Publication of Methodology Useful for Research in DM

Methods in Molecular Biology is a long-running series disseminating research protocols and methodologies. A strength is the ability to precisely reproduce methodologies based upon step-by-step protocols.

In the latest release of Methods in Molecular Biology, six protocols from the myotonic dystrophy field are included. These include:

• FISH techniques to detect expanded CUG repeat RNA or DMPK transcripts in DM1 plus a combined FISH/immunofluorescence protocol to visualize the colocalization of expanded repeat RNA and MBNL1 (Klein et al., 2020);
• Methodology for in vitro synthesis and structure analysis of expanded CUG repeat RNA (van Cruchten and Wansink, 2020);
• Technology for a tractable cell culture system to study instability of expanded CUG repeats; (Kononenko et al., 2020);
• Use of FLP recombinase to construct HeLa clonal cell lines containing CNG repeats of various lengths at a single ectopic recombination acceptor site (Gadgil et al., 2020);
• Design of PCR-based assays, using the model system Saccharomyces cerevisiae, to determine the behavior of expanded CTG tracts in stable and DM1 threshold ranges (Williams et al., 2020); and
• Protocols for expanded repeat-primed PCR and restriction enzyme-digested PCR to detect/identify interrupted expanded repeat alleles in DM1 (Tomé and Gourdon, 2020).

MDF is including this item in the Research News to highlight the availability of these now-published research protocols.

References:

FISH Protocol for Myotonic Dystrophy Type 1 Cells.
Klein AF, Arandel L, Marie J, Furling D.
Methods Mol Biol. 2020;2056:203-215. doi: 10.1007/978-1-4939-9784-8_13.

In Vitro Synthesis and RNA Structure Probing of CUG Triplet Repeat RNA.
van Cruchten RTP, Wansink DG.
Methods Mol Biol. 2020;2056:187-202. doi: 10.1007/978-1-4939-9784-8_12.

Experimental System to Study Instability of (CGG)_n Repeats in Cultured Mammalian Cells.
Kononenko AV, Ebersole T, Mirkin SM.
Methods Mol Biol. 2020;2056:137-150. doi: 10.1007/978-1-4939-9784-8_9.

Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site.
Gadgil RY, Rider SD Jr, Lewis T, Barthelemy J, Leffak M.
Methods Mol Biol. 2020;2056:121-136. doi: 10.1007/978-1-4939-9784-8_8.

Tracking Expansions of Stable and Threshold Length Trinucleotide Repeat Tracts In Vivo and In Vitro Using Saccharomyces cerevisiae.
Williams GM, Petrides AK, Balakrishnan L, Surtees JA.
Methods Mol Biol. 2020;2056:25-68. doi: 10.1007/978-1-4939-9784-8_3.

Fast Assays to Detect Interruptions in CTG.CAG Repeat Expansions.
Tomé S, Gourdon G.
Methods Mol Biol. 2020;2056:11-23. doi: 10.1007/978-1-4939-9784-8_2.

While CDM is clearly associated with long progenitor repeat expansions, recent evidence suggests that repeat length is not the only operative mechanism in differentiating this more severe form of DM1. Although the predictive value of CTG repeat tract length can be improved by including specific phenotypic and genotypic parameters (see A Tighter Relationship Between CTG Repeat Length and DM1 Phenotype), this ‘correction’ alone cannot explain CDM. By contrast, evidence is building that epigenetic variations around the DMPK locus are a putative determinant of CDM vs. DM1 phenotypes. Moreover, an epigenetic effect may play a role in the biased inheritance pattern of CMD (see Epigenetics Underlying the Parent of Origin Effect in CDM).

Drs. Stella Lanni and Christopher Pearson (Hospital for Sick Children and University of Toronto) have examined the molecular genetics evidence underlying CDM and published a synthesis and disease model in Neurobiology of Disease (Lanni and Pearson, 2019). This review offers a conceptual framework to build upon in understanding the molecular mechanisms that determine phenotypic severity in CDM and DM1.

Modulation of Disease Severity in CDM and DM1

Inherited DMPK CTG expanded repeat length is a correlate but not an absolute determinant of the phenotypic expression of DM1. Since genetic testing reflects current level of somatic expansion, evidence now points toward utilizing estimated progenitor allele measurements in studies of genotype-phenotype correlations (Overend et al., 2019). Focusing on repeat length alone poses a trap—Drs. Lanni and Pearson note the extensive literature establishing that CDM cannot be treated as an early-onset and more severe DM1, but has distinctive clinical features of its own.

The authors note the extreme parent-of-origin effect in CDM (88-91% maternal bias) that strongly contrasts with the roughly equal transmission in childhood/infantile-onset DM1 and the paternal transmission bias in juvenile, adult, and late-onset DM1. Paternal transmission of CMD rarely occurs, even in cases of transmission of very long CTG repeats. Maternal factors other than genetics have largely been excluded, as has maternal transmission of mitochondrial mutations that could influence phenotype. To date, the data suggest that hypermethylation of CpG islands at the CTCF1 site upstream of maternally-inherited DMPK repeat expansions contributes toward CDM severity and also explains its maternal inheritance bias (Barbé et al., 2017).

