Leigh syndrome is the main clinical characteristic of PTCD3 deficiency

Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1G>C];[1918C>G] and c.[710del];[902C>T]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.© 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology

Lancet

BACKGROUND: In 2015, the second cycle of the CONCORD programme established global surveillance of cancer survival as a metric of the effectiveness of health systems and to inform global policy on cancer control. CONCORD-3 updates the worldwide surveillance of cancer survival to 2014. METHODS: CONCORD-3 includes individual records for 37.5 million patients diagnosed with cancer during the 15-year period 2000-14. Data were provided by 322 population-based cancer registries in 71 countries and territories, 47 of which provided data with 100% population coverage. The study includes 18 cancers or groups of cancers: oesophagus, stomach, colon, rectum, liver, pancreas, lung, breast (women), cervix, ovary, prostate, and melanoma of the skin in adults, and brain tumours, leukaemias, and lymphomas in both adults and children. Standardised quality control procedures were applied; errors were rectified by the registry concerned. We estimated 5-year net survival. Estimates were age-standardised with the International Cancer Survival Standard weights. FINDINGS: For most cancers, 5-year net survival remains among the highest in the world in the USA and Canada, in Australia and New Zealand, and in Finland, Iceland, Norway, and Sweden. For many cancers, Denmark is closing the survival gap with the other Nordic countries. Survival trends are generally increasing, even for some of the more lethal cancers: in some countries, survival has increased by up to 5% for cancers of the liver, pancreas, and lung. For women diagnosed during 2010-14, 5-year survival for breast cancer is now 89.5% in Australia and 90.2% in the USA, but international differences remain very wide, with levels as low as 66.1% in India. For gastrointestinal cancers, the highest levels of 5-year survival are seen in southeast Asia: in South Korea for cancers of the stomach (68.9%), colon (71.8%), and rectum (71.1%); in Japan for oesophageal cancer (36.0%); and in Taiwan for liver cancer (27.9%). By contrast, in the same world region, survival is generally lower than elsewhere for melanoma of the skin (59.9% in South Korea, 52.1% in Taiwan, and 49.6% in China), and for both lymphoid malignancies (52.5%, 50.5%, and 38.3%) and myeloid malignancies (45.9%, 33.4%, and 24.8%). For children diagnosed during 2010-14, 5-year survival for acute lymphoblastic leukaemia ranged from 49.8% in Ecuador to 95.2% in Finland. 5-year survival from brain tumours in children is higher than for adults but the global range is very wide (from 28.9% in Brazil to nearly 80% in Sweden and Denmark). INTERPRETATION: The CONCORD programme enables timely comparisons of the overall effectiveness of health systems in providing care for 18 cancers that collectively represent 75% of all cancers diagnosed worldwide every year. It contributes to the evidence base for global policy on cancer control. Since 2017, the Organisation for Economic Co-operation and Development has used findings from the CONCORD programme as the official benchmark of cancer survival, among their indicators of the quality of health care in 48 countries worldwide. Governments must recognise population-based cancer registries as key policy tools that can be used to evaluate both the impact of cancer prevention strategies and the effectiveness of health systems for all patients diagnosed with cancer. FUNDING: American Cancer Society; Centers for Disease Control and Prevention; Swiss Re; Swiss Cancer Research foundation; Swiss Cancer League; Institut National du Cancer; La Ligue Contre le Cancer; Rossy Family Foundation; US National Cancer Institute; and the Susan G Komen Foundation

Modeling fragment counts improves single-cell ATAC-seq analysis

Single-cell ATAC-sequencing (scATAC-seq) coverage in regulatory regions is typically binarized as an indicator of open chromatin. Here we show that binarization is an unnecessary step that neither improves goodness-of-fit, clustering, cell type identification, nor batch integration. Fragment counts, but not read counts, should instead be modeled, which preserves quantitative regulatory information. These results have immediate implications for scATAC-seq analysis

OCR-Stats: Robust estimation and statistical testing of mitochondrial respiration activities using Seahorse XF Analyzer.

