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

A tale of two lakes: droughts, El Ninos and major cultural change during the last 5000 years in the Cuzco region of Peru.

No description supplie

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

Using the West Midlands CONCERT to characterise regional incidence of acute-onset post cataract surgery endophthalmitis.

BACKGROUND Whilst research and innovation is embedded within the UK's National Health Service (NHS) constitution, Doctors-in-training have little opportunity to contribute to designing, leading and recruiting into clinical trials or cohort studies. We formed the West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (The West Midlands CONCERT) and undertook a characterisation of post cataract surgery endophthalmitis as a proof-of-concept study to test the feasibility of the CONCERT model. METHODS Doctors-in-training formed a collaborative working group to test the concept of delivering a pan-regional clinical effectiveness study across multiple hospital sites by performing retrospective analyses of post cataract endophthalmitis over a 6-year period. RESULTS Overall, 157,653 cataract surgeries were performed by participating centres accredited to deliver the Royal College of Ophthalmologists training curriculum. Thirty-eight cases of post cataract endophthalmitis were identified, giving an incidence of 2.41 per 10,000 cases (0.0241%). A further 15 endophthalmitis cases presented who had surgery in non-training centres, giving a total of 53 cases. The most common organisms were S. epidermidis (14 (51.9%)) and P. aeruginosa (5 (18.5%)). Anterior-chamber and vitreous sampling yielded positive culture in 33.3% (6/18) and 50.9% (27/53), respectively. At 6 months follow-up, 19 (51.4%) patients achieved visual acuities of ≤0.5 LogMAR. Repeat intravitreal injections (11 (20.8%)) and vitrectomy (n = 22 (41.5%)) were not associated with better outcomes. CONCLUSIONS Using post cataract endophthalmitis as a pilot cohort, this study highlights the feasibility of using the CONCERT model for studies across multiple sites. A UK-CONCERT could provide a powerful infrastructure enabling characterisation of patient cohorts and a platform for high-quality interventional studies, improving patient care