Altered Gene Expression and CDM

Literature supporting altered gene expression and DMPK transcript processing in CDM was reviewed by Dr. Lanni and Pearson—of note was the observation that DMPK transcript levels are elevated in CDM fetal muscles and muscle precursor cells. Consistent with the MBNL sequestration model of DM, the elevated expanded repeat transcript levels are associated with the most severe phenotypes. Moreover, abundance of the DMPK antisense transcript is decreased in hypermethylated CDM muscles; the reduced antisense transcript, in turn, may reduce abundance of a protective 21nt RNA. Collectively, these events support the notion that hypermethylation contributes to CDM severity. Any potential contribution of RAN translation products to CDM is currently unknown.

Molecular Modeling of CDM1

Taken together, Dr. Lanni and Pearson suggest that current evidence supports a CDM1 model whereby CTCF1 site hypermethylation disrupts CTCF protein binding--this, in turn, alters gene expression. The subsequent increased transcription of the DMPK sense strand and decreased transcription of both the antisense and the neighboring SIX5 then contribute toward the more severe mis-splicing and phenotypic severity that are characteristic of CDM. Altered SIX5 expression may explain the maternal bias of CDM, via its impact on spermatogenesis.

The authors acknowledge that there are more questions remaining, but the existence of an integrated and testable molecular genetics model is a vital step toward understanding the disease mechanisms of and developing targeted therapies for CDM.

References:

Molecular genetics of congenital myotonic dystrophy.
Lanni S, Pearson CE.
Neurobiol Dis. 2019 Jul 18:104533. doi: 10.1016/j.nbd.2019.104533. [Epub ahead of print] Review.

Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes.
Overend G, Légaré C, Mathieu J, Bouchard L, Gagnon C, Monckton DG.
Hum Mol Genet. 2019 Jul 1;28(13):2245-2254. doi: 10.1093/hmg/ddz055.

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.
Barbé L, Lanni S, López-Castel A, Franck S, Spits C, Keymolen K, Seneca S, Tomé S, Miron I, Letourneau J, Liang M, Choufani S, Weksberg R, Wilson MD, Sedlacek Z, Gagnon C, Musova Z, Chitayat D, Shannon P, Mathieu J, Sermon K, Pearson CE.
Am J Hum Genet. 2017 Mar 2;100(3):488-505. doi: 10.1016/j.ajhg.2017.01.033.

The New Frontier?

While at first glance genome editing strategies offer considerable excitement for treating monogenetic disorders, on closer examination there are considerable hurdles to be overcome. Appropriately, the initial clinical testing of CRISPR/Cas9 is focused upon ex vivo editing or delivery to the restricted environment of the eye, since in vivo genome editing presents order of magnitude greater challenges of delivery and safety. To explore the path forward for DM, MDF’s Genome Editing Workshop report carefully evaluated the opportunities and challenges of this novel technology.

A Current Status Report: CRISPR/Cas for DM1

As of mid-August 2019, there have been nine original research publications on a CRISPR/Cas strategy for DM1 (see listing below). While academic research findings require considerable expertise, time, and effort to translate into effective therapies, MDF strives to monitor these technological advances. A new article by Dr. Derick Wansink (Radboud University Medical Center) and colleagues reviews eight of these published efforts using CRISPR/Cas genome editing in DM1, compares these efforts with others in neurological disorders with microsatellite expansions, and discusses some of the translational questions to achieve a therapy for DM1.

The authors describe application of CRISPR/Cas to excise the expanded repeat at the DMPK locus as the most straightforward application of the technology—as this strategy would permanently halt the primary genetic disease mechanism. They note preclinical validation of the approach achieved in three peer-reviewed publications (Dastidar et al., 2018; Provenzano et al., 2017; Wang et al., 2018) and in one on a preprint server (Yanovsky-Dagan et al., 2019); although one report (van Agtmaal et al., 2017) showed detrimental destabilization of the repeat tract. Challenges in expanded repeat excision include the inability to distinguish short and long repeats and that its double strand breaks may produce uncontrolled deletion of large repeats, resulting in an inability to predict residual repeat length. Whether repeats can be excised in terminally differentiated cells is also unclear.

Based upon studies in other microsatellite expansion disorders, the authors raised the possibility of reducing expanded repeat length via homology-directed repair, but note that this has not yet been tried in DM and raise doubts about its value. Likewise, allele-specific editing is under investigation for Huntington’s, but the authors note that identification of SNPs required in this approach have not been achieved for the DMPK mutant locus.

There are CRISPR/Cas alternatives to DNA deletions. One study (Wang et al., 2018) has shown the utility of a homology-directed recombination strategy to insert a premature polyA signal, thereby interfering with transcription of repeat-expanded DMPK.

Inactivated Cas9 (dCas9) is being touted as a way to exploit many of the advantages of CRISPR/Cas technology while reducing off-target safety concerns. Dr. Wansink and colleagues point to the work of Pinto et al. (2017), who achieved transcriptional block targeted to the mutant DMPK allele. If DMPK knockdown proves to not be deleterious (thus far it hasn’t), such an allele-specific strategy would have additional advantages.

