# Discriminating Pathogenic from Benign Alleles of Myelodysplastic Syndrome Predisposition Genes

> **NIH NIH R21** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $233,250

## Abstract

Patient genome sequencing has revealed germline genetic variation in DDX41 as one of the most frequent
genomic alterations implicated in creating a predisposition to myelodysplastic syndrome (MDS) and acute
myeloid leukemia (AML). DDX41 encodes an RNA helicase that regulates RNA splicing, senses double-stranded
DNA, operates in the cGAS-Sting pathway and promotes innate immunity. Many questions remain regarding
DDX41 mechanisms and how genetic variants impact its functions. To gain fundamental and translational
insights, we engineered the genome of HoxB8-immortalized murine hematopoietic progenitor cells, which
recapitulate the phenotype of primary progenitors, to yield Ddx41+/- cells. We innovated a rescue system to
compare human DDX41 activity with that of clinical variants. Using an unbiased genomic strategy, we identified
DDX41-regulated mRNAs and membrane proteins as activity metrics. We will use our foundation and machine
learning to construct a matrix that informs the relationship between DDX41 and clinical variants of uncertain
significance (VUS) or those deemed pathogenic. Although DDX41 represents one of >60 DEAD box domain
(DDX) proteins, unifying principles are not established. Aim 1 will innovate a system to discriminate
pathogenic from benign human DDX41 clinical genetic variants. Using a prioritization strategy involving
genetic variation attributes, an ensemble of variants was assembled for analysis. We will use our DDX41 activity
metrics to create a matrix that establishes the functional signature of any variant. This will enable a classification
strategy to predict whether a variant resembles DDX41 (“DDX41-like”) or pathogenic variants (“path-DDX41”).
Activity metrics will be extended by quantitative proteomics to identify additional DDX41-regulated proteins and
advanced RNA-seq analyses to identify transcript isoforms. Loss-of-function and rescue studies will determine
if activity metrics can be extrapolated to primary hematopoietic stem/progenitor cells. Aim 2 will conduct
pilot/exploratory studies on a mechanism involving DDX41-dependent alternative splicing at a locus
encoding an RNA splicing factor-regulatory kinase. Our results revealed that DDX41, but not a pathogenic
variant, promotes intron retention in Clk3 RNA, and DDX41 elevates the CDC-like Kinase-3 (CLK3) protein level
in myeloid cells. The CLK3 kinase phosphorylates splicing factor (SR) proteins SRSF1-12, some of which are
implicated in MDS and AML. We hypothesize that DDX41-induced intron retention and elevated CLK3 protein
have important functional consequences. We will test models to explain the consequences of Clk3 intron
retention and CLK3 protein elevation. The studies will establish a foundation to understand the DDX41-CLK3
mechanism, which may have considerable physiological and pathological impact. The rules governing DDX41
function and dysfunction that emerge will advance patient genetic curation and mechanistic logic to inform future
lines of biological...

## Key facts

- **NIH application ID:** 10999782
- **Project number:** 1R21AI186406-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Emery H. Bresnick
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $233,250
- **Award type:** 1
- **Project period:** 2024-05-20 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10999782

## Citation

> US National Institutes of Health, RePORTER application 10999782, Discriminating Pathogenic from Benign Alleles of Myelodysplastic Syndrome Predisposition Genes (1R21AI186406-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10999782. Licensed CC0.

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