Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1

Abstract

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

Molecular and Clinical Implications of Variant Repeats in Myotonic Dystrophy Type 1

“…The occurrence of different types and patterns of variant repeats indicates that they probably originate by rare base substitutions within the pure (CTG)n array then spread along the expansion during processes involved in DNA metabolism. Rare findings of de novo occurring CTC [ 40 ] and CCG [ 43 , 44 ] variant repeats support this hypothesis of their origin.…”

“…Although Aci I digestion was more commonly used for patient screening [ 36 , 43 , 46 ], it is of note that studies relying on RP-PCR also identified CCG repeat as the most common interruption [ 37 , 40 ]. Proof-of-concept studies have demonstrated the potential of PCR-based long-read PacBio sequencing technology to overcome the aforementioned limitations [ 43 , 44 ]. Harnessing long-read sequencing technologies will certainly bring a more complete picture of structural variations of not only DMPK expansions, but other expansions as well, including DM2.…”

“…Both expansion and contraction events are possible during intergenerational transmission of DMPK expansions with variant repeats in DM1 families. However, contractions or, at least, stable transmissions are more likely, both in paternal and maternal transmissions [ 36 , 37 , 38 , 40 , 44 ]. For examples of maternal and paternal transmission of interrupted DMPK expansions we are referring readers to Figures 3 and 4 (subjects DF1-1, DF1-2, DF1-3, DF2-1 and DF2-2) in Pesovic et al [ 40 ].…”

“…Interestingly, even a DMPK expansion carrying a single CAG interruption was characterized by stable transmission or contraction in successive generations [ 42 ], suggesting that a single base substitution within CTG repeat may be sufficient to increase the meiotic stability of DMPK expansion. Although transmissions of interrupted DMPK expansions showed no specificity related to the sex of the transmitting parent [ 40 ], it is worth mentioning that all de novo occurring variant DMPK expansions, described so far, were transmitted by fathers, whether it was an expansion carrying a single CTC repeat [ 40 ], or different patterns of CCG repeats [ 43 , 44 ].…”

Identification de nouveaux facteurs entraînant des contractions CTG.CAG dans la dystrophie myotonique de type 1

“…Des fibroblastes contenant plusieurs centaines de répétitions CTG seront traités par ces molécules candidates ou du DMSO sur une période de trois mois, temps nécessaire pour observer et quantifier la mosaïque somatique (temps estimé sur la base de nos expériences précédentes). La mosaïque somatique des répétitions CTG dans les différentes cultures cellulaires sera mesurée en utilisant la dernière technologie de séquençage à longue lecture développée par Pacific Biosciences (Figure 3) [13,14] Cette technologie de séquençage permet de mesurer la taille des répétitions molécule par molécule et ainsi d'étudier la distribution de la mosaïque somatique dans les cellules traitées et leurs contrôles respectifs. Parallèlement, la prolifération cellulaire et la cytotoxicité pour différentes doses de drogues seront testées en utilisant un système d'imagerie automatisée qui permet de suivre l'évolution des cellules en temps réel (Incucyte ® , Sartorius).…”