Finally, the review’s authors discuss the RNA-targeted CRISPR/dCas9 work of Batra et al. (2017), findings of which have moved toward clinical development with the biotech company Locana. This approach, also potentially safer than DNA excision, is based upon the efficient targeting of RNA by dCas9 and subsequent blocking/displacement of MBNL from expanded repeat RNA hairpin structures.

Opportunities and Challenges for CRISPR/Cas in DM

Dr. Wansink and colleagues provided a nice summary of the path forward for genome editing in DM. A synopsis of their presentation, as well as MDF's observations, are presented here.

Opportunities facilitating translation of genome editing in DM include:

• Availability of splicing event biomarkers (and, potentially, methylation status in CDM) capable of rapid readout of molecular target engagement and modulation;
• Leveraging lessons from ongoing work in other repeat expansion disorders, including fragile X syndrome, Friedreich’s ataxia, and Huntington’s disease;
• With the regulatory approval of Zolgensma and advances for other diseases, some of the hurdles for AAV delivered genetic therapies may be mitigated; and
• The expanding interest and commitments to exploring genome editing for DM helps ensure that the field can take advantage of new technological advancements in editing reagents, delivery, and safety that are already on the horizon.

Challenges facing genome editing in DM included:

• Safety issues with off-target editing that must be resolved for any candidate therapy where CRISPR/Cas reagents are delivered in vivo. These issues increase if the in vivo expressed reagents are not under control of an on/off regulator;
• Delivery represents a major challenge for CRISPR/Cas9 utility in DM, as noted in MDF’s Genome Editing Workshop report, Unless alternative delivery using nanoparticles, antibodies, or other means proves feasible, the field will be looking at the additive challenges of AAV delivery for genome editing strategies;
• Studies suggest that pre-existing immunity to bacterial Cas proteins affects a large percentage of the population, thus effective strategies will be needed to mitigate;
• The review’s authors point toward the potential of ex vivo editing and cell therapy. While considerably safer, this approach poses considerable delivery issues and it’s difficult to see how multi-system consequences of DM1 could be addressed by this strategy; and
• If CRISPR/Cas strategies mature sufficiently to move into clinical evaluation, careful choices of DM1 patient cohort, organ systems to be targeted and evaluated, and efficacy and safety endpoints will need to be made.

Reference:

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities.
Raaijmakers RHL, Ripken L, Ausems CRM, Wansink DG.
Int J Mol Sci. 2019 Jul 27;20(15). pii: E3689. doi: 10.3390/ijms20153689. Review.

Original CRISPR/Cas Research Articles on DM1(PubMed August 2019):

CRISPR/Cas9-Induced (CTG⋅CAG)_n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.
van Agtmaal EL, André LM, Willemse M, Cumming SA, van Kessel IDG, van den Broek WJAA, Gourdon G, Furling D, Mouly V, Monckton DG, Wansink DG, Wieringa B.
Mol Ther. 2017 Jan 4;25(1):24-43. doi: 10.1016/j.ymthe.2016.10.014. Epub 2017 Jan 4.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.
Batra R, Nelles DA, Pirie E, Blue SM, Marina RJ, Wang H, Chaim IA, Thomas JD, Zhang N, Nguyen V, Aigner S, Markmiller S, Xia G, Corbett KD, Swanson MS, Yeo GW.
Cell. 2017 Aug 24;170(5):899-912.e10. doi: 10.1016/j.cell.2017.07.010. Epub 2017 Aug 10.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.
Pinto BS, Saxena T, Oliveira R, Méndez-Gómez HR, Cleary JD, Denes LT, McConnell O, Arboleda J, Xia G, Swanson MS, Wang ET.
Mol Cell. 2017 Nov 2;68(3):479-490.e5. doi: 10.1016/j.molcel.2017.09.033. Epub 2017 Oct 19.

CRISPR/Cas9-Mediated Deletion of CTG Expansions Recovers Normal Phenotype in Myogenic Cells Derived from Myotonic Dystrophy 1 Patients.
Provenzano C, Cappella M, Valaperta R, Cardani R, Meola G, Martelli F, Cardinali B, Falcone G.
Mol Ther Nucleic Acids. 2017 Dec 15;9:337-348. doi: 10.1016/j.omtn.2017.10.006. Epub 2017 Oct 14.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophypatient-derived iPS and myogenic cells.
Dastidar S, Ardui S, Singh K, Majumdar D, Nair N, Fu Y, Reyon D, Samara E, Gerli MFM, Klein AF, De Schrijver W, Tipanee J, Seneca S, Tulalamba W, Wang H, Chai YC, In't Veld P, Furling D, Tedesco FS, Vermeesch JR, Joung JK, Chuah MK, VandenDriessche T.
Nucleic Acids Res. 2018 Sep 19;46(16):8275-8298. doi: 10.1093/nar/gky548.

Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9.
Wang Y, Hao L, Wang H, Santostefano K, Thapa A, Cleary J, Li H, Guo X, Terada N, Ashizawa T, Xia G.
Mol Ther. 2018 Nov 7;26(11):2617-2630. doi: 10.1016/j.ymthe.2018.09.003. Epub 2018 Sep 11.

