MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats

Incidence and phenotypes of childhood-onset genetic epilepsies:a prospective population-based national cohort

Epilepsy is common in early childhood. In this age group it is associated with high rates of therapy-resistance, and with cognitive, motor, and behavioural comorbidity. A large number of genes, with wide ranging functions, are implicated in its aetiology, especially in those with therapy-resistant seizures. Identifying the more common single-gene epilepsies will aid in targeting resources, the prioritization of diagnostic testing and development of precision therapy. Previous studies of genetic testing in epilepsy have not been prospective and population-based. Therefore, the population-incidence of common genetic epilepsies remains unknown. The objective of this study was to describe the incidence and phenotypic spectrum of the most common single-gene epilepsies in young children, and to calculate what proportion are amenable to precision therapy. This was a prospective national epidemiological cohort study. All children presenting with epilepsy before 36 months of age were eligible. Children presenting with recurrent prolonged (>10 min) febrile seizures; febrile or afebrile status epilepticus (>30 min); or with clusters of two or more febrile or afebrile seizures within a 24-h period were also eligible. Participants were recruited from all 20 regional paediatric departments and four tertiary children’s hospitals in Scotland over a 3-year period. DNA samples were tested on a custom-designed 104-gene epilepsy panel. Detailed clinical information was systematically gathered at initial presentation and during follow-up. Clinical and genetic data were reviewed by a multidisciplinary team of clinicians and genetic scientists. The pathogenic significance of the genetic variants was assessed in accordance with the guidelines of UK Association of Clinical Genetic Science (ACGS). Of the 343 patients who met inclusion criteria, 333 completed genetic testing, and 80/333 (24%) had a diagnostic genetic finding. The overall estimated annual incidence of single-gene epilepsies in this well-defined population was 1 per 2120 live births (47.2/100 000; 95% confidence interval 36.9–57.5). PRRT2 was the most common single-gene epilepsy with an incidence of 1 per 9970 live births (10.0/100 000; 95% confidence interval 5.26–14.8) followed by SCN1A: 1 per 12 200 (8.26/100 000; 95% confidence interval 3.93–12.6); KCNQ2: 1 per 17 000 (5.89/100 000; 95% confidence interval 2.24–9.56) and SLC2A1: 1 per 24 300 (4.13/100 000; 95% confidence interval 1.07–7.19). Presentation before the age of 6 months, and presentation with afebrile focal seizures were significantly associated with genetic diagnosis. Single-gene disorders accounted for a quarter of the seizure disorders in this cohort. Genetic testing is recommended to identify children who may benefit from precision treatment and should be mainstream practice in early childhood onset epilepsy

Isolated short CTG/CAG DNA slip-outs are repaired efficiently by hMutSβ, but clustered slip-outs are poorly repaired

Expansions of CTG/CAG trinucleotide repeats, thought to involve slipped DNAs at the repeats, cause numerous diseases including myotonic dystrophy and Huntington's disease. By unknown mechanisms, further repeat expansions in transgenic mice carrying expanded CTG/CAG tracts require the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSβ complex. Using an in vitro repair assay, we investigated the effect of slip-out size, with lengths of 1, 3, or 20 excess CTG repeats, as well as the effect of the number of slip-outs per molecule, on the requirement for human MMR. Long slip-outs escaped repair, whereas short slip-outs were repaired efficiently, much greater than a G-T mismatch, but required hMutSβ. Higher or lower levels of hMutSβ or its complete absence were detrimental to proper repair of short slip-outs. Surprisingly, clusters of as many as 62 short slip-outs (one to three repeat units each) along a single DNA molecule with (CTG)50•(CAG)50 repeats were refractory to repair, and repair efficiency was reduced further without MMR. Consistent with the MutSβ requirement for instability, hMutSβ is required to process isolated short slip-outs; however, multiple adjacent short slip-outs block each other's repair, possibly acting as roadblocks to progression of repair and allowing error-prone repair. Results suggest that expansions can arise by escaped repair of long slip-outs, tandem short slip-outs, or isolated short slip-outs; the latter two types are sensitive to hMutSβ. Poor repair of clustered DNA lesions has previously been associated only with ionizing radiation damage. Our results extend this interference in repair to neurodegenerative disease-causing mutations in which clustered slip-outs escape proper repair and lead to expansions

Representative CAG repeat distributions, and <i>Msh3</i> variations in B6 and CBy mice.

