Abstract
Multisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including DMPK, its neighboring genes (SIX5 or DMWD) and downstream MBNL1. However, direct evidence is lacking. Here, we develop a new strategy to generate mice carrying multigene heterozygous mutations to mimic dosage reduction in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, the quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness, providing a unique model for screening small molecules for treatment of DM1. Our results suggest that the complex symptoms of DM1 result from the reduced dosage of multiple genes.
Highlights
Myotonic dystrophy type 1 (DM1) is a complex disease with variable pathological phenotypes, disease severities, and onset ages.[1,2,3] The major symptoms include myotonia, muscle wasting, muscle weakness, cardiac conduction defects, cataracts and insulin resistance.[3]
Generation of a novel DM1 model carrying mutations in Dmpk, Six[5] and Mbnl[1] We first examined the feasibility of semi-cloning technology to generate mouse models of DM1 carrying a single mutant gene by injection of haploid cells carrying a mutation in Dmpk, Six[5] or Mbnl[1], the three well-studied DM1-related genes
S1–3) and found that these cells efficiently supported the generation of live SC pups carrying heterozygous mutant Dmpk, Six[5] or Mbnl[1] by ICAHCI (Supplementary information, Table S1)
Summary
Myotonic dystrophy type 1 (DM1) is a complex disease with variable pathological phenotypes, disease severities, and onset ages.[1,2,3] The major symptoms include myotonia, muscle wasting, muscle weakness, cardiac conduction defects, cataracts and insulin resistance.[3] DM1 is a genetic disease caused by the expansion of a CTG repeat in the 3′-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene.[4,5,6] Usually, with an increasing number of the repeats, respiratory failure and mental retardation can be observed in the most severe form of the disease (congenital DM1, CDM).[7,8,9,10] A large number of studies have shown that the triple repeat expansion reduces the protein or mRNA levels of DMPK (haploinsufficiency model),[11,12,13] and alters the adjacent chromatin structure and reduces the expression of the neighboring genes,[14,15,16,17] including downstream SIX518–22 and upstream DMWD23 in DM1 cells or patients (local chromatin structure change model), albeit with controversial observations.[24,25,26] numerous studies have shown that nuclear-accumulated RNA containing the expanded CUG repeats aberrantly recruits splicing regulators, such as MBNL1, and forms the ribonuclear aggregates (foci) in nucleus (RNA toxicity model), leading to misregulation of alternative splicing.[27,28,29,30,31,32] Mouse models carrying one mutation in
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