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.
Angelbello AJ, Rzuczek SG, Mckee KK, Chen JL, Olafson H, Cameron MD, Moss WN, Wang ET, Disney MD.
Proc Natl Acad Sci U S A. 2019 Apr 16;116(16):7799-7804. doi: 10.1073/pnas.1901484116. Epub 2019 Mar 29.

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.
Lo Scrudato M, Poulard K, Sourd C, Tomé S, Klein AF, Corre G, Huguet A, Furling D, Gourdon G, Buj-Bello A.
Mol Ther. 2019 Aug 7;27(8):1372-1388. doi: 10.1016/j.ymthe.2019.05.021. Epub 2019 Jun 5.

Deletion of the CTG Expansion in Myotonic Dystrophy Type 1 Reverses DMPK Aberrant Methylation in Human Embryonic Stem Cells but not Affected Myoblasts
Yanovsky-Dagan S, Bnaya E, Diab MA, Handal T, Zahdeh F, van den Broek,WJAA, Epsztejn-Litman S, Wansink DG, Eiges R.
bioRxiv. 2019, 631457.

The Essentiality of Communication

For any disease with autosomal dominant inheritance, the distributed risks across a family pedigree are a harsh reality. Moreover, genetic anticipation—the inherited increase in expanded CTG tract length—represents an added risk in DM1, increasing impact upon generations beyond the affected individual. Despite the lack of an approved therapy, many of the multi-organ system sequelae of DM1 can be managed through early diagnosis and treatment. Thus, family communication, or lack of communication, around a diagnosis can easily impact diagnostic odyssey, access to adequate care, and both medical and non-medical decision-making. Any consideration of who to tell and when to tell family members about a DM1 diagnosis raises a host of interpersonal relationship and ethical questions.

Data on How to Talk about a DM1 Diagnosis

The issue of how families communicate regarding a DM diagnosis has received little attention to date—although it’s a difficult topic to search in PubMed, there appear to be few or no refereed publications. Although not an exact parallel, there has been considerable discussion of this issue in Huntington’s disease (where genetic testing often becomes a very personal issue), and lessons there may potentially inform families living with DM1.

Recruiting subjects from the New Zealand nationwide MD Prev study, Dr. Shelby Taylor (University of Melbourne and The Royal Melbourne Hospital) and colleagues examined family communication following a DM1 diagnosis (Taylor et al., 2019). They introduce the topic stating a disease-independent observation: “it is clear that most individuals at genetic risk do not receive sufficient information from their family members to be able to make an informed decision about accessing genetic testing.” The study involved a small cohort (17 patients, unaffected parents, and spouses), so sample size is a confounder to definitive conclusions—but the study provides important insights that can be built upon and ways to support familial communication around the diagnosis.

While communication patterns ran the gamut from open discussion through active avoidance of the diagnosis with family members, the majority of interviewees reported diagnosis disclosures at some level. Some extended family members, often female, took responsibility for communication even when an affected parent would not tell their own adult children (majority of males did not disclose their diagnosis to at-risk relatives). There was clear sentiment among interviewees that ‘to tell or not tell’ decisions altered family relationships, both positively and negatively—non-disclosures, among this cohort, invariably had negative connotations for family relationships.

Ensuring knowledge of the diagnosis for the purposes of medical or reproductive autonomy was a communication motivator detected by the research team. Extreme polarization could result when diagnosis non-disclosures were associated with birth of children with CDM. Minimization of potential disease consequences was a communication strategy that was reported by the majority of study participants. Finally, those who initially communicated information about a diagnosis assumed, indeed expected—often wrongly—that the information would be disseminated across the extended family.

Study Implications

Dr. Taylor and colleagues acknowledge the small cohort and heterogeneity of responses are consistent with an exploratory first study on the family communication topic in DM1. The research team concluded that reproductive considerations were a powerful motivator to communication of a DM1 diagnosis within families. The observed differences in communication patterns of males and females need to be recognized by medical practitioners. The team views genetic counselors as having a critical role in fostering communication among families; in this role, the counselors need to have greater awareness of the dynamic range of family communications around this diagnosis.

Finally, the authors' use of many exact quotations from interviews makes the publication feel a bit anecdotal, but this also enhances its power as a piece to both understand and to communicate the importance of communication to patients and family members.

Reference:

Family communication following a diagnosis of myotonic dystrophy: To Tell or not to tell?
Taylor S, Rodrigues M, Poke G, Wake S, McEwen A.
J Genet Couns. 2019 Aug 5. doi: 10.1002/jgc4.1156. [Epub ahead of print]

Hypermethylation and Transcriptional Alterations Drive CDM

Increasing evidence has established a role for epigenetic events in pathogenesis of CDM. Methylation at CpG sites upstream of the DMPK locus has been linked to the etiology of CDM, as well as to the maternal bias of its inheritance (Barbé et al., 2017). Moreover, hypermethylation at this locus has been modeled as a key differentiator of the CDM vs. DM1 clinical phenotype (Lanni and Pearson, 2019).

Does DMPK Methylation Also Modulate DM1 Phenotypes?

