Biology subjects

Lo, R. S.

Publications and source records attributed to Lo, R. S..

4 recordsLinked to original sources

bioRxiv2025

Functional Profiling of 2,193 ASS1 Missense Variants: Insights into Variant Pathogenicity and Epistatic Interactions in Citrullinemia Type I

Sequence variants in the urea cycle gene argininosuccinate synthase (ASS1) cause Citrullinemia type 1 (CTLN1), a rare autosomal recessive disease. Mechanistically, reduction in argininosuccinate synthetase (ASS) enzyme activity impairs the urea cycle, leading to an accumulation of citrulline and neurotoxic ammonia. Disease severity varies according to the degree of enzyme impairment, ranging from severe neonatal forms (classic citrullinemia) to milder, late-onset forms that may manifest in childhood or adulthood. We established a high-throughput yeast functional assay of human ASS and individually measured the impact of 2193 amino acid substitutions, representing 90% of all single nucleotide variant (SNV)-accessible substitutions. When benchmarked against existing clinical variant annotation, our assay distinguishes known benign variants from strong loss of function pathogenic variants, enabling identification of a functional score threshold below which variants show clinically relevant impairment of ASS activity. Using the ACMG OddsPath framework, our assay meets PS3_supporting criteria for pathogenicity classification and achieves full PS3-level strength when variants observed as homozygotes in other primates are used as benign proxies for calibration. These results provide direct functional evidence to inform reclassification of ASS1 missense variants, supporting their clinical interpretation and diagnostic utility. Mapping functional scores onto the protein structure, we confirmed that residues involved in catalysis are highly sensitive to substitution. In addition, we identified residues from adjacent subunits of the ASS homotetramer that form compound active sites. Assaying these positions revealed a capacity for intragenic complementation consistent with a variant sequestration model: a form of positive epistasis in which deleterious variants from different subunits are sequestered into only a subset of active sites, restoring function in the remaining variant-free sites. The discovery of intragenic complementation in ASS reveals a novel mode of functional interaction with clinical implications for interpreting variant combinations in heterozygous individuals.

genetics↗

bioRxiv2023

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

BackgroundPathogenic 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. MethodsRecently, 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. ResultsResults 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. ConclusionsTaken 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.

genetics↗

bioRxiv2022

The functional impact of 1,570 SNP-accessible missense variants in human OTC

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare, but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Neonatal onset patients appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma and death, outcomes ameliorated by rapid diagnosis and treatment. Existing biochemical assays have limitations, including the sensitivity of the citrulline assays used in newborn screening panels. With prior knowledge of variant pathogenicity, DNA sequence-based diagnostics would provide an alternative screening method. Here, we develop a high throughput functional assay for human OTC and measure the impact of 1,570 variants, 84% of all SNP-accessible missense mutations. Our assay scores agree well with existing clinical significance calls, distinguishing known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. Further, use of an intronless expression construct allows us to measure the impact of amino acid changes at splice sites independent of their effect on splicing, thereby separating the contribution of splicing and protein coding changes to aid the analysis of molecular mechanisms underlying pathogenicity. Finally, we assess the utility of our functional data on OTC variant curation by using the current ACMG/AMP guidelines to reclassify variants. Inclusion of our data as PS3/BS3 substantially improves variant interpretation. Thus, our dataset is of high clinical utility and illustrates the power of functional assays to inform interpretation of existing and novel genetic variation.

genetics↗

bioRxiv2019

Multi-model preclinical platform predicts clinical response of melanoma to immunotherapy

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrates durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models representing the main molecular and phenotypic subtypes of human melanomas and exhibiting their range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify new mechanisms and treatment strategies to improve patient care.

cancer biology↗