# Massively parallel experimental measurement of variant functional impacts

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $430,822

## Abstract

The commoditization of exome and genome sequencing is reshaping basic research and clinical practice
in genetics. Despite successes in implicating casual disease loci through large-scale sequencing, there are no
similarly straightforward means to distinguish between pathogenic and neutral alleles for a given gene. Even
for genes with long-standing disease associations, a substantial fraction of clinically observed alleles are
classified as “variants of unknown significance”, or VUS. For instance, mutations in DNA mismatch repair
(MMR) genes strongly predispose to colorectal and other cancers and are frequently screened for in the clinic,
yet >30% of all known MMR gene mutations are classified as VUS (Thompson et al., Nature Genetics, 2013).
This uncertainty poses a considerable obstacle to the goal of genotype-driven treatment. We propose to
address this challenge by developing scalable technologies to synthesize and functionally screen mutations in
a massively parallel fashion. We anticipate that these tools will be broadly useful, and we will apply them first to
human MMR gene mutations, with the following specific aims: (1) to profile the functional activity of every
possible missense variant for three human MMR genes (MSH2, MLH1, and MSH6), and to predict the resulting
likelihood of pathogenicity across alleles. We will construct comprehensive allelic series for these genes,
introduce them into relevant MMR-null mammalian tissue culture models, select en masse for restoration of
MMR activity, and count the resulting allelic depletion by deep sequencing; (2) to survey the prevalence and
degree of epistatic effects between and within MMR genes by introducing libraries of double mutants and
screening as in Aim 1; and, (3) to directly characterize the types and frequencies of mutations caused by
inactivation of MMR using whole-genome sequencing in isogenic cell lines following repeated passaging.
Completion of these aims will clarify sequence-structure-function relationships for human MMR genes, and will
allow derivation of a risk score for clinically observed mutations. The methodological advances proposed here
will provide a foundation for generalized approaches to dissect the allelic heterogeneity of human oligogeneic
disorders, and a path toward functional annotation of the rapidly growing VUS catalogs.

## Key facts

- **NIH application ID:** 10439616
- **Project number:** 5R01GM129123-05
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Jacob Otto Kitzman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $430,822
- **Award type:** 5
- **Project period:** 2018-08-06 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10439616

## Citation

> US National Institutes of Health, RePORTER application 10439616, Massively parallel experimental measurement of variant functional impacts (5R01GM129123-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10439616. Licensed CC0.

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