The degree to which epigenetic events that have been linked to CDM could also play a role in DM1 severity is unclear. Dr. Luigi Bouchard (Université de Sherbrooke) and colleagues sought to determine whether DNA methylation around DMPK-expanded repeat tracts could impact DM1 phenotypes (Légaré et al., 2019).

The research team studied 90 patients with the adult form of DM. Expanded CTG repeat length, DMPK methylation at 8 distinct CpG island loci (designated L1-L8), muscle strength (by dynamometer), and pulmonary function (FVC, PEF, MIP, and MEP) were evaluated. Relationships between patient molecular and functional parameters were assessed using stepwise multiple linear regression analyses.

CTG repeat length correlated with methylation level, but in opposite directions for upstream vs. downstream CpG sites. The presence of variant repeats in the CTG tract was associated with higher methylation at downstream, but not at upstream, sites. Data showed that CpG site methylation at the expanded repeat DMPK locus explained variability in muscle strength and respiratory function profiles independent of expanded CTG repeat length. Specifically, a downstream (L6 site) epigenetic modification contributed to variability in ankle dorsiflexion, grip, and pinch strengths, as well as to FVC and MIP. Moreover, methylation levels were associated with somatic instability of the CTG repeat (difference of average and progenitor repeat length), but not with symptom onset age.

Potential Mechanism of Action of Methylation at DM1 Locus

Due to variability in affected muscle groups, the research team suggested that methylation at specific CpG sites might alter affinity of specific transcription factors, but using binding site analysis could not identify any particular transcription factor. They suggest that methylation could underlie variability of DM1 phenotypes via: (a) blockage of CTCF binding, (b) reduction of DMPK and SIX5 transcription, and/or (c) alteration of CTG repeat instability. Finally, the authors suggest that assessment of DNA methylation at the pathogenic DMPK locus may have prognostic value to guide patient care.

References:

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.
Barbé L, Lanni S, López-Castel A, Franck S, Spits C, Keymolen K, Seneca S, Tomé S, Miron I, Letourneau J, Liang M, Choufani S, Weksberg R, Wilson MD, Sedlacek Z, Gagnon C, Musova Z, Chitayat D, Shannon P, Mathieu J, Sermon K, Pearson CE.
Am J Hum Genet. 2017 Mar 2;100(3):488-505. doi: 10.1016/j.ajhg.2017.01.033.

Molecular genetics of congenital myotonic dystrophy.
Lanni S, Pearson CE.
Neurobiol Dis. 2019 Jul 18:104533. doi: 10.1016/j.nbd.2019.104533. [Epub ahead of print] Review.

DMPK gene DNA methylation levels are associated with muscular and respiratory profiles in DM1
Légaré C, Overend G, Guay SP, Monckton DG, Mathieu J, Gagnon C, Bouchard L.
Neurol Genet. 2019 May 23;5(3):e338. doi: 10.1212/NXG.0000000000000338. eCollection 2019 Jun.

When Carl Shotwell was exploring graduate programs, he came across a fellow in Andy Berglund’s lab at the Center for NeuroGenetics at the University of Florida, who described the work she was doing on myotonic dystrophy. He became fascinated with the disease and later joined Berglund’s lab. “What was interesting to me was the complexity of the disease. There are problems at the DNA level, the RNA level, and the protein level,” he said. “What excited me was trying to wrap my head around it because it has such a devastating effect on patients.”

Carl, now a third-year Ph.D. candidate at UFL's Center for NeuroGenetics, continues to work in Berglund’s lab, which is now part of The RNA Institute at the University at Albany in New York. MDF, in partnership with MDF UK, awarded Carl a 2019 Fellowship to support his research. Carl’s research focuses on the role muscleblind-like (MBNL) proteins play in myotonic dystrophy and to explore whether the synthetic proteins he has developed to tease out the function of MBNL may represent a potential therapeutic strategy to disrupt the disease process. The two-year fellowship will provide $55,000 in total funding to support his work.

MBNL proteins are master regulators of RNA, which carries instructions from DNA for controlling the synthesis of proteins. DM1 is caused by a triplet repeat expansion in the non-coding portion of the DMPK gene, which produces the myotonic dystrophy protein kinase.but it is believed to play a critical role in muscle, heart and brain cells. DM2 is caused by a different repeat expansion in the CNBP gene. The CNBP protein is found through out the body but is abundant in heart and skeletal muscles.

The repeats produce toxic RNA that interferes with the normal function of MBNL. These repeats act like a sponge and soak up the available MBNL and prevent it from acting as it should. The first part of Carl’s research is to better understand how MBNL works by creating a variety of synthetic proteins that can mimic parts of MBNL to tease out their function.

The second part of Carl’s work will build on that to test the synthetic proteins he has engineered to see if they will preferentially link to the toxic repeats and prevent them from interfering with the proper functioning of MBNL. If the early work is successful, he hopes to be able to advance the synthetic proteins into mouse models of DM to see if they exhibit therapeutic benefits.

