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In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant

Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patien...

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Autores principales: Cohen, Michal, Pignatti, Emanuele, Dines, Monica, Mory, Adi, Ekhilevitch, Nina, Kolodny, Rachel, Flück, Christa E., Tiosano, Dov
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461554/
https://www.ncbi.nlm.nih.gov/pubmed/32824094
http://dx.doi.org/10.3390/ijms21165857
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author Cohen, Michal
Pignatti, Emanuele
Dines, Monica
Mory, Adi
Ekhilevitch, Nina
Kolodny, Rachel
Flück, Christa E.
Tiosano, Dov
author_facet Cohen, Michal
Pignatti, Emanuele
Dines, Monica
Mory, Adi
Ekhilevitch, Nina
Kolodny, Rachel
Flück, Christa E.
Tiosano, Dov
author_sort Cohen, Michal
collection PubMed
description Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patient, 46 XX newborn, was diagnosed with classical salt wasting CAH in the neonatal period after initially presenting with ambiguous genitalia. Multiplex ligation-dependent probe analysis demonstrated a full deletion of the paternal CYP21A2 gene, and Sanger sequencing revealed a novel de novo CYP21A2 variant c.694–696del (E232del) in the other allele. This variant resulted in the deletion of a non-conserved single amino acid, and its functional relevance was initially undetermined. We used both in silico and in vitro methods to determine the mechanistic significance of this mutation. Computational analysis relied on the solved structure of the protein (Protein-data-bank ID 4Y8W), structure prediction of the mutated protein, evolutionary analysis, and manual inspection. We predicted impaired stability and functionality of the protein due to a rotatory disposition of amino acids in positions downstream of the deletion. In vitro biochemical evaluation of enzymatic activity supported these predictions, demonstrating reduced protein levels to 22% compared to the wild-type form and decreased hydroxylase activity to 1–4%. This case demonstrates the potential of combining in-silico analysis based on evolutionary information and structure prediction with biochemical studies. This approach can be used to investigate other genetic variants to understand their potential effects.
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spelling pubmed-74615542020-09-04 In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant Cohen, Michal Pignatti, Emanuele Dines, Monica Mory, Adi Ekhilevitch, Nina Kolodny, Rachel Flück, Christa E. Tiosano, Dov Int J Mol Sci Communication Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patient, 46 XX newborn, was diagnosed with classical salt wasting CAH in the neonatal period after initially presenting with ambiguous genitalia. Multiplex ligation-dependent probe analysis demonstrated a full deletion of the paternal CYP21A2 gene, and Sanger sequencing revealed a novel de novo CYP21A2 variant c.694–696del (E232del) in the other allele. This variant resulted in the deletion of a non-conserved single amino acid, and its functional relevance was initially undetermined. We used both in silico and in vitro methods to determine the mechanistic significance of this mutation. Computational analysis relied on the solved structure of the protein (Protein-data-bank ID 4Y8W), structure prediction of the mutated protein, evolutionary analysis, and manual inspection. We predicted impaired stability and functionality of the protein due to a rotatory disposition of amino acids in positions downstream of the deletion. In vitro biochemical evaluation of enzymatic activity supported these predictions, demonstrating reduced protein levels to 22% compared to the wild-type form and decreased hydroxylase activity to 1–4%. This case demonstrates the potential of combining in-silico analysis based on evolutionary information and structure prediction with biochemical studies. This approach can be used to investigate other genetic variants to understand their potential effects. MDPI 2020-08-14 /pmc/articles/PMC7461554/ /pubmed/32824094 http://dx.doi.org/10.3390/ijms21165857 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Cohen, Michal
Pignatti, Emanuele
Dines, Monica
Mory, Adi
Ekhilevitch, Nina
Kolodny, Rachel
Flück, Christa E.
Tiosano, Dov
In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant
title In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant
title_full In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant
title_fullStr In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant
title_full_unstemmed In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant
title_short In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant
title_sort in silico structural and biochemical functional analysis of a novel cyp21a2 pathogenic variant
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461554/
https://www.ncbi.nlm.nih.gov/pubmed/32824094
http://dx.doi.org/10.3390/ijms21165857
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