Heterozygosity for a Hypomorphic Polβ Mutation Reduces the Expansion Frequency in a Mouse Model of the Fragile X-Related Disordersby Rachel Adihe Lokanga, Alireza Ghodsi Senejani, Joann Balazs Sweasy, Karen Usdin

PLoS Genet



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Heterozygosity for a Hypomorphic Polβ

Mutation Reduces the Expansion Frequency in a Mouse Model of the Fragile X-Related


Rachel Adihe Lokanga1,2, Alireza Ghodsi Senejani3, Joann Balazs Sweasy3,

Karen Usdin1* 1 Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of

Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of

America, 2 Department of Biochemistry, University of Cape TownMedical School, Cape Town, South Africa, 3 Departments of Therapeutic Radiology and Human Genetics, Yale University, School of Medicine, New

Haven, Connecticut, United States of America * ku@helix.nih.gov


The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat.

The FXDs result from expansion of a CGG/CCG repeat tract in the 5’ UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion

Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMRmachinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

Author Summary

Unstable microsatellites are responsible for a number of debilitating human diseases known as the Repeat Expansion Diseases. The unstable microsatellites, which consist of tandem arrays of short repeat units, are prone to increase in length (expand) on intergenerational transmission and during the lifetime of the individual. Unlike the typical

PLOSGenetics | DOI:10.1371/journal.pgen.1005181 April 17, 2015 1 / 16


Citation: Lokanga RA, Senejani AG, Sweasy JB,

Usdin K (2015) Heterozygosity for a Hypomorphic

Polβ Mutation Reduces the Expansion Frequency in a Mouse Model of the Fragile X-Related Disorders.

PLoS Genet 11(4): e1005181. doi:10.1371/journal. pgen.1005181

Editor: Sue Jinks-Robertson, Duke University,


Received: January 16, 2015

Accepted: March 27, 2015

Published: April 17, 2015

Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files.

Funding: Funding for this work was made possible by a grant from the Intramural program (IRP) of the

National Institute of Diabetes, Digestive and Kidney

Diseases to KU (DK057808-07). The research was also supported by R01ES019179 from the National

Institute of Environmental Health Sciences to JBS.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. microsatellite instability seen in disorders like Lynch syndrome that arise from mutations in mismatch repair (MMR) genes, expansions of these microsatellites are abolished when

MMR is lost. However, how MMR, which normally protects the genome against microsatellite instability, actually promotes microsatellite expansions in these diseases is unknown.

There is evidence to suggest that a second DNA repair process, base excision repair (BER), may be involved, but whether the nicks generated early in the BER-process are subverted by an MMR-dependent pathway that generates expansions or whether some MMR proteins contribute to a BER-based expansion process is unclear. Here we show that a mutation that reduces the activity of Polβ, an essential BER enzyme, also reduces the expansion frequency. Since Polβ is essential for key events in BER downstream of the generation of nicks, our data favor a model in which expansions occur via a BER-dependent pathway in which MMR participates.


The Fragile X-related disorders (FXDs) are members of the group of diseases known as the

Repeat Expansion Diseases. This group of diseases, which includes Huntington disease (HD) and Myotonic dystrophy type 1, are all caused by an increase in the number of repeats in an expansion-prone tandem repeat tract [1,2]. In the case of the FXDs the repeat is CGG/CCG and it is located in the 5’ untranslated region of the FMR1 gene (MIM 309550; reviewed in [3]).

The FXDs include Fragile X-associated primary ovarian insufficiency and Fragile X-associated tremor/ataxia syndrome (MIM# 300623) that occur in carriers of alleles with 54–200 repeats, so-called premutation (PM) alleles. Fragile X syndrome (MIM# 300624), the leading heritable cause of intellectual disability is seen in carriers of full mutation alleles (>200 repeats).

The repeats responsible for the Repeat Expansion Diseases share the ability to form unusual secondary structures of one sort or another [1,2]. In the case of the FXDs, the repeats have the potential to form hairpins containing a mixture of Watson-Crick and Hoogsteen base pairs, as well as a variety of quadruplex structures [4,5,6,7,8,9,10]. Many of these sequences also form persistent RNA:DNA hybrids [11,12,13]. Current thinking in the field is that these structures are the substrates upon which the expansion and contraction processes act. However, the mechanism involved is unclear.