As Carl’s began working on DM, he attended the the International Myotonic Dystrophy Consortium 11 and the MDF Annual Conference in 2017. He said those were life-changing experiences for him because he met with patients and their families, which helped him to see the suffering caused by the disease and connect his research to actual patients. It was something the researcher could relate to from his personal experiences. When he was 13, Carl fell 46 feet in a rock-climbing accident that resulted in multiple broken bones. Surgery repaired his shattered knees, but he spent four months in a wheelchair before progressing to a walker for three months and crutches for four more months. During that time, he relied on his mother’s help to do everyday tasks from getting into bed to using the bathroom.

“I felt a connection with patients as I have some personal, albeit less devastating, experience with muscle loss and having to depend on others to take care of you,” he wrote in his fellowship proposal. He said meeting patients of myotonic dystrophy helped him realize that the problems he had faced were nothing compared to the lifelong challenges that DM patients face. That, he said, has increased his drive for his research.

“We are going to need a lot of people to continue researching this to find creative solutions,” he said.

Carl is not the first MDF Fellow to come from the Berglund Lab. Past Berglund Lab recipients include Kaalak Reddy (2017).

Molecular Diagnosis of DM1

Accurate sizing of expanded triplet repeat sequences is essential in establishing diagnosis of DM1. The Genetic Testing Registry at NCBI/NIH currently lists 50 facilities that provide molecular genetics tests for DM1. Current commercially available testing includes the Athena Diagnostics’ Myotonic Syndrome Advanced Evaluation (detection of abnormal splicing of ATP2A1, CAV3, CLCN1, HSPG2, and SCN4A and repeat expansions in DMPK and CNBP), GeneDx’s Myotonic Dystrophy, Type 1 (detection of DMPK repeat expansion), and MNG Laboratories’ Myotonic Dystrophy 1 (DMPK) Genetic Testing (detection of DMPK repeat expansion).

Commercial tests offer considerable value for rapid diagnosis, but all have limited research value due to inability to accurately size repeat sizes above 150. More accurate repeat sizing is essential for understanding of genotype/phenotype relationships and likely needed for interventional clinical trials. Continued efforts to develop and validate robust and broadly available molecular diagnostics for DM carry considerable importance.

Assessing a New, Commercially Available Test for DM1

Drs. Lonneke Haer-Wigman (Radboud University), Ming Guan (The BioFactory Pte Ltd, Singapore), and colleagues have published work in Science Reports evaluating the performance of a new commercially available triplet-repeat primed PCR (TP-PCR) methodology for sizing expanded CTG repeats, the FastDM1^TM DMPK sizing kit (Leferink et al., 2019). The new kit sought to overcome limitations in existing TP-PCR diagnostics, including inabilities to distinguish between intermediate (150>x >51) and large (x>150) pathogenic alleles and to identify and size samples from patients with interrupted repeats.

In an assessment of sizing accuracy, 19 reference samples of established expanded repeat length (consensus from three independent labs for 53% of samples and from one independent lab for the remainder) were sized with the FastDM1^TM kit—all determinations were within 3 repeats of the reference samples. To evaluate assay sensitivity, testing of a 1-200 ng input DNA range showed that sample sizes from 25-200 ng retained sizing accuracy. Mosaic samples (artificially created by mixing reference samples) could accurately detect up to 2.5% mosaicism, but that repeat size could be underestimated at under 20% mosaicism.

The specificity of the FastDM1^TM kit for DMPK repeat expansions was demonstrated by the failure to detect and size CTG expansions in TCF4 in DNA samples containing either only the TCF4 repeat or samples containing expanded repeat TCF4 mixed with reference samples with DMPK expanded repeats. The research team also established intra-assay consistency and intra- and inter-kit batch (including kits from separate batches produced over a two-year period) reproducibility in use of the FastDM1^TM kit.

Use of the FastDM1^TM DMPK Sizing Kit with Patient Samples

Performance of the FastDM1^TM kit was evaluated using 225 postnatal and 10 prenatal clinical samples. The research team reported 100% clinical sensitivity and specificity in classifying samples tested into four genotypic groups (normal: 5-35 repeats, non-pathogenic pre-expansion: 36-50, unstable intermediate sized: 51-150, and unstable pathogenic: >150) recognized by the European Molecular Genetics Quality Network. In contrast to prior technologies, the kit showed the ability to distinguish between unstable intermediate and unstable pathogenic alleles (over 150 repeats)—a distinction of considerable diagnostic importance. Finally, the research team evaluated eight samples with known interrupted repeats and found that the kit had the ability to detect such repeats, albeit with less accuracy in sizing. These findings suggest that the kit may be an important new tool for the DM1 field.

Reference:

Robust and accurate detection and sizing of repeats within the DMPK gene using a novel TP-PCR test.
Leferink M, Wong DPW, Cai S, Yeo M, Ho J, Lian M, Kamsteeg EJ, Chong SS, Haer-Wigman L, Guan M.
Sci Rep. 2019 Jun 4;9(1):8280. doi: 10.1038/s41598-019-44588-3.

Value of Registries?