<p>A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the <i>Msh2</i>−/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the <i>Msh3</i> locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen-1003280-g001" target="_blank">Figure 1B</a>). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern (<i>e.g. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Mangiarini1" target="_blank">[10]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Manley1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Wheeler1" target="_blank">[15]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-vanDenBroek1" target="_blank">[16]</a>. This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by <i>Taq</i> polymerase slippage <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Zhang1" target="_blank">[62]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Hauge1" target="_blank">[63]</a>. The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the <i>Msh3</i> locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) <i>Msh3</i> polymorphisms in <i>Msh3</i> gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in <i>dbSNP</i> by sequencing the <i>Msh3</i> gene.</p

MSH3 coding polymorphisms and protein expression in different mouse strains.

<p>A) <i>Msh3</i> polymorphisms in <i>Msh3</i> gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in <i>dbSNP</i> by sequencing the <i>Msh3</i> gene. In DBA/2J, exon 8, AA#392 was correctly identified to be T/Valine. For a given amino acid the same codon was used for the variants. The complete set of MSH3 protein polymorphisms in 14 mouse strains is in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280.s009" target="_blank">Table S2</a>. B) MSH3 expression in spleen extract from different background using two different MSH3 antibodies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Holt1" target="_blank">[65]</a>. The faster migrating band for 5A5 was a non-specific cross-reacting product, as described for 5A5 but not 2F11 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Holt1" target="_blank">[65]</a>. All other figures in this study used 2F11. C) Typical GeneScan traces for sizing of the CAG repeat as outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen-1003280-g001" target="_blank">Figure 1B</a>. Representative CAG repeat distributions from liver of F1 progeny between CBy and other inbred strains of mice. The top, bottom and second panel show the controls CBy (stable), B6 (unstable), and CBy X B6 (intermediate) CAG profiles, respectively. Note: Western blot data comes from inbred mice. The higher levels of MSH3 in C3H and B6 are halved in the cross to CBy.</p

Structural and sequence analysis of MSH3.

<p>A: Multiple sequence alignment of MSH3. Jalview created visualization <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Clamp1" target="_blank">[120]</a> using the first 500 amino acids of the mouse B6 MSH3 (NP_034959.2). Conservation values and consensus sequence are based on alignment of <i>S. cerevisiae</i> Msh3p, <i>E. coli</i> MutS and 17 mammalian MSH3 homologs; values range from 0–9, where 0 is lowest and 9 is the highest. Protein interacting domains indicated pertain to those regions of the human MSH3 protein. This panel only shows an abbreviated set of the species of MSH3 sequence, the full set analysed is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280.s005" target="_blank">Figure S5</a>. B: MSH3 variant within β-turn. The T321I variant occurs within a Type I β-turn, as determined by specific backbone turn angles <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Kabsch1" target="_blank">[117]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Venkatachalam1" target="_blank">[118]</a> from the human MSH3 structure (3THW_B). Top left: hMSH3 tube diagram of Cα atoms of β-turn (blue), <i>i</i>+2 (T) residue (red) and additional three residues on N- and C-terminal ends (green). Bottom left table shows the β-turn propensity is relatively strong throughout MutS/MSH3 homologs, while the CBy variant (Isoleucine at <i>i+2</i> position) is extremely disfavored (table bottom left) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Hutchinson1" target="_blank">[69]</a>. Right: Ball and stick diagram of contact sites of Asp (D) and Thr (T) residues in β-turn with residues 194 and 214 respectively. Line diagram of Thr (T) hydroxyl group contact with neighbouring Threonine residue at position 365. The absence of the Threonine hydroxyl group may be important to stabilizing the β-turn itself, and/or may change the conformation of the turn, potentially disrupting distant contacts important for proper protein folding. MSH3 visualizations created using PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).</p

Representative CAG repeat distributions from reciprocal <i>Msh3</i> congenic lines of mice.

<p>Typical GeneScan traces for sizing of the CAG repeat as outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen-1003280-g001" target="_blank">Figure 1B</a>. Liver (A) and Striatum (B) from 16–20 week old R6/1 transgenic mice showing the effect of homozygosity at the <i>Msh3</i> locus on the pattern of expansion in the reciprocal congenic mice. Regardless of genetic background, CBy homozygosity at the congenic locus results in loss of somatic expansion, while B6 homozygosity is permissive of somatic expansion.</p

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

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