Three-dimensional imaging in myotonic dystrophy type 1

Abstract

ObjectiveWe aimed to determine whether 3D imaging reconstruction allows identifying molecular:clinical associations in myotonic dystrophy type 1 (DM1).MethodsWe obtained myoblasts from 6 patients with DM1 and 6 controls. We measured cytosine-thymine-guanine (CTG) expansion and detected RNA foci and muscleblind like 1 (MBNL1) through 3D reconstruction. We studied dystrophia myotonica protein kinase (DMPK) expression and splicing alterations of MBNL1, insulin receptor, and sarcoplasmic reticulum Ca(2+)-ATPase 1.ResultsThree-dimensional analysis showed that RNA foci (nuclear and/or cytoplasmic) were present in 45%–100% of DM1-derived myoblasts we studied (range: 0–6 foci per cell). RNA foci represented <0.6% of the total myoblast nuclear volume. CTG expansion size was associated with the number of RNA foci per myoblast (r = 0.876 [95% confidence interval 0.222–0.986]) as well as with the number of cytoplasmic RNA foci (r = 0.943 [0.559–0.994]). Although MBNL1 colocalized with RNA foci in all DM1 myoblast cell lines, colocalization only accounted for 1% of total MBNL1 expression, with the absence of DM1 alternative splicing patterns. The number of RNA foci was associated with DMPK expression (r = 0.967 [0.079–0.999]). On the other hand, the number of cytoplasmic RNA foci was correlated with the age at disease onset (r = −0.818 [−0.979 to 0.019]).ConclusionsCTG expansion size modulates RNA foci number in myoblasts derived from patients with DM1. MBNL1 sequestration plays only a minor role in the pathobiology of the disease in these cells. Higher number of cytoplasmic RNA foci is related to an early onset of the disease, a finding that should be corroborated in future studies.

Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1

“…Our study population consisted of ten DM1 patients, of which the clinical characteristics have been, in part, described previously ( Table 1 , [ 12 , 13 ]). Eight out of ten DM1 patients provided skin and muscle biopsies, whereas from the other two only lymphoblastoid cell lines were available.…”

Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity

“…A direct association between CTG size and foci number in muscle cells has been reported before by our group. 23 We hypothesize that although the CTG size would determine the number of RNA foci at the single cell level, the instability of the CTG repeat in the cells of a certain tissue, would determine the mean of the RNA foci in this particular tissue. 18 , 24 , 30 …”

“… 20 We and others have found correlation between RNA foci formation and CTG expansion size, and RNA foci formation and age of onset. 22 , 23 It is known that foci can bind to important cellular proteins, such as the splicing regulator, muscleblind-like 1 (MBNL1). 24 MBNL1 sequestration and downregulation causes the aberrant splicing of several genes, including insulin receptor ( INSR ), 25 sarcoplasmic reticulum Ca(2+)-ATPase 1 ( ATP2A1 ), 26 LIM Domain Binding 3 ( LDB3 ), 27 dystrophin ( DMD ), 28 bridging Integrator 1 ( BIN1 ), 29 and MBNL1 (which regulates its own splicing).…”

Sustainable recovery of MBNL activity in autoregulatory feedback loop in myotonic dystrophy

“…In muscles of patients with DM1 and DM2, the size of CTG or CCTG repeat expansion differs significantly between patients but also between different parts of the same muscle of the same individual, due to somatic instability of repeat tract. 12 , 18 , 21 , 22 Hence, the sequestration of MBNL proteins is highly heterogeneous. Therefore, we hypothesized that unequal sequestration of the MBNL pool can be buffered by self-regulated overexpression of MBNL1auto.…”

“…18 , 19 , 20 The length of repeats can be different even in nuclei of the same muscle fiber, which reflects somatic mosaicism of CUG exp . 21 , 22 Moreover, the length of CCUG exp may be also deeply heterogeneous in the same patient, as indicated by recent studies showing variability in repeat length and repeat composition. 23 , 24 Hence, the sequestration of MBNL proteins is highly heterogeneous in both DM forms, even in the same tissue of the same patient.…”