Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with a novel de novo mutation (m.8969G>A) in the mitochondrial encoded ATP6 gene.

Mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA) is a rare mitochondrial disorder that has previously been associated with mutations in PUS1 and YARS2. In the present report, we describe a 6-year old male with an MLASA plus phenotype. This patient had features of MLASA in the setting of developmental delay, sensorineural hearing loss, epilepsy, agenesis of the corpus callosum, failure to thrive, and stroke-like episodes. Sequencing of the mitochondrial genome identified a novel de novo, heteroplasmic mutation in the mitochondrial DNA (mtDNA) encoded ATP6 gene (m.8969G>A, p.S148N). Whole exome sequencing did not identify mutations or variants in PUS1 or YARS2 or any known nuclear genes that could affect mitochondrial function and explain this phenotype. Studies of fibroblasts derived from the patient revealed a decrease in oligomycin-sensitive respiration, a finding which is consistent with a complex V defect. Thus, this mutation in MT-ATP6 may represent the first mtDNA point mutation associated with the MLASA phenotype.

Transition to next generation analysis of the whole mitochondrial genome: a summary of molecular defects.

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity. Conventional analysis of the mitochondrial genome often starts with a screening panel for common mitochondrial DNA (mtDNA) point mutations and large deletions (mtScreen). If negative, it has been traditionally followed by Sanger sequencing of the entire mitochondrial genome (mtWGS). The recently developed "Next-Generation Sequencing" (NGS) technology offers a robust high-throughput platform for comprehensive mtDNA analysis. Here, we summarize the results of the past 6 years of clinical practice using the mtScreen and mtWGS tests on 9,261 and 2,851 unrelated patients, respectively. A total of 344 patients (3.7%) had mutations identified by mtScreen and 99 (3.5%) had mtDNA mutations identified by mtWGS. The combinatorial analyses of mtDNA and POLG revealed a diagnostic yield of 6.7% in patients with suspected mitochondrial disorders but no recognizable syndromes. From the initial mtWGS-NGS cohort of 391 patients, 21 mutation-positive cases (5.4%) have been identified. The mtWGS-NGS provides a one-step approach to detect common and uncommon point mutations, as well as deletions. Additionally, NGS provides accurate, sensitive heteroplasmy measurement, and the ability to map deletion breakpoints. A new era of more efficient molecular diagnosis of mtDNA mutations has arrived.

Age and gestural differences in the ease of rotating a virtual 3D image on a large, multi-touch screen.

Providing a natural mapping between multi-touch gestures and manipulations of digital content is important for user-friendly interfaces. Although there are some guidelines for 2D digital content available in the literature, a guideline for manipulation of 3D content has yet to be developed. In this research, two sets of gestures were developed for experiments in the ease of manipulating 3D content on a touchscreen. As there typically are large differences between age groups in the ease of learning new interfaces, we compared a group of adults with a group of children. Each person carried out three tasks linked to rotating the digital model of a green turtle to inspect major characteristics of its body. Task completion time, subjective evaluations, and gesture changing frequency were measured. Results showed that using the conventional gestures for 2D object rotation was not appropriate in the 3D environment. Gestures that required multiple touch points hampered the real-time visibility of rotational effects on a large screen. While the cumulative effects of 3D rotations became complicated after intensive operations, simpler gestures facilitated the mapping between 2D control movements and 3D content displays. For rotation in Cartesian coordinates, moving one fingertip horizontally or vertically on a 2D touchscreen corresponded to the rotation angles of two axes for 3D content, while the relative movement between two fingertips was used to control the rotation angleof the third axis. Based on behavior analysis, adults and children differed in the diversity of gesture types and in the touch points with respect to the object's contours. Offering a robust mechanism for gestural inputs is necessary for universal control of such a system.

Molecular characterization of CPS1 deletions by array CGH.

CPSI deficiency usually results in severe hyperammonemia presenting in the first days of life warranting prompt diagnosis. Most CPS1 defects are non-recurrent, private mutations, including point mutation, small insertions and deletions. In this study, we report the detection of large deletions varying from 1.4 kb to >130 kb in the CPS1 gene of 4 unrelated patients by targeted array CGH. These results underscore the importance of analysis of large deletions when only one mutation or no mutations are identified in cases where CPSI deficiency is strongly indicated.

Leigh syndrome caused by a novel m.4296G>A mutation in mitochondrial tRNA isoleucine.

