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A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening

The lysosomal storage disorder, mucopolysaccharidosis I (MPSI), results from mutations in IDUA, the gene that encodes the glycosaminoglycan-degrading enzyme α-L-iduronidase. Newborn screening efforts for MPSI have greatly increased the number of novel IDUA variants identified, but with insufficient...

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Autores principales: Yu, Seok-Ho, Pollard, Laura, Wood, Tim, Flanagan-Steet, Heather, Steet, Richard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711455/
https://www.ncbi.nlm.nih.gov/pubmed/33198351
http://dx.doi.org/10.3390/ijns6040088
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author Yu, Seok-Ho
Pollard, Laura
Wood, Tim
Flanagan-Steet, Heather
Steet, Richard
author_facet Yu, Seok-Ho
Pollard, Laura
Wood, Tim
Flanagan-Steet, Heather
Steet, Richard
author_sort Yu, Seok-Ho
collection PubMed
description The lysosomal storage disorder, mucopolysaccharidosis I (MPSI), results from mutations in IDUA, the gene that encodes the glycosaminoglycan-degrading enzyme α-L-iduronidase. Newborn screening efforts for MPSI have greatly increased the number of novel IDUA variants identified, but with insufficient experimental evidence regarding their pathogenicity, many of these variants remain classified as variants of uncertain significance (VUS). Defining pathogenicity for novel IDUA variants is critical for decisions regarding medical management and early intervention. Here, we describe a biochemical platform for the characterization of IDUA variants that relies on viral delivery of IDUA DNA into IDUA-deficient HAP1 cells and isolation of single cell expression clones. The relative specific activity of wild-type and variant α-iduronidase was determined using a combination of Western blot analysis and α-iduronidase activity assays. The specific activity of each variant enzyme was consistent across different single cell clones despite variable IDUA expression and could be accurately determined down to 0.05–0.01% of WT α-iduronidase activity. With this strategy we compared the specific activities of known pseudodeficiency variants (p.His82Gln, p.Ala79Thr, p.Val322Glu, p.Asp223Asn) or pathogenic variants (p.Ser633Leu, p.His240Arg) with variants of uncertain significance (p.Ser586Phe, p.Ile272Leu). The p.Ser633Leu and p.His240Arg variants both show very low activities consistent with their association with Scheie syndrome. In our experiments, however, p.His240Arg exhibited a specific activity five times higher than p.Ser633Leu in contrast to other reports showing equivalent activity. Cell clones expressing the p.Ser586Phe and p.Ile272Leu variants had specific activities in the range of other pseudodeficiency variants tested. Our findings show that pseudodeficiency and pathogenic variants can be distinguished from each other with regard to specific activity, and confirms that all the pseudodeficiency variants variably reduce α-iduronidase activity. We envision this platform will be a valuable resource for the rigorous assessment of the novel IDUA variants emerging from the expansion of newborn screening efforts.
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spelling pubmed-77114552020-12-04 A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening Yu, Seok-Ho Pollard, Laura Wood, Tim Flanagan-Steet, Heather Steet, Richard Int J Neonatal Screen Article The lysosomal storage disorder, mucopolysaccharidosis I (MPSI), results from mutations in IDUA, the gene that encodes the glycosaminoglycan-degrading enzyme α-L-iduronidase. Newborn screening efforts for MPSI have greatly increased the number of novel IDUA variants identified, but with insufficient experimental evidence regarding their pathogenicity, many of these variants remain classified as variants of uncertain significance (VUS). Defining pathogenicity for novel IDUA variants is critical for decisions regarding medical management and early intervention. Here, we describe a biochemical platform for the characterization of IDUA variants that relies on viral delivery of IDUA DNA into IDUA-deficient HAP1 cells and isolation of single cell expression clones. The relative specific activity of wild-type and variant α-iduronidase was determined using a combination of Western blot analysis and α-iduronidase activity assays. The specific activity of each variant enzyme was consistent across different single cell clones despite variable IDUA expression and could be accurately determined down to 0.05–0.01% of WT α-iduronidase activity. With this strategy we compared the specific activities of known pseudodeficiency variants (p.His82Gln, p.Ala79Thr, p.Val322Glu, p.Asp223Asn) or pathogenic variants (p.Ser633Leu, p.His240Arg) with variants of uncertain significance (p.Ser586Phe, p.Ile272Leu). The p.Ser633Leu and p.His240Arg variants both show very low activities consistent with their association with Scheie syndrome. In our experiments, however, p.His240Arg exhibited a specific activity five times higher than p.Ser633Leu in contrast to other reports showing equivalent activity. Cell clones expressing the p.Ser586Phe and p.Ile272Leu variants had specific activities in the range of other pseudodeficiency variants tested. Our findings show that pseudodeficiency and pathogenic variants can be distinguished from each other with regard to specific activity, and confirms that all the pseudodeficiency variants variably reduce α-iduronidase activity. We envision this platform will be a valuable resource for the rigorous assessment of the novel IDUA variants emerging from the expansion of newborn screening efforts. MDPI 2020-11-12 /pmc/articles/PMC7711455/ /pubmed/33198351 http://dx.doi.org/10.3390/ijns6040088 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 Article
Yu, Seok-Ho
Pollard, Laura
Wood, Tim
Flanagan-Steet, Heather
Steet, Richard
A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening
title A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening
title_full A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening
title_fullStr A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening
title_full_unstemmed A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening
title_short A Biochemical Platform to Define the Relative Specific Activity of IDUA Variants Identified by Newborn Screening
title_sort biochemical platform to define the relative specific activity of idua variants identified by newborn screening
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711455/
https://www.ncbi.nlm.nih.gov/pubmed/33198351
http://dx.doi.org/10.3390/ijns6040088
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