In silico and in vivo models for Qatari-specific classical homocystinuria as basis for development of novel therapies

Hesham M. Ismail, Navaneethakrishnan Krishnamoorthy, Nader Al-Dewik, Hatem Zayed, Nura A. Mohamed, Valeria Di Giacomo, Sapna Gupta, Johannes Häberle, Beat Thöny, Henk J. Blom, Waren D. Kruger, Tawfeg Ben-Omran, Gheyath K. Nasrallah

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Abstract

Homocystinuria is a rare inborn error of methionine metabolism caused by cystathionine β-synthase (CBS) deficiency. The prevalence of homocystinuria in Qatar is 1:1,800 births, mainly due to a founder Qatari missense mutation, c.1006C>T; p.R336C (p.Arg336Cys). We characterized the structure–function relationship of the p.R336C-mutant protein and investigated the effect of different chemical chaperones to restore p.R336C-CBS activity using three models: in silico, ΔCBS yeast, and CRISPR/Cas9 p.R336C knock-in HEK293T and HepG2 cell lines. Protein modeling suggested that the p.R336C induces severe conformational and structural changes, perhaps influencing CBS activity. Wild-type CBS, but not the p.R336C mutant, was able to restore the yeast growth in ΔCBS-deficient yeast in a complementation assay. The p.R336C knock-in HEK293T and HepG2 cells decreased the level of CBS expression and reduced its structural stability; however, treatment of the p.R336C knock-in HEK293T cells with betaine, a chemical chaperone, restored the stability and tetrameric conformation of CBS, but not its activity. Collectively, these results indicate that the p.R336C mutation has a deleterious effect on CBS structure, stability, and activity, and using the chemical chaperones approach for treatment could be ineffective in restoring p.R336C CBS activity.

Original languageEnglish
JournalHuman Mutation
DOIs
Publication statusAccepted/In press - 1 Jan 2018

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Keywords

  • CBS
  • chemical chaperones
  • Homocystinuria
  • in silico
  • in vivo models
  • p.R336C mutation
  • Qatar

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Ismail, H. M., Krishnamoorthy, N., Al-Dewik, N., Zayed, H., Mohamed, N. A., Giacomo, V. D., Gupta, S., Häberle, J., Thöny, B., Blom, H. J., Kruger, W. D., Ben-Omran, T., & Nasrallah, G. K. (Accepted/In press). In silico and in vivo models for Qatari-specific classical homocystinuria as basis for development of novel therapies. Human Mutation. https://doi.org/10.1002/humu.23682