Dataset Information

Challenging popular tools for the annotation of genetic variations with a real case, pathogenic mutations of lysosomal alpha-galactosidase.

ABSTRACT: BACKGROUND:Severity gradation of missense mutations is a big challenge for exome annotation. Predictors of deleteriousness that are most frequently used to filter variants found by next generation sequencing, produce qualitative predictions, but also numerical scores. It has never been tested if these scores correlate with disease severity. RESULTS:wANNOVAR, a popular tool that can generate several different types of deleteriousness-prediction scores, was tested on Fabry disease. This pathology, which is caused by a deficit of lysosomal alpha-galactosidase, has a very large genotypic and phenotypic spectrum and offers the possibility of associating a quantitative measure of the damage caused by mutations to the functioning of the enzyme in the cells. Some predictors, and in particular VEST3 and PolyPhen2 provide scores that correlate with the severity of lysosomal alpha-galactosidase mutations in a statistically significant way. CONCLUSIONS:Sorting disease mutations by severity is possible and offers advantages over binary classification. Dataset for testing and training in silico predictors can be obtained by transient transfection and evaluation of residual activity of mutants in cell extracts. This approach consents to quantitative data for severe, mild and non pathological variants.

SUBMITTER: Cimmaruta C

PROVIDER: S-EPMC6266955 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Challenging popular tools for the annotation of genetic variations with a real case, pathogenic mutations of lysosomal alpha-galactosidase.

Cimmaruta Chiara C Citro Valentina V Andreotti Giuseppina G Liguori Ludovica L Cubellis Maria Vittoria MV Hay Mele Bruno B

BMC bioinformatics 20181130 Suppl 15

<h4>Background</h4>Severity gradation of missense mutations is a big challenge for exome annotation. Predictors of deleteriousness that are most frequently used to filter variants found by next generation sequencing, produce qualitative predictions, but also numerical scores. It has never been tested if these scores correlate with disease severity.<h4>Results</h4>wANNOVAR, a popular tool that can generate several different types of deleteriousness-prediction scores, was tested on Fabry disease. ...[more]

PMID: 30497360

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Project description:BACKGROUND: The pharmacological chaperones therapy is a promising approach to cure genetic diseases. It relies on substrate competitors used at sub-inhibitory concentration which can be administered orally, reach difficult tissues and have low cost. Clinical trials are currently carried out for Fabry disease, a lysosomal storage disorder caused by inherited genetic mutations of alpha-galactosidase. Regrettably, not all genotypes respond to these drugs. RESULTS: We collected the experimental data available in literature on the enzymatic activity of ninety-six missense mutants of lysosomal alpha-galactosidase measured in the presence of pharmacological chaperones. We associated with each mutation seven features derived from the analysis of 3D-structure of the enzyme, two features associated with their thermo-dynamic stability and four features derived from sequence alone. Structural and thermodynamic analysis explains why some mutants of human lysosomal alpha-galactosidase cannot be rescued by pharmacological chaperones: approximately forty per cent of the non responsive cases examined can be correctly associated with a negative prognostic feature. They include mutations occurring in the active site pocket, mutations preventing disulphide bridge formation and severely destabilising mutations. Despite this finding, prediction of mutations responsive to pharmacological chaperones cannot be achieved with high accuracy relying on combinations of structure- and thermodynamic-derived features even with the aid of classical and state of the art statistical learning methods.We developed a procedure to predict responsive mutations with an accuracy as high as 87%: the method scores the mutations by using a suitable position-specific substitution matrix. Our approach is of general applicability since it does not require the knowledge of 3D-structure but relies only on the sequence. CONCLUSIONS: Responsiveness to pharmacological chaperones depends on the structural/functional features of the disease-associated protein, whose complex interplay is best reflected on sequence conservation by evolutionary pressure. We propose a predictive method which can be applied to screen novel mutations of alpha galactosidase. The same approach can be extended on a genomic scale to find candidates for therapy with pharmacological chaperones among proteins with unknown tertiary structures.

Project description:The aberrant accumulation of alpha-synuclein (?-syn) is believed to contribute to the onset and pathogenesis of Parkinson's disease (PD). The autophagy-lysosome pathway (ALP) is responsible for the high capacity clearance of ?-syn. ALP dysfunction is documented in PD and pre-clinical evidence suggests that inhibiting the ALP promotes the pathological accumulation of ?-syn. We previously identified the pathological accumulation of ?-syn in the brains of mice deficient for the soluble lysosomal enzyme alpha-Galactosidase A (?-Gal A), a member of the glycosphingolipid metabolism pathway. In the present study, we quantified ?-Gal A activity and levels of its glycosphingolipid metabolites in postmortem temporal cortex specimens from control individuals and in PD individuals staged with respect to ?-syn containing Lewy body pathology. In late-state PD temporal cortex we observed significant decreases in ?-Gal A activity and the 46kDa "active" species of ?-Gal A as determined respectively by fluorometric activity assay and western blot analysis. These decreases in ?-Gal A activity/levels correlated significantly with increased ?-syn phosphorylated at serine 129 (p129S-?-syn) that was maximal in late-stage PD temporal cortex. Mass spectrometric analysis of 29 different isoforms of globotriaosylceramide (Gb3), a substrate of ?-Gal A indicated no significant differences with respect to different stages of PD temporal cortex. However, significant correlations were observed between increased levels of several Gb3 isoforms and with decreased ?-Gal A activity and/or increased p129S-?-syn. Deacylated Gb3 (globotriaosylsphingosine or lyso-Gb3) was also analyzed in PD brain tissue but was below the limit of detection of 20pmol/g. Analysis of other lysosomal enzymes revealed a significant decrease in activity for the lysosomal aspartic acid protease cathepsin D but not for glucocerebrosidase (GCase) or cathepsin B in late-stage PD temporal cortex. However, a significant correlation was observed between decreasing GCase activity and increasing p129S-?-syn. Together our findings indicate ?-Gal A deficiency in late-stage PD brain that correlates significantly with the pathological accumulation of ?-syn, and further suggest the potential for ?-Gal A and its glycosphingolipid substrates as putative biomarkers for PD.

Dataset Information

Challenging popular tools for the annotation of genetic variations with a real case, pathogenic mutations of lysosomal alpha-galactosidase.

Publications

Challenging popular tools for the annotation of genetic variations with a real case, pathogenic mutations of lysosomal alpha-galactosidase.

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