Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation

Warren van Loggerenberg; Shahin Sowlati-Hashjin; Jochen Weile; Rayna Hamilton; Aditya Chawla; Dayag Sheykhkarimli; Marinella Gebbia; Nishka Kishore; Laure Frésard; Sami Mustajoki; Elena Pischik; Elena Di Pierro; Michela Barbaro; Ylva Floderus; Caroline Schmitt; Laurent Gouya; Alexandre Colavin; Robert Nussbaum; Edith C H Friesema; Raili Kauppinen; Jordi To-Figueras; Aasne K Aarsand; Robert J Desnick; Michael Garton; Frederick P Roth

doi:10.1016/j.ajhg.2023.08.012

Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation

Warren van Loggerenberg, Shahin Sowlati-Hashjin, Jochen Weile, Rayna Hamilton, Aditya Chawla, Dayag Sheykhkarimli, Marinella Gebbia, Nishka Kishore, Laure Frésard, Sami Mustajoki, Elena Pischik, Elena Di Pierro, Michela Barbaro, Ylva Floderus, Caroline Schmitt, Laurent Gouya, Alexandre Colavin, Robert Nussbaum, Edith C H Friesema, Raili KauppinenJordi To-Figueras, Aasne K Aarsand, Robert J Desnick, Michael Garton, Frederick P Roth

Internal Medicine

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Original language	English
Pages (from-to)	1769-1786
Number of pages	18
Journal	American Journal of Human Genetics
Volume	110
Issue number	10
Early online date	19 Sept 2023
DOIs	https://doi.org/10.1016/j.ajhg.2023.08.012
Publication status	Published - 5 Oct 2023

Bibliographical note

Funding Information:
We gratefully acknowledge funding for this project from Alnylam Pharmaceuticals. We further acknowledge the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI) Center of Excellence in Genomic Science Initiative (HG010461), the NIH/NHGRI Impact of Genomic Variation on Function (IGVF) Initiative (UM1HG011989), the Canada Excellence Research Chairs Program, and a Canadian Institutes of Health Research Foundation Grant to F.P.R. Computational resources for the molecular dynamics simulations were provided by Compute Canada and SharcNet. We gratefully acknowledge Dr. Sharon D. Whatley for providing clinically annotated variants for use in our positive reference set. F.P.R. is an investor in Ranomics, Inc. and is an investor in and advisor for SeqWell, Inc. BioSymetrics, Inc. and Constantiam Biosciences, Inc. and has accepted conference travel support from Illumina, Inc. L.F. A.C. and R.N. are employed by and invested in Invitae. R.J.D. has received both a grant and royalties and has also served as a consultant for Alnylam Pharmaceuticals.

Funding Information:
We gratefully acknowledge funding for this project from Alnylam Pharmaceuticals . We further acknowledge the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI) Center of Excellence in Genomic Science Initiative ( HG010461 ), the NIH/NHGRI Impact of Genomic Variation on Function (IGVF) Initiative ( UM1HG011989 ), the Canada Excellence Research Chairs Program, and a Canadian Institutes of Health Research Foundation Grant to F.P.R. Computational resources for the molecular dynamics simulations were provided by Compute Canada and SharcNet. We gratefully acknowledge Dr. Sharon D. Whatley for providing clinically annotated variants for use in our positive reference set.

Publisher Copyright:
© 2023 The Authors

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PIIS0002929723002884Final published version, 2.14 MBLicence: CC BY

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van Loggerenberg, W., Sowlati-Hashjin, S., Weile, J., Hamilton, R., Chawla, A., Sheykhkarimli, D., Gebbia, M., Kishore, N., Frésard, L., Mustajoki, S., Pischik, E., Di Pierro, E., Barbaro, M., Floderus, Y., Schmitt, C., Gouya, L., Colavin, A., Nussbaum, R., Friesema, E. C. H., ... Roth, F. P. (2023). Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation. American Journal of Human Genetics, 110(10), 1769-1786. https://doi.org/10.1016/j.ajhg.2023.08.012

@article{d7031e3d5d17489c810b11e181ed78f9,

title = "Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation",

abstract = "Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as {"}variants of uncertain significance{"} (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.",

author = "{van Loggerenberg}, Warren and Shahin Sowlati-Hashjin and Jochen Weile and Rayna Hamilton and Aditya Chawla and Dayag Sheykhkarimli and Marinella Gebbia and Nishka Kishore and Laure Fr{\'e}sard and Sami Mustajoki and Elena Pischik and {Di Pierro}, Elena and Michela Barbaro and Ylva Floderus and Caroline Schmitt and Laurent Gouya and Alexandre Colavin and Robert Nussbaum and Friesema, {Edith C H} and Raili Kauppinen and Jordi To-Figueras and Aarsand, {Aasne K} and Desnick, {Robert J} and Michael Garton and Roth, {Frederick P}",

