title: Surrogate genetics and metabolic profiling for characterization of human disease alleles.
abstract: Cystathionine-β-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.
authors.affiliation: Department of Molecular and Cell Biology, California Institute of Quantitative Biosciences, University of California, Berkeley, California 94720, USA.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Mayfield
authors.forename: Meara W
authors.initials: MW
authors.lastname: Davies
authors.forename: Dago
authors.initials: D
authors.lastname: Dimster-Denk
authors.forename: Nick
authors.initials: N
authors.lastname: Pleskac
authors.forename: Sean
authors.initials: S
authors.lastname: McCarthy
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Boydston
authors.forename: Logan
authors.initials: L
authors.lastname: Fink
authors.forename: Xin Xin
authors.initials: XX
authors.lastname: Lin
authors.forename: Ankur S
authors.initials: AS
authors.lastname: Narain
authors.forename: Michael
authors.initials: M
authors.lastname: Meighan
authors.forename: Jasper
authors.initials: J
authors.lastname: Rine
mesh_terms: D000483:Alleles; D002853:Chromatography, Liquid / Q000379:methods; D003067:Coenzymes / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D015894:Genome, Human; D005978:Glutathione / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006712:Homocystinuria / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D055442:Metabolome; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D025101:Vitamin B 6 / Q000378:metabolism / Q000494:pharmacology
chemical_list: D003067:Coenzymes; D003470:Culture Media; D006418:Heme; D025101:Vitamin B 6; D003541:Cystathionine beta-Synthase; D005978:Glutathione