The existence of patient registries is essential to establishing clinical trial readiness for any rare disease. Without the ability to identify and characterize patients, it is virtually impossible to develop and validate biomarkers and clinical outcome measures necessary for decision-making in interventional clinical trials. Any advances in a highly heterogeneous disease such as DM require standardized, longitudinal data collection and analysis from as well-powered a cohort as possible. While safe and effective therapies are under development, an understanding of disease progression and assessment of the efficacy of existing care management strategies gained through well-powered registry studies can make a meaningful difference in patient care. It then is important to capture lessons learned from all existing DM patient registries, as well as those ongoing for other neuromuscular diseases, to both improve patient care now and gain knowledge to guide the development of novel, life-changing therapies.

The DM-Scope Registry Model

Over the last several years, the Myotonic Research News has highlighted several important new findings in DM from the French national registry, DM-Scope. These have included identification of gender as a phenotypic modifier in DM1, establishment of a more detailed DM1 classification scheme, and the characterization of pediatric DM1 subgroups and their potential use in improving evidence-based care.

In the process of their studies to improve understanding of the DM patient, leadership of DM-Scope realized an opportunity to “promote DM epidemiology, clinical research and patient care management simultaneously.” Dr. Guillaume Bassez (Pitié-Salpêtrière Hospital and INSERM/Sorbonne University) and his 10 co-authors and 82 collaborators in DM-Scope have articulated this concept and their progress over the 11 years since the registry's founding in a new publication (De Antonio et al., 2019).

The authors describe the development and implementation of an information technology platform that facilitates clinical research in DM using data collected in regular, routine clinical visits by DM1 (n = 2828) and DM2 (n = 142) patients at 55 neuromuscular centers throughout France. Details are provided as to the governance and oversight of research studies, ethical/legal considerations, data breadth/depth/quality control/security, and design of the database, user interfaces, and statistical package.

DM-Scope Accomplishments and Lessons

DM-Scope has established nationwide coverage for annual visits and data input from the nearly 3,000 DM enrollees (enrollment continues to increase linearly since 2009), >77% with genetic diagnosis, at the pediatric and adult clinical sites, facilitating 10 published clinical studies to date. Capability and efficiency of DM-Scope to support clinical trials was demonstrated in the recruitment of 71 patients for OPTIMISTIC in the timespan of six months. Patient demographics, clinical presentation, and education/employment data suggest that a broad spectrum of clinical data is being captured. Kaplan-Meier survival analysis identified heterogeneity between the 20 DM-Scope centers that recorded death status.

The authors conclude that DM-Scope has been able to overcome key challenges that are generally experienced by rare disease registries, including optimizing the standardization and comparability of patient data, patient retention and longitudinal follow-up, facilitating interoperability across existing registries, limiting gaps in data, and improving data quality. This model both facilitates critical research efforts and, through longitudinal comparisons of outcomes, provides practitioners with knowledge to optimize patient care.

Reference:

The DM-scope registry: a rare disease innovative framework bridging the gap between research and medical care.
De Antonio M, Dogan C, Daidj F, Eymard B, Puymirat J, Mathieu J, Gagnon C, Katsahian S; Filnemus Myotonic Dystrophy Study Group, Hamroun D, Bassez G.
Orphanet J Rare Dis. 2019 Jun 3;14(1):122. doi: 10.1186/s13023-019-1088-3.

Need to Identify Genetic Modifiers for DM

One need look no further than Duchenne muscular dystrophy to understand the power of genetic modifiers, where variants in SPP1, LTBP4, CD40, ACTN3, and THBS1 impact disease phenotype. At least two of these modifiers, SPP1 and LTBP4, impact patient phenotypes to the extent that they should be controlled for in interventional clinical trials. By contrast, the literature around putative genetic modifiers of the pathogenesis of DM1 is sparse. Most recently, data suggest that the rbFOX1 RNA binding protein can compete with MBNL1 for binding to expanded CCUG repeats, potentially mitigating the phenotype of DM2 versus DM1 flies (Sellier et al., 2018). The relative dearth of knowledge is an important gap in mechanistic understanding of DM, as genetic modifiers may prove important in stratification of subjects in clinical trials, as well as representing putative therapeutic development targets.

Extensive literature supports a key genetic modifier role for DNA mismatch repair (MMR) proteins in microsatellite expansion disorders, as these proteins are essential for triplet repeat instability. Previously, a variant of the MMR, MSH3, was associated with somatic expansion rate in DM1 (Morales et al., 2016). The validation of a putative genetic modifier acting at such an early stage in the pathogenic cascade in DM1 would have considerable value.

MSH3 Variant: A Modifier of Somatic Instability and Disease Severity

Studies by Drs. Sarah Tabrizi (University College London), Darren Monckton (University of Glasgow), and colleagues have examined whether an MSH3 variant may indeed modify the instability of the CAG·CTG expansions that underlie Huntington’s disease (HD) and DM1 (Flower et al., 2019). The research team conducted Illumina sequencing of DNA from HD (n = 218) and DM1 (two cohorts, n = 247 and n = 199, as well as a meta-analysis that included the Morales et al. cohort) subjects and showed that a three-repeat allele in MSH3 exon 1 (3a allele) is associated with a reduced relative rate of somatic CAG·CTG expansion and delayed disease age of onset for HD and DM1. These data were supported by further studies in HD subjects.

