spine. The decreased ROM of flexion, extension and lateral bending was slightly more in BPSS model than in TPTD model, but TPTD model had similar ROM of axial rotation with BPSS model. The screws' peak stress of TPTD screw focused on the L4-L5 intervertebral space region, and more caution should be put at this site for the fatigue breakage. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our finite element study provides the biomechanical properties of novel TPTD screw fixation, and promotes this novel transpedicular transdiscal screw fixation with interbody arthrodesis technique be used clinically.

Correction: A novel NDUFS3 mutation in a Chinese patient with severe Leigh syndrome.

The originally published version of this article contained an error in Figure 1. The correct figure of this article should have read as below. This has now been corrected in the PDF and HTML versions of the article. The authors apologize for any inconvenience caused.

A Novel NDUFS3 mutation in a Chinese patient with severe Leigh syndrome.

Leigh syndrome is one of the most common subtypes of mitochondrial disease. Mutations in encoding genes of oxidative phosphorylation complexes have been frequently reported, of which, MTATP6 was one of the most frequently reported genes for Leigh syndrome. In this study, by using next-generation sequencing targeted to MitoExome in a patient with clinical manifestations of Leigh syndrome, two missense mutations of NDUFS3 (c.418 C > T/p.R140W and c.595 C > T/p.R199W) were identified, of which c.418 C > T was novel. Functionally, the patient derived lymphoblastoid cells showed decreased amount of NDUFS3 and complex I assembly when compared with two control cells. Although NDUFS3 mutations have been related to late onset Leigh syndrome, we found that the patient carrying these two mutations developed an early onset Leigh syndrome. To our knowledge, this is the second study on patient carrying NDUFS3 mutations. In conclusion, we identified a novel Leigh syndrome causing NDUFS3 mutation and expanded the clinical spectrum caused by NDUFS3 mutations in this study.