Surrogate Genetics and Metabolic Profiling for Characterization of Human Disease Alleles

Abstract

Cystathionine-b-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.T HE first complete human genome sequence seeded the defining challenge of human genetics for the foreseeable future: interpreting the impact of variations in the sequences of individual human genomes. Comparative genome sequencing reveals an average of one single-nucleotide change per 1200 bp between any two individuals. In the absence of strong Mendelian inheritance and linkage, confirming that any human genotype actually caused a phenotype is a significant challenge given the approximately 3 million genetic variants per person. Indeed, 4000 traits of medical interest show evidence for inheritance but lack a clear determinant (Online Mendelian Inheritance in Man 2012). Next-generation sequencing within small pedigrees (Ng et al. 2010a,b;Fan et al. 2011), or a more narrowly defined clinical phenotype (Schubert et al. 1997), can sometimes disentangle the underlying contribution of a gene to disease. In this work we have taken an approach that complements both increased sequencing capacity and expanded phenotypic description. We used surrogate genetics to assay directly the function of allelic variants and then evaluate their potential contribution to phenotypes of clinical importance.Homocystinuria, elevated levels of the sulfur-containing metabolite homocystine in the urine, illustrates several Reference numbers for publicly available data; GenBank: L14577.1 (CBS); dbSNP: rs17849313 (A69P), rs2229413 (P70L), rs11700812 (R369P); SGD: YGR155W (CYS4) and YDR232W (HEM1 challenges inherent to elucidating the molecular bases of human geneti...

A formal perturbation equation between genotype and phenotype determines the Evolutionary Action of protein-coding variations on fitness

“…(B) The average rank of current methods (bars), from different groups (letters), to predict the activity of cystathionine beta-synthase (CBS) mutants was assessed by the Critical Assessment of Genome Interpretation (CAGI) of 2011. The CBS activity was assayed for the ability of each mutant to restore growth in yeast cells lacking the normal CYS4 ortholog under two different growth conditions (high and low concentrations of pyridoxine cofactor) (Mayfield et al 2012). Twenty methods from nine groups were assessed over nine criteria (precision, recall, accuracy, harmonic mean f1, Spearman's rank correlation coefficient, Student's t-test P-value, root mean square deviation [RMSD], RMSD over Z-scores, and the AUC) for each cofactor concentration, and then their rank was averaged.…”

Inferring causality and functional significance of human coding DNA variants

“…For example, a yeast system was successfully used to functionally characterize 84 human variants observed in patients with cystathionineb-synthase deficiency (58).…”

Nutritional Control of Epigenetic Processes in Yeast and Human Cells

“…Cell debris was pelleted for 10 min at 10,000 3 g and then the supernatants were adjusted to pH 6 using a solution of 2 M KOH/0.02 M K 2 HPO 4 , after which the precipitated salt debris was pelleted and removed. Metabolites were quantified by liquid chromatography-mass spectrometry analysis (LC-MS), as described previously (Mayfield et al 2012).…”

“…Cell debris was pelleted for 10 min at 10,000 3 g and then the supernatants were adjusted to pH 6 using a solution of 2 M KOH/0.02 M K 2 HPO 4 , after which the precipitated salt debris was pelleted and removed. Metabolites were quantified by liquid chromatography-mass spectrometry analysis (LC-MS), as described previously (Mayfield et al 2012).For detection of SAM by the Pi SAM-I riboswitch aptamer, extracts were diluted 1:50, and 1 ml was applied to 9 ml radiolabeled RNA in TBMK buffer (90 mM Tris, 89 mM boric acid, 10 mM MgCl 2 , 10 mM KCl), as described previously (Karns et al 2013). Before mixing with lysate, the RNA had been renatured at 70°for 3 min and then allowed to return to room temperature for 10 min.…”