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. 2003 May 1;22(9):2264-73.
doi: 10.1093/emboj/cdg202.

CTG repeat instability and size variation timing in DNA repair-deficient mice

Cédric Savouret  1 , Edith Brisson, Jeroen Essers, Roland Kanaar, Albert Pastink, Hein te Riele, Claudine Junien, Geneviève Gourdon
Affiliations

CTG repeat instability and size variation timing in DNA repair-deficient mice

Cédric Savouret et al. EMBO J. .

Abstract

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining). Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.

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Figures

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Fig. 1. Changes in CTG repeat size between transgenic parent and offspring for the various transmissions. The x-axis shows CTG repeat length change and the y-axis shows the percentage corresponding to each size change for all transmissions in each line. The frequency (%) and mean size (m) of expansions are indicated on the right. The frequency (%) and mean size (m) of contractions are indicated on the left. The number of transmissions assayed for each genotype was as follows: DM300–328, 299; Msh2 +/– to +/–, 82; Msh2 –/– to –/–, 82.
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Fig. 2. CTG repeat size variations following transgene transmission between parent and offspring carrying different Msh2 genotypes. The upper genotype indicates parental Msh2 status and the lower genotype indicates that of the offspring. + is the wild-type allele and – is the mutant allele. In each column, the left bar corresponds to male transmissions and the right bar to female transmissions. The frequencies of expansions (Exp.) and contractions (Cont.) are indicated for each transmission. n is the number of transmissions analyzed.
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Fig. 3. Distribution of alleles with different CTG repeat sizes in spermatozoa from 10-month-old male transgenic DM300–328, Msh2 +/– or Msh2 –/– mice, as determined by single-cell PCR. The y axis shows the percentage of each size allele with respect to all single genomes analyzed. The dotted line indicates the CTG repeat size in tail DNA at weaning: 372 CTG for DM300–328, 373 CTG for Msh2 +/– and 372 CTG for Msh2 –/–. The frequency and mean size of expansions are indicated on the right and the frequency and mean size of contractions are indicated on the left. The number of single genomes analyzed for each tissue was as follows: DM300–328, 113; Msh2 +/–, 93; Msh2 –/–, 92.
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Fig. 4. Somatic instability in 10-month-old male and female DM300–328, Rad54 +/–, Rad54 –/–, DNA-PKcs +/–, DNA-PKcs –/–, Msh2 +/– and Msh2 –/– transgenic mice. The same instability profiles were observed between Rad52 +/– and Rad52 –/– transgenic mice (data not shown). h, heart; b, brain; l, liver; p, pancreas; k, kidney; q, quadriceps; o, ovary; t, testis; bl, blood; c, cerebellum; lu, lung. CTG repeat length in blood is indicated for each mouse. m, male; f, female.
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Fig. 5. Distribution of alleles with different CTG repeat sizes in quadriceps from 10-month-old male transgenic DM300–328, Rad52 –/–, Rad54 –/– and DNA-PKcs –/– mice, as determined by single-cell PCR. The dotted line is the CTG repeat size in blood: 372 CTG for DM300–328, 369 CTG for Rad52 –/–, 387 CTG for Rad54 –/– and 373 CTG for DNA-PKcs –/–. The number of single molecules analyzed for each male was as follows: DM300–328: 87; Rad52 –/–, 105; Rad54 –/–, 106; DNA-PKcs –/–, 86.
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Fig. 6. Distribution of alleles with different CTG repeat sizes in quadriceps and cerebellum from 10-month-old transgenic male DM300–328, Msh2 +/– and Msh2 –/– mice, as determined by single-cell PCR. Dotted lines represent the CTG repeat size measured in blood: 372 CTG for DM300–328, 373 CTG for Msh2 +/– and 372 CTG for Msh2 –/–. The number of single molecules analyzed for each tissue was as follows, DM300–328: quadriceps, 87; cerebellum, 80; Msh2 +/–: quadriceps, 106; cerebellum, 83; Msh2 –/–: quadriceps, 101; cerebellum, 88.
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