The accurate quantification of cellular and mitochondrial bioenergetic activity is of great interest in medicine and biology. Mitochondrial stress tests performed with Seahorse Bioscience XF Analyzers allow the estimation of different bioenergetic measures by monitoring the oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of the statistical best practices for determining aggregated OCR measurements and comparisons have been lacking. Therefore, to understand how OCR behaves across different biological samples, wells, and plates, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines. We show that the noise of OCR is multiplicative, that outlier data points can concern individual measurements or all measurements of a well, and that the inter-plate variation is greater than the intra-plate variation. Based on these insights, we developed a novel statistical method, OCR-Stats, that: i) robustly estimates OCR levels modeling multiplicative noise and automatically identifying outlier data points and outlier wells; and ii) performs statistical testing between samples, taking into account the different magnitudes of the between- and within-plate variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 45% and of maximal respiration by 29%. Moreover, using positive and negative controls, we show that our statistical test outperforms the existing methods, which suffer from an excess of either false positives (within-plate methods), or false negatives (between-plate methods). Altogether, this study provides statistical good practices to support experimentalists in designing, analyzing, testing, and reporting the results of mitochondrial stress tests using this high throughput platform

Levadura viva (Saccharomyces cerevisiae) en la degradabilidad in vitro de una dieta para bovinos basada en forraje más concentrado

Four levels of live yeast (Saccharomyces cerevisiae) culture in the in vitro digestibility of a more concentrated forage based cattle diet were evaluated. Ruminal fluid of two fistulated bulls was used, as a bacterial inoculum. Two Ankom® incubators were used for in vitro digestibility, where F57 bags (Ankom Technology) containing the samples of experimental treatments were deposited. The treatments were; T1: control diet with more concentrated forage, T2: T1 plus 1 g of CL (Yeast Culture) kg-1 of feed, T3: T1 plus 2 g of CL kg-1 of feed and T4: T1 plus 3 g of CL kg-1 of food. The experiment was developed in the Laboratory of Nutrition Rumiology and Metabolism with a duration of 90 days. In vitro dry matter degradability (DIMS) showed no changes at 3, 12 and 72 h of ruminal incubation because of CL supplementation. However, although in a non-linear manner, this CL caused differences for the incubation periods of 0, 6, 24 and 48 hours. The DRIV of organic matter (DIMO) did not present a linear behavior because of the increasing addition of CL to the base diet. There were differences only in incubation times of 3, 24 and 48 hours, but not in the longest incubation time (72 h). CLs do not cause clear changes in the degradability of the diets studied.Se evaluaron cuatro niveles de cultivo de levadura viva (Saccharomyces cerevisiae) en la digestibilidad in vitro de una dieta para bovinos basada en forraje más concentrado. Se utilizó líquido ruminal de dos toros fistulados, como inoculo bacteriano. Se emplearon dos incubadores Ankom® para digestibilidad in vitro, donde se depositaron bolsas F57 (Ankom Technology) que contenían las muestras de tratamientos experimentales. Los tratamientos fueron; T1: dieta testigo a base de forraje más concentrado, T2: T1 más 1 g de CL (Cultivo de levadura) kg-1 de alimento, T3: T1+2 g de CL kg-1 de alimento y T4: T1+3 g de CL kg-1 de alimento. La incubación se efectuó en siete tiempos; y la degradación se detuvo mediante refrigeración a 4 ºC. El experimento duró 90 días. Se evidenció a las 3, 12 y 72 h de incubación ruminal, la degradabilidad in vitro (DRIV) de la materia seca (DIMS), no presentó cambios por efecto de la suplementación del CL. No obstante, aunque de una manera no lineal, este CL provocó diferencias para los periodos de incubación de 0, 6, 24 y 48 horas. La DRIV de la materia orgánica (DIMO) tampoco presentó un comportamiento lineal por efecto de la adición creciente del CL a la dieta base, existiendo diferencias sólo en los tiempos de incubación de 3, 24 y 48 horas, pero no en el mayor tiempo de incubación (72 h). Los CL no provocan cambios claros en la degradabilidad de las dietas estudiadas

Diagnostic odyssey in an adult patient with ophthalmologic abnormalities and hearing loss: Contribution of RNA-seq to the diagnosis of a PEX1 deficiency.

