Downloads – Frameshift Mutator Assay (FSMA)

Frameshift Mutator Assay (FSMA)

Strong evidence from human genome-wide association studies (GWAS) of people with Huntington’s Disease (HD) have linked components of the mismatch repair (MMR) pathway, particularly MSH3, as key modulators of the impact of CAG repeat length in Exon 1 of HTT on age at onset¹.  Inhibition of MSH3 activity is able to slow or halt somatic expansion of CAG repeats in both human cellular models^2,3 and animal models^4,5 with expanded CAG repeats.  In order to study the structure-function relationship of MSH3 and other MMR proteins in a cellular context we sought to develop an assay with a more rapid and robust readout relative to measuring HTT Exon 1 CAG expansion, which typically takes weeks to months.  To this end, a GFP frameshift mutator assay (FSMA)^6,7 was developed in HeLa cell lines, exploiting the observation that tetranucleotide repeat motifs, adapted from Elevated Microsatellite Alterations at Selected Tetranucleotide (EMAST) motifs, can expand or contract during mitosis in a matter of days.  A stretch of 28 AAAG repeats were cloned upstream of an eGFP reporter to render GFP out of frame for transcription. Upon repeat slippage events, GFP becomes in-frame and is expressed. GFP-positive cells were quantified by fluorescence-activated cell-sorting (FACS), and levels of relevant MMR-associated proteins were quantified by simple Western blotting (Jess). GFP-positive cell fraction can be correlated with MMR pathway activity which, under these conditions, primarily relies on MutSβ (MSH2/MSH3) and MutLa (MLH1/PMS2). To investigate MMR-protein interactions and genome-wide association study (GWAS)-relevant mutations, selected pools with specific knock-out (KO) and over-expression (OE) combinations were generated and analyzed in comparison to controls, such as WT cells (in the following indicated as “no KO, no OE”) and KO of MMR components, alone or in combination.

In this document, specific aspects of the studies are shown per page, and summaries of FACS and Jess data are described; on subsequent pages the FACS data from all single experiments are presented. Each summary page has a unique identifier so that the respective data set can be identified from the associated master data table.

• GeM-HD Consortium, 2019, Cell 178:  887-900
• Ferguson et al., 2024,  Am J Human Gen, 111:  1165-1183.
• Bunting et al., 2025, Sci Transl Med 17: eadn4600.
• Mouro Pinto et al., 2025, Nat Gen., 57(2):314-322
• O’Reilly et al., 2023, Molecular Therapy 31: 1-14.
• Slebos et al., 2002, Cancer Research 62: 6052-6060.
• Raeker et al., 2020, Cell Mol Gastroenterol Hepatol 9: 689-704.