Leigh syndrome is a severe neurodegenerative disease with heterogeneous genetic etiology. We report a novel m.4296G>A variant in the mitochondrial tRNA isoleucine gene in a child with Leigh syndrome, mitochondrial proliferation, lactic acidosis, and abnormal respiratory chain enzymology. The variant is present at >75% heteroplasmy in blood and cultured fibroblasts from the proband, <5% in asymptomatic maternal relatives, and is absent in 3000 controls. It is located in the highly conserved anticodon region of tRNA(Ile) where three other pathogenic changes have been described. We conclude that there is strong evidence to classify m.4296G>A as a pathogenic mutation causing Leigh syndrome.

Mitochondrial myopathy due to novel missense mutation in the cytochrome c oxidase 1 gene.

We report a novel heteroplasmic mutation p.Y440C in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX) gene in a patient with late onset progressive painless weakness. Her muscle biopsy showed scattered COX-negative fibers and several small collections of inflammatory cells. The mutation was detected in the patient's muscle but not in her blood. The low mutant load in muscle could explain the patient's late onset of the myopathy and milder phenotype when compared to the previously published cases with MTCO1 mutations.

Novel POLG splice site mutation and optic atrophy.

OBJECTIVE: To investigate the molecular etiology of 2 unrelated patients with a multisystem mitochondrial disorder accompanied by optic atrophy in one of them. DESIGN: Clinical examination and neurophysiological, radiological, morphological, and molecular analyses. SETTING: Tertiary care neuromuscular clinic and molecular genetics laboratory. PATIENTS: A 65-year-old man (patient 1) with dyschromatopsia and vision loss since childhood developed progressive external ophthalmoplegia, ptosis, and myopathy in the seventh decade of life and was found to have optic atrophy. A 63-year-old man (patient 2) with a similar phenotype, without visual symptoms, experienced also hearing loss and parkinsonism. MAIN OUTCOME MEASURES: Description of the clinical and molecular findings. RESULTS: A muscle biopsy specimen showed ragged-red, ragged-blue, and cytochrome c oxidase-negative fibers in both patients. Because optic atrophy in patient 1 suggested an autosomal dominant OPA1-related disorder, the OPA1 gene was first sequenced, the results of which did not detect any mutations. Southern blot and polymerase chain reaction analyses of muscle mitochondrial DNA revealed multiple deletions. Sequencing of POLG detected a novel variant, c.3104 + 3A>T, in both patients. Patient 1 was compound heterozygous for a known p.F749S mutation; patient 2 had p.G848S as the second mutation. Analysis of POLG complementary DNA showed that c.3104 + 3A>T results in skipping of exon 18. CONCLUSION: Early-onset dyschromatopsia and optic atrophy can occur not only in OPA1-related but also in POLG-related disorders with significant impact on genetic counseling.

Doxycycline- and tetracycline-regulated transcriptional silencer enhance the expression level and transactivating performance of rtTA.

BACKGROUND: The tetracycline-regulated transcriptional silencer (tTS) has been demonstrated to mitigate leaky expression of the tetracycline-inducible promoter under uninduced condition, and, when conjugated with reverse-type tetracycline-controlled transactivator (rtTA), shows great promise for gene therapy. This effect was attributed to the effectiveness of tTS as a repressor of transcription at the tetracycline-regulated promoter. However, we observed an unexpected increase in transactivational activity by rtTA in the presence of tTS under inducible condition. METHODS: To explore the nature of this co-activational effect of tTS on rtTA, we examined the expression patterns of rtTA by Western blotting analysis of total cellular lysates or an enriched ubiquitinated pool of proteins under various conditions, including the one when proteasomal degradation is inhibited. RESULTS: We demonstrate tTS, in addition to its established role as a transcriptional silencer, can enhance rtTA expression level by salvaging rtTA from the ubiquitin-dependent proteasomal degradation pathway. Along with this finding, we also demonstrate that doxycycline, a commonly used tetracycline analogue, inhibits the susceptibility of rtTA to ubiquitin/proteasome-mediated degradation and enhances the expression level of rtTA. CONCLUSIONS: Taken together, our data establish an unappreciated role of doxycycline and tTS in tetracycline-regulated gene expression and the functionality of rtTA, and should shed light on the design of gene therapy vectors based on tetracycline-controlled transcriptional regulation systems.