Constructing and interpreting a large-scale variant effect map for an ultrarare disease gene: Comprehensive prediction of the functional impact of PSAT1 genotypes.

Reduced activity of the enzymes encoded by PHGDH, PSAT1, and PSPH causes a set of ultrarare, autosomal recessive diseases known as serine biosynthesis defects. These diseases present in a broad phenotypic spectrum: at the severe end is Neu-Laxova syndrome, in the intermediate range are infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end is childhood disease with intellectual disability. However, L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms. Therefore, knowledge of pathogenic variants can improve clinical outcomes. Here, we use a yeast-based assay to individually measure the functional impact of 1,914 SNV-accessible amino acid substitutions in PSAT. Results of our assay agree well with clinical interpretations and protein structure-function relationships, supporting the inclusion of our data as functional evidence as part of the ACMG variant interpretation guidelines. We use existing ClinVar variants, disease alleles reported in the literature and variants present as homozygotes in the primAD database to define assay ranges that could aid clinical variant interpretation for up to 98% of the tested variants. In addition to measuring the functional impact of individual variants in yeast haploid cells, we also assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. Results from our diploid assay successfully distinguish the genotypes of affected individuals from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements to predict the biallelic function of ~1.8 million allele combinations corresponding to potential human genotypes. Taken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of ultrarare diseases.

Predicting the functional effect of compound heterozygous genotypes from large scale variant effect maps.

Background: Pathogenic variants in PHGDH, PSAT1 , and PSPH cause a set of rare, autosomal recessive diseases known as serine biosynthesis defects. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately in the form of infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, as childhood disease with intellectual disability. However, because L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is highly actionable. Methods: Recently, our laboratory established a yeast-based assay for human PSAT1 function. We have now applied it at scale to assay the functional impact of 1,914 SNV-accessible amino acid substitutions. In addition to assaying the functional impact of individual variants in yeast haploid cells, we can assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. Results: Results of our assays of individual variants (in haploid yeast cells) agree well with clinical interpretations and protein structure-function relationships, supporting the use of our data as functional evidence under the ACMG interpretation guidelines. Results from our diploid assay successfully distinguish patient genotypes from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements (in haploid yeast cells) to accurately predict the biallelic function (in diploid yeast cells) of ~ 1.8 million allele combinations corresponding to potential human genotypes. Conclusions: Taken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of a rare disease.