Complex patterns of male germline instability and somatic mosaicism in myotonic dystrophy type 1

The genetic basis of myotonic dystrophy type 1 (DM1) is the expansion of a CTG repeat in the 3' untranslated region of DM1PK . Once into the disease range, the repeat becomes highly unstable and is biased toward expansion in both somatic and germline tissues. Intergenerational differences usually reveal an increase in allele length, concordant with the clinical anticipation characteristic of DM1, but there have also been cases with intergenerational contractions of the repeat length, accompanied by apparent anticipation. In order to gain a better understanding of this intergenerational behaviour, we have obtained semen samples from six DM males and used single molecule analyses to compare the allele distributions present in their sperm and blood with those of their offspring. We have confirmed that the male germline mutational pathway is distinct from that of the soma, but the extent of variation is highly variable from one individual to another and not obviously correlated with progenitor allele length. Nonetheless, in all cases the alleles present in the father's sperm overlap with those observed in their offspring. These data also provide further indications that the interpretation of intergenerational transmissions by standard analyses is frequently compromised by the masking of germline differences by age-dependent somatic expansion in the parent

CRISPR/Cas9-induced (CTG⋅CAG)n repeat instability in the myotonic dystrophy type 1 locus: implications for therapeutic genome editing

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1

A genetic association study of glutamine-encoding DNA sequence structures, somatic CAG expansion, and DNA repair gene variants, with Huntington disease clinical outcomes

Background: Huntington disease (HD) is caused by an unstable CAG/CAA repeat expansion encoding a toxic polyglutamine tract. Here, we tested the hypotheses that HD outcomes are impacted by somatic expansion of, and polymorphisms within, the HTT CAG/CAA glutamine-encoding repeat, and DNA repair genes. Methods: The sequence of the glutamine-encoding repeat and the proportion of somatic CAG expansions in blood DNA from participants inheriting 40 to 50 CAG repeats within the TRACK-HD and Enroll-HD cohorts were determined using high-throughput ultra-deep-sequencing. Candidate gene polymorphisms were genotyped using kompetitive allele-specific PCR (KASP). Genotypic associations were assessed using time-to-event and regression analyses. Findings: Using data from 203 TRACK-HD and 531 Enroll-HD participants, we show that individuals with higher blood DNA somatic CAG repeat expansion scores have worse HD outcomes: a one-unit increase in somatic expansion score was associated with a Cox hazard ratio for motor onset of 3·05 (95% CI = 1·94 to 4·80, p = 1·3 × 10−6). We also show that individual-specific somatic expansion scores are associated with variants in FAN1 (pFDR = 4·8 × 10-6), MLH3 (pFDR = 8·0 × 10−4), MLH1 (pFDR = 0·004) and MSH3 (pFDR = 0·009). We also show that HD outcomes are best predicted by the number of pure CAGs rather than total encoded-glutamines. Interpretation: These data establish pure CAG length, rather than encoded-glutamine, as the key inherited determinant of downstream pathophysiology. These findings have implications for HD diagnostics, and support somatic expansion as a mechanistic link for genetic modifiers of clinical outcomes, a driver of disease, and potential therapeutic target in HD and related repeat expansion disorders

Unstable triplet repeat diseases

Seven inherited human disorders are now associated with the intragenic expansion of triplet repeat DNA sequences. These repeats demonstrate extreme instability in both germline and somatic tissue, accounting for the unusual genetic inheritance patterns and symptom variability associated with these diseases

Unstable triplet repeat diseases

Seven inherited human disorders are now associated with the intragenic expansion of triplet repeat DNA sequences. These repeats demonstrate extreme instability in both germline and somatic tissue, accounting for the unusual genetic inheritance patterns and symptom variability associated with these diseases

Attempts to detect retrotransposition and de novo deletion of Alus and other dispersed repeats at specific loci in the human genome

Dispersed repeat elements contribute to genome instability by de novo insertion and unequal recombination between repeats. To study the dynamics of these processes, we have developed single DNA molecule approaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci in human genomic DNA. Validation experiments showed these approaches could detect insertions and deletions at frequencies below 10(-6) per cell. However, bulk analysis of germline (sperm) and somatic DNA showed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of 2 x 10(-7) and 3 x 10(-7), respectively, in the individuals tested. Such re-arrangements at these loci therefore occur at a rate lower than that detectable by the most sensitive methods currently available.Peer Reviewe

Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands

An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels of instability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis -acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats

Myotonic dystrophy: An unstable CTG repeat in a protein kinase gene

Myotonic dystrophy (DM) is caused by the amplification of CTG repeats in the 3’ untranslated region of a gene encoding a protein homologous to serine/threonine protein kinases. In DM patients the CTG repeats are extremely unstable, varying in length from patient to patient and generally increasing in length in successive generations. There is a strong correlation between the size of the repeats and the age of onset and severity of the disease. The molecular basis of the effect of the CTG expansion on the development of the DM phenotype continues to be investigated. The first working hypothesis of the molecular mechanism of DM was a reduction in steady-state myotonin-protein kinase (Mt-PK) mRNA and protein levels. However, although the consensus finding is that the Mt PK mRNA and protein levels are decreased in DM patients, it is still not clear if this reduction leads directly to the DM phenotype. In this short review we discuss the molecular aspects of CTG instability and the expression of the myotonin-protein kinase gene in normal and DM populations