Peroxisomal biogenesis disorders (PBDs) are a heterogeneous group of genetic diseases. Multiple peroxisomal pathways are impaired, and very long chain fatty acids (VLCFA) are the first line biomarkers for the diagnosis. The clinical presentation of PBDs may range from severe, lethal multisystemic disorders to milder, late-onset disease. The vast majority of PBDs belong to Zellweger Spectrum Disordes (ZSDs) and represents a continuum of overlapping clinical symptoms, with Zellweger syndrome being the most severe and Heimler syndrome the less severe disease. Mild clinical conditions frequently present normal or slight biochemical alterations, making the diagnosis of these patients challenging. In the present study we used a combined WES and RNA-seq strategy to diagnose a patient presenting with retinal dystrophy as the main clinical symptom. Results showed the patient was compound heterozygous for mutations in PEX1. VLCFA were normal, but retrospective analysis of lysosphosphatidylcholines (LPC) containing C22:0-C26:0 species was altered. This simple test could avoid the diagnostic odyssey of patients with mild phenotype, such as the individual described here, who was diagnosed very late in adult life. We provide functional data in cell line models that may explain the mild phenotype of the patient by demonstrating the hypomorphic nature of a deep intronic variant altering PEX1 mRNA processin

An Integrated Transcriptomics and Genomics Approach Detects an X/Autosome Translocation in a Female with Duchenne Muscular Dystrophy

Duchenne and Becker muscular dystrophies, caused by pathogenic variants in DMD, are the most common inherited neuromuscular conditions in childhood. These diseases follow an X-linked recessive inheritance pattern, and mainly males are affected. The most prevalent pathogenic variants in the DMD gene are copy number variants (CNVs), and most patients achieve their genetic diagnosis through Multiplex Ligation-dependent Probe Amplification (MLPA) or exome sequencing. Here, we investigated a female patient presenting with muscular dystrophy who remained genetically undiagnosed after MLPA and exome sequencing. RNA sequencing (RNAseq) from the patient’s muscle biopsy identified an 85% reduction in DMD expression compared to 116 muscle samples included in the cohort. A de novo balanced translocation between chromosome 17 and the X chromosome (t(X;17)(p21.1;q23.2)) disrupting the DMD and BCAS3 genes was identified through trio whole genome sequencing (WGS). The combined analysis of RNAseq and WGS played a crucial role in the detection and characterisation of the disease-causing variant in this patient, who had been undiagnosed for over two decades. This case illustrates the diagnostic odyssey of female DMD patients with complex structural variants that are not detected by current panel or exome sequencing analysis

Principle of the mitochondrial stress test assay.

<p>(<b>A</b>) Cartoon illustration of OCR levels (y-axis) versus time (x-axis). Injection of the three compounds oligomycin, FCCP, and rotenone delimits four time intervals within each of which OCR is roughly constant. (<b>B</b>) Targets of each compound in the electron transport chain. (<b>C</b>) Typical layout of a mitochondrial stress test 96-well plate.</p

Power analysis.

<p>Standard deviation of the residuals from the model in Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199938#pone.0199938.e011" target="_blank">2</a>) (left y-axis) against number of wells per biological sample and per plate (x-axis) for each OCR log-ratio difference (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199938#pone.0199938.t001" target="_blank">Table 1</a>). The right y-axis corresponds to the minimal detectable relative differences using three plates at 5% significance level (Materials and methods). For every number of wells, the 10 data points correspond to each of the 10 random samplings without replacement of the wells per biological sample and per plate.</p

OCR behavior over time.

<p>(<b>A</b>) Typical time series replicates inside a plate. Behavior of OCR expressed in pmol/min (y-axis) of Fibro_VY_017 over time (x-axis). Colors indicate the row and shape the column of 12-well replicates. Variation increases for larger OCR values, OCR has a systematic well effect, and there are two types of outliers: well-level and single-point. (<b>B</b>) Scatterplot of standard deviation (y-axis) vs. mean (x-axis) of all three time replicates of each interval, well, and plate of OCR of NHDF only shows a positive correlation (<i>n</i> = 409). (<b>C</b>) The same as (B) but for the logarithm of OCR, where the correlation disappears.</p