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Saturation functional assays of MUTYH resolve clinical variants of uncertain significance

ABSTRACT: Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder mutations and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,122 existing clinical missense VUS in MUTYH and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.

ORGANISM(S): synthetic construct

PROVIDER: GSE290379 | GEO | 2025/03/01

REPOSITORIES: GEO

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	GSE290379_aa_counts.tsv.gz	Tabular

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Project description:Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>5000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 99% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of HAP1 haploid cells endogenously targeted using a CRISPR/cas9 knockin approach. A total of 6959 SNVs were characterized for effects on cell survival. The assay was validated relative to nonsense and synonymous variants, ClinVar pathogenic/likely pathogenic and benign/likely benign variants and homology directed repair cell based DNA repair assay results, all of which showed >94% sensitivity and specificity. Variants were assigned posterior probabilities of pathogenicity using a VarCall two component Bayesian mixture model and were further grouped according to ACMG strength of evidence under the PS3/BS3 rule resulting in Benign Strong (n=5430), Benign Moderate (n=190), Benign Supporting (n=61), VUS (122), Pathogenic Strong (n=1021), Pathogenic Moderate (n=88), and Pathogenic Supporting (n=47). Breast cancer case-control association studies showed that pooled SNVs encoding functionally pathogenic missense variants were associated with increased risks of breast (odds ratio (OR)3.81, 95%CI: 2.88-5.07) and ovarian cancer (OR 5.93, 95%CI: 4.12-8.52). The functional data were also combined with other sources of information in the ClinGen BRCA1/2 VCEP ACMG/AMP-classification model. A total of 785 SNVs, including 261 missense SNVs, were classified as pathogenic or likely pathogenic, while 5566 SNVs, including 3786 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers.

Project description:Despite widespread advances in DNA sequencing in the past decade, the functional consequences of most rare genetic variants remain poorly understood, severely limiting our ability to connect variants to their consequences on protein function, identify biochemical mechanisms by which variation causes disease, and interpret variant pathogenicity. Multiplexed Assays of Variant Effect (MAVEs), which can measure the function of tens of thousands variants, are beginning to address this problem. However, existing MAVEs cannot be applied to the approximately 10% of human genes encoding secreted proteins, about a quarter of which are associated with disease. We developed a flexible and scalable human cell surface display method, Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), that can simultaneously measure the functional effects of tens of thousands of variants in secreted proteins. We used MultiSTEP to study the consequences of missense variation in coagulation factor IX (FIX), a vitamin K-dependent plasma serine protease where variation can cause FIX deficiency and the bleeding disorder hemophilia B. We used a panel of antibodies to detect FIX secretion or FIX post-translational modification, measuring a total of 45,024 effects for 9,007 variants. 43.8% of all possible F9 missense variants impact FIX secretion, post-translational modification or both. We also identify new signals of functional constraint on secretion including within the signal peptide, folded domains, and for nearly all variants that caused gain or loss of cysteine. FIX secretion scores correlate strongly with FIX levels in patient plasma and also reveal that most F9 missense variants causing severe hemophilia do so by profoundly impacting secretion. We integrate the secretion and post-translational modification data to develop a F9 variant classifier that can identify loss of function variants with high specificity. We use the resulting classifications to reinterpret and upgrade 62 of 97 F9 variants of uncertain significance (VUS) in the MyLifeOurFuture hemophilia genotyping project to likely pathogenic. Lastly, we show that MultiSTEP can be applied to a wide variety of secreted proteins, ranging from small signaling proteins like insulin to large proteins like factor VIII. Thus, we establish a multiplexed, multimodal, and generalizable method for systematically assessing variant effects for secreted proteins at scale, paving the way for improved understanding of biochemical mechanisms of disease and clinical variant interpretation.

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Saturation functional assays of MUTYH resolve clinical variants of uncertain significance

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