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.

Figure 1

Growth of CBS-complemented yeast on solid media. Cultures grown to saturation in liquid minimal medium containing glutathione and lacking B6 were plated in a fivefold dilution series onto solid medium ± glutathione. Growth was imaged after 3 days at 30°. The growth of the major allele and representative alleles of the nonfunctional and B6-responsive classes are shown.

Figure 2

CBS protein levels in yeast whole cell extracts. (A) Immunoblotting of yeast cells with the CBS major allele (MA), a B6-responsive allele (A226T), an AdoMet-domain mutation (Q526K), or an empty expression vector (EV) were grown in minimal medium with 400 ng/ml B6 alone, with glutathione alone, or with glutathione and 400 ng/ml B6. (B) Yeast cells with the CBS MA and five variant alleles were grown in minimal medium with glutathione alone or with glutathione and 400 ng/ml B6. Representative alleles from the nonfunctional (T87N and P88S) and sick (P145L, V168M, and M126V) phenotypic classes were processed for immunoblotting. 3-Phosphoglycerate kinase (PGK) was detected as a loading control.

Figure 3

CBS yeast exhibited B6-dependent growth. (A) Representative growth curves of yeast with the major allele of human CBS cultures supplemented with six different levels of B6 (colored lines). Average growth rate (±SD) is shown for each B6 level (n = 84–90). (B) The growth of each mutant (n ≥ 4) was expressed as the percentage of average growth rate of yeast with the major allele of human CBS at each B6 level (±SD).

Figure 4

CBS yeast growth responses to B6 and heme grouped alleles into distinct classes. Heat maps of growth rates normalized to the growth of the major allele after titration of (A) B6 in HEM1 yeast or (B) B6 and heme in hem1 yeast. The column Z-score indicates the mean growth rate (Z-score of 0) and standard deviation (Z-score of ±1) of all alleles per column, with positive Z-scores indicating higher than average growth. Arrowheads indicate alleles that respond to cofactor titration more strongly than other alleles in their cluster. Asterisks (*) denote alleles that failed to grow in HEM1 yeast but were capable of growth in hem1 yeast.

Figure 5

Metabolite profiles of CBS yeast grown under nutrient replete or limiting conditions. Heat map of amino acid or derivative metabolite levels in cell extracts from yeast grown with either the major CBS allele (MA) or the G307S (nonfunctional) allele, as measured by mass spectrometry. Each column represents the average of four biological replicates. B6 was supplemented at doses that produced robust growth of the major allele (400 ng/ml) or measurable, but compromised, growth (1 ng/ml). Metabolite levels were scaled for each row and both metabolites and experimental conditions were subject to hierarchical clustering. The row Z-score indicates the mean and standard deviations for each metabolite, such that the mean metabolite level has as a Z-score of 0. Duplicate columns were independent cell extracts and demonstrated trial-to-trial variation that was not significant in any of the known metabolites (t-test P > 0.05). The oxidizing conditions used for extraction strongly favored isolation of homocystine over homocysteine. Similarly cystathionine and cysteine were not detected because of limitations in sample processing or because intracellular pools are small.

Figure 6

Levels of metabolites critical in CBS function. Scatter plots of the levels of four different metabolites measured by mass spectrometry. The average of four biological replicates (bars) and their individual measurements (squares) are shown. Duplicated columns show trial-to-trial variation in independent cell extracts. (A) Methionine, (B) AdoMet, (C) homocystine, and (D) glutathione. The levels of all four metabolites are significantly different (ANOVA P < 0.0001); all significant differences between the MA at high B6 and other classes are indicated (Tukey’s honest significance test **, P < 0.005; ****, P < 0.0001).

Figure 7

CBS phenotypes in relation to primary structure. Diagram of the domain structure of the CBS protein with the location of the 84 alleles used in this analysis represented by colored bars above the diagram. Each bar represents an allele; colors indicate the affect of the allele on growth. The Robust row reports the position of alleles indistinguishable from the predominant allele.