note = "Funding Information: We gratefully acknowledge funding for this project from Alnylam Pharmaceuticals. We further acknowledge the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI) Center of Excellence in Genomic Science Initiative (HG010461), the NIH/NHGRI Impact of Genomic Variation on Function (IGVF) Initiative (UM1HG011989), the Canada Excellence Research Chairs Program, and a Canadian Institutes of Health Research Foundation Grant to F.P.R. Computational resources for the molecular dynamics simulations were provided by Compute Canada and SharcNet. We gratefully acknowledge Dr. Sharon D. Whatley for providing clinically annotated variants for use in our positive reference set. F.P.R. is an investor in Ranomics, Inc. and is an investor in and advisor for SeqWell, Inc. BioSymetrics, Inc. and Constantiam Biosciences, Inc. and has accepted conference travel support from Illumina, Inc. L.F. A.C. and R.N. are employed by and invested in Invitae. R.J.D. has received both a grant and royalties and has also served as a consultant for Alnylam Pharmaceuticals. Funding Information: We gratefully acknowledge funding for this project from Alnylam Pharmaceuticals . We further acknowledge the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI) Center of Excellence in Genomic Science Initiative ( HG010461 ), the NIH/NHGRI Impact of Genomic Variation on Function (IGVF) Initiative ( UM1HG011989 ), the Canada Excellence Research Chairs Program, and a Canadian Institutes of Health Research Foundation Grant to F.P.R. Computational resources for the molecular dynamics simulations were provided by Compute Canada and SharcNet. We gratefully acknowledge Dr. Sharon D. Whatley for providing clinically annotated variants for use in our positive reference set. Publisher Copyright: {\textcopyright} 2023 The Authors",

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doi = "10.1016/j.ajhg.2023.08.012",

language = "English",

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van Loggerenberg, W, Sowlati-Hashjin, S, Weile, J, Hamilton, R, Chawla, A, Sheykhkarimli, D, Gebbia, M, Kishore, N, Frésard, L, Mustajoki, S, Pischik, E, Di Pierro, E, Barbaro, M, Floderus, Y, Schmitt, C, Gouya, L, Colavin, A, Nussbaum, R, Friesema, ECH, Kauppinen, R, To-Figueras, J, Aarsand, AK, Desnick, RJ, Garton, M & Roth, FP 2023, 'Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation', American Journal of Human Genetics, vol. 110, no. 10, pp. 1769-1786. https://doi.org/10.1016/j.ajhg.2023.08.012

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T1 - Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation

AU - van Loggerenberg, Warren

AU - Sowlati-Hashjin, Shahin

AU - Weile, Jochen

AU - Hamilton, Rayna

AU - Chawla, Aditya

AU - Sheykhkarimli, Dayag

AU - Gebbia, Marinella

AU - Kishore, Nishka

AU - Frésard, Laure

AU - Mustajoki, Sami

AU - Pischik, Elena

AU - Di Pierro, Elena

AU - Barbaro, Michela

AU - Floderus, Ylva

AU - Schmitt, Caroline

AU - Gouya, Laurent

AU - Colavin, Alexandre

AU - Nussbaum, Robert

AU - Friesema, Edith C H

AU - Kauppinen, Raili

AU - To-Figueras, Jordi

AU - Aarsand, Aasne K

AU - Desnick, Robert J

AU - Garton, Michael

AU - Roth, Frederick P

N1 - Funding Information: We gratefully acknowledge funding for this project from Alnylam Pharmaceuticals. We further acknowledge the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI) Center of Excellence in Genomic Science Initiative (HG010461), the NIH/NHGRI Impact of Genomic Variation on Function (IGVF) Initiative (UM1HG011989), the Canada Excellence Research Chairs Program, and a Canadian Institutes of Health Research Foundation Grant to F.P.R. Computational resources for the molecular dynamics simulations were provided by Compute Canada and SharcNet. We gratefully acknowledge Dr. Sharon D. Whatley for providing clinically annotated variants for use in our positive reference set. F.P.R. is an investor in Ranomics, Inc. and is an investor in and advisor for SeqWell, Inc. BioSymetrics, Inc. and Constantiam Biosciences, Inc. and has accepted conference travel support from Illumina, Inc. L.F. A.C. and R.N. are employed by and invested in Invitae. R.J.D. has received both a grant and royalties and has also served as a consultant for Alnylam Pharmaceuticals. Funding Information: We gratefully acknowledge funding for this project from Alnylam Pharmaceuticals . We further acknowledge the National Institutes of Health National Human Genome Research Institute (NIH/NHGRI) Center of Excellence in Genomic Science Initiative ( HG010461 ), the NIH/NHGRI Impact of Genomic Variation on Function (IGVF) Initiative ( UM1HG011989 ), the Canada Excellence Research Chairs Program, and a Canadian Institutes of Health Research Foundation Grant to F.P.R. Computational resources for the molecular dynamics simulations were provided by Compute Canada and SharcNet. We gratefully acknowledge Dr. Sharon D. Whatley for providing clinically annotated variants for use in our positive reference set. Publisher Copyright: © 2023 The Authors

PY - 2023/10/5

Y1 - 2023/10/5

N2 - Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

AB - Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

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U2 - 10.1016/j.ajhg.2023.08.012

DO - 10.1016/j.ajhg.2023.08.012

M3 - Article

C2 - 37729906

SN - 0002-9297

VL - 110

SP - 1769

EP - 1786

JO - American Journal of Human Genetics

JF - American Journal of Human Genetics

IS - 10

ER -

Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation

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Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation

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