The mechanism of action of the MSH3 variant in regulating triplet repeat stability is not yet known. The MSH3 3a allele might alter the MMR protein expression level or, alternatively, compromise its recognition and repair functions and thereby impact repeat length. Overall, the team suggests that a common MMR mechanism operating in HD and DM1 mediates trinucleotide repeat expansion and that this mechanism may be a viable target in therapeutic development for these two diseases, if not additional diseases.

References:

rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophytype 1/type 2 differences.
Sellier C, Cerro-Herreros E, Blatter M, Freyermuth F, Gaucherot A, Ruffenach F, Sarkar P, Puymirat J, Udd B, Day JW, Meola G, Bassez G, Fujimura H, Takahashi MP, Schoser B, Furling D, Artero R, Allain FHT, Llamusi B, Charlet-Berguerand N.
Nat Commun. 2018 May 22;9(1):2009. doi: 10.1038/s41467-018-04370-x.

A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients.
Morales F, Vásquez M, Santamaría C, Cuenca P, Corrales E, Monckton DG.
DNA Repair (Amst). 2016 Apr;40:57-66. doi: 10.1016/j.dnarep.2016.01.001. Epub 2016 Mar 8.

MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1.
Flower M, Lomeikaite V, Ciosi M, Cumming S, Morales F, Lo K, Hensman Moss D, Jones L, Holmans P; TRACK-HD Investigators; OPTIMISTIC Consortium, Monckton DG, Tabrizi SJ.
Brain. 2019 Jun 19. pii: awz115. doi: 10.1093/brain/awz115. [Epub ahead of print]

Known Value of CTG Repeat Length as a Biomarker

The identification and validation of a prognostic biomarker can have considerable value for DM1 stakeholders, from improving the ability to provide informed patient care to stratifying subjects in an interventional clinical trial. Theoretically, CTG progenitor allele length at birth should provide such value because of the relationship between repeat length, somatic instability, MBNL sequestration, and mis-splicing and, in turn, determining DM1 symptomatology. Yet, to date, expanded CTG repeat length has been strongly correlated only with patient age at symptom onset. Its value as a predictive biomarker lies in the establishment of stronger ties to measures of disease progression and, subsequently, to the efficacy of a candidate therapeutic. Due to the heterogeneity of DM1, achieving this goal requires detailed genotypic and phenotypic characterization of well-powered cohorts.

Controlling for Confounders Improves Predictive Value

The presence of variants in expanded CTG tracts in DMPK is associated with delayed age of DM1 onset, supporting the need to distinguish pure CTG from interrupted expansions (Braida et al., 2010). Drs. Darren Monckton (University of Glasgow), Cynthia Gagnon (Université de Sherbrooke), and colleagues sought to determine whether taking into account interrupting variant repeats in CTG expansions and other potential confounding factors could improve the predictive value of CTG repeat length for a broader array of patient phenotypic traits (Overend et al., 2019).

Using phenotypic data from 192 subjects in the Saguenay-Lac-Saint-Jean population of DM1 patients, the research team determined progenitor CTG expanded allele length, determined presence/absence of interrupted repeats, and looked for correlations with a range of objective measures of respiratory function and skeletal muscle power. Nine percent of subjects were found to exhibit interrupted repeat tracts (with runs of CCG or CGG).

Correlations of age at onset (available for 77% of the cohort) confirmed that progenitor expanded CTG length was a key modifier of age at DM1 symptom onset. The presence of variant repeat tracts was associated with later age of symptom onset. Somatic instability (difference between progenitor allele length and length at time of evaluation for this study) showed that instability was strongly correlated with progenitor allele length and whether the repeat tract included non-CTG interruptions. Measures of respiratory and skeletal muscle function were strongly correlated with progenitor allele length. Finally, the presence/absence of interrupted CTG tracts in DMPK was the most important factor in all measures of skeletal muscle function.

Synthesis

Taken together, the presence of interruptions among DMPK CTG expansions significantly decreased somatic instability and, in turn, increased the age of symptom onset. Moreover, these patients were less severely affected than would be expected given their CTG expansion length. The research team notes that it is likely that these patients also show slower progression, although that remains to be confirmed in longitudinal studies.

Finally, taking the presence/absence of repeat interruptions into account, modal CTG length may prove to be an improved predictor of time of onset and rate of progression of DM1. Thus, complete genotype assessments are essential in establishing prognosis and patient management plans, as well as important stratification factors for interventional clinical trials. The team concludes that outcome measures that correlate best with genotype are potentially the best decision-making measures for interventional clinical trials in DM1.

References:

Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients.
Braida C, Stefanatos RK, Adam B, Mahajan N, Smeets HJ, Niel F, Goizet C, Arveiler B, Koenig M, Lagier-Tourenne C, Mandel JL, Faber CG, de Die-Smulders CE, Spaans F, Monckton DG.
Hum Mol Genet. 2010 Apr 15;19(8):1399-412. doi: 10.1093/hmg/ddq015. Epub 2010 Jan 15

Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes.
Overend G, Légaré C, Mathieu J, Bouchard L, Gagnon C, Monckton DG.
Hum Mol Genet. 2019 Jul 1;28(13):2245-2254. doi: 10.1093/hmg/ddz055.