Evaluating clinical mitochondrial respiratory chain enzymes from biopsy specimens presenting skewed probability distribution of activity data

Hautakangas, Milla‐Riikka; Hinttala, Reetta; Rantala, Heikki; Nieminen, Pentti; Uusimaa, Johanna; Hassinen, Ilmo E.

doi:10.1016/j.mito.2016.05.004

Cited by 3 publications

(3 citation statements)

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“…Therefore, we propose modeling OCR on a logarithmic scale, where the dependence between the variance and the mean disappears ( Fig 2B and 2C ). The activities of respiratory chain enzymes such as NADH-ubiquinone reductase also obey log-normal distributions [ 33 ].…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…Further, the activities of mitochondrial respiratory chain enzyme complexes were determined from skeletal muscle biopsy samples, as described previously. 14…”

Section: Methodsmentioning

confidence: 99%

Biallelic loss-of-function P4HTM gene variants cause hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy, and eye abnormalities (HIDEA syndrome)

Rahikkala

Myllykoski

Hinttala

et al. 2019

Genetics in Medicine

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PurposeA new syndrome with hypotonia, intellectual disability, and eye abnormalities (HIDEA) was previously described in a large consanguineous family. Linkage analysis identified the recessive disease locus, and genome sequencing yielded three candidate genes with potentially pathogenic biallelic variants: transketolase (TKT), transmembrane prolyl 4-hydroxylase (P4HTM), and ubiquitin specific peptidase 4 (USP4). However, the causative gene remained elusive.MethodsInternational collaboration and exome sequencing were used to identify new patients with HIDEA and biallelic, potentially pathogenic, P4HTM variants. Segregation analysis was performed using Sanger sequencing. P4H-TM wild-type and variant constructs without the transmembrane region were overexpressed in insect cells and analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot.ResultsFive different homozygous or compound heterozygous pathogenic P4HTM gene variants were identified in six new and six previously published patients presenting with HIDEA. Hypoventilation, obstructive and central sleep apnea, and dysautonomia were identified as novel features associated with the phenotype. Characterization of three of the P4H-TM variants demonstrated yielding insoluble protein products and, thus, loss-of-function.ConclusionsBiallelic loss-of-function P4HTM variants were shown to cause HIDEA syndrome. Our findings enable diagnosis of the condition, and highlight the importance of assessing the need for noninvasive ventilatory support in patients.

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“…2B, 2C). Respiratory chain enzyme activities such as NADH-ubiquinone reductase have also been shown to obey log-normal distributions (33).…”

Section: Resultsmentioning

confidence: 99%

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

Kremer

Iuso

et al. 2017

Preprint

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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 estimating different bioenergetic measures by monitoring oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of statistical best practices for determining OCR measurements and comparisons have been lacking so far. Therefore, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines to understand how OCR behaves across different biosamples, wells, and plates. 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 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-plates variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 36% and of maximal respiration by 32%. Moreover, using positive and negative controls, we show that our statistical test outperforms existing methods, which either suffer from an excess of false positives (within-plates methods), or of false negatives (between-plates methods). Altogether, the aim of this study is to propose statistical good practices to support experimentalists in designing, analyzing, testing and reporting results of mitochondrial stress tests using this high throughput platform.

show abstract

Evaluating clinical mitochondrial respiratory chain enzymes from biopsy specimens presenting skewed probability distribution of activity data

Cited by 3 publications

References 32 publications

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

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

Biallelic loss-of-function P4HTM gene variants cause hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy, and eye abnormalities (HIDEA syndrome)

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

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