Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient’s age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells

Genetic and epigenetic characterization of variant DMPK expansions as a modifier of phenotype in myotonic dystrophy type 1

Povećanje broja ponovljenih motiva u mikrosatelitskim lokusima ili dinamične mutacije uzrokuju skoro 50 neuroloških oboljenja, označenih kao bolesti ekspanzija ponovljenih motiva. Kvantitativan efekat broja ponovljenih motiva na fenotip i inheretno nestabilna priroda dinamičnih mutacija objašnjavaju jedinstvene karakteristike ove grupe bolesti, kao što su genetička anticipacija – ranije i teže ispoljavanje bolesti u narednim generacijama, i izrazito varijabilni klinički fenotipovi. Miotonična distrofija tip 1 (DM1) je uzrokovana ekspanzijama CTG tripleta u DMPK genu, čiji broj predstavlja glavnu determinantu ekstremno varijabilne kliničke prezentacije koja otežava predviđanje toka bolesti. Deo fenotipske varijabilnosti, neobjašnjene veličinom ekspanzije, ukazuje na postojanje dodatnih modifikatora bolesti. Kod ~5% DM1 bolesnika opisane su ekspanzije sa varijantnim tripletima (CCG, CTC, CAG), koji su dovedeni u vezu sa neobičnim i/ili blažim simptomima nego što bi se očekivalo na osnovu veličine ekspanzije datog bolesnika. Ovaj rad daje pregled dosadašnjih znanja o tipovima varijantnih tripleta u DMPK ekspanzijama, kliničkim karakteristikama bolesnika i mehanizmima kojim varijantni tripleti ostvaruju svoj modifikujući efekat na fenotip. Ključna uloga pripisuje se stabilišućem efektu varijantnih tripleta na DMPK ekspanzije u somatskim ćelijama, što objaš - njava kasniji uzrast početka simptoma bolesti, kao i stabilišućem efektu u polnim ćelijama što objašnjava odsustvo najteže forme bolesti u porodicama sa varijantnim tripletima. Takođe, rad predstavlja naše originalno otkriće metilacije varijantnih CCG tripleta, koje otvara pitanje uloge epigenetičkih mehanizama u stabilizaciji DMPK lokusa. Dosadašnja znanja ističu kliničku relevantnost varijantnih tripleta u pogledu pružanja adekvatnog genetičkog saveta i regrutovanja bolesnika za kliničke studije, i podržavaju somatsku nestabilnost ekspanzija kao metu za nove terapeutike.Repeat expansions in microsatellites or dynamic mutations cause ~50 neurological diseases, known as repeat expansion diseases. Quantitative effect of the repeat number and inherent instability of dynamic mutations underlie unique features of these diseases, such as genetic anticipation – an earlier and more severe disease presentation in successive generations, and a pronounced variability in clinical presentation. Myotonic dystrophy type 1 (DM1) is caused by expansion of CTG repeats in DMPK gene, whose number is the main determinant of an extremely variable phenotype, making prognosis of the disease course challenging. A part of unexplained phenotype variability suggests the existence of disease modifiers. Variant repeats (CCG, CTC, CAG) are present in ~5% of patients and have been associated with unusual and/or milder symptoms than expected for a given expansion size. In this review, we provide an overview of types of variant repeats, clinical characteristics of patients, and mechanisms by which variant repeats modify DM1 phenotype. The key role is attributed to their stabilizing effect on DMPK expansions in the somatic cells, explaining a later age at symptoms onset, as well as in the germline cells, clarifying the absence of the most severe DM1 form in families with variant repeats. Additionally, we present our discovery of the methylation of CCG variant repeats, raising the question of the role of epigenetic mechanisms in the stabilization of DMPK locus. Current knowledge highlights the clinical relevance of variant repeats for genetic counseling and patient recruitment for clinical studies, and supports somatic instability of expansions as therapeutic targets

Značaj direktnog genetičkog testiranja za utvrđivanje žena prenosioca kod distrofinopatija

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene. They are X-linked recessive diseases, where males are affected and females are mostly healthy carriers of the mutation. It is estimated that 2/3 mothers of DMD probands are carriers, while 1/3 of patients have de novo mutations. The aim was to confirm the carrier status of females in the families of DMD/BMD probands, using direct genetic methods. Methods. We tested 38 females from 31 families of DMD/BMD probands with deletion/duplication in the dystrophin gene. Also, 4 cases of prenatal diagnosis of DMD/BMD were included. We preformed the polymerase chain reaction (PCR) and the multiplex ligation-dependent method (MLPA) for deletion detection, i.e. deletion/duplication in the dystrophin gene. Results. In 31 DMD/BMD probands, we identified 87.1% deletions and 12.9% duplications of one or more exons. Of the 29 tested mothers, mutations were found in 17 (14 deletions and 3 duplications). Mutations were found in 57.9% (11/19) mothers of DMD and in 60% (6/10) mothers of BMD, respectively. Also, in probands with deletions 56% (14/25) of mothers were carries and in probands with duplications 3 mothers of 4 (75%). Of the 9 other female relatives, mutations were found in 4. In prenatal diagnosis, we identified deletion in one male and one female foetus of one mother. Conclusion. The study showed that mothers were carriers in almost 60% of sporadic cases of DMD/BMD with deletions and duplication. Also, the carrier frequency tended to be higher in mothers of the probands with duplication (75%) then in probands with deletions (56%). In the case of a mother who was confirmed as a carrier, deletion was detected in 2 of 3 foetuses.Dišenova mišićna distrofija (DMD) i Bekerova mišićna distrofija (BMD) su uzrokovane mutacijama u genu za distrofin. To su X-vezane recesivne bolesti, gde oboljevaju muškarci a žene su uglavnom zdravi prenosioci mutacije. Procenjeno je da su kod DMD probanada 2/3 majki nosioci, dok 1/3 pacijenata ima novu mutaciju. Cilj rada je bio da se utvrdi status prenosioca kod žena u porodicama obolelih od DMD/BMD, primenom direktne genetičke metode. Metode. Testirali smo 38 žena iz 31 porodice DMD/BMD probanada sa delecijama i duplikacijama u genu za distrofin. Takođe, u studiju su bila uključena i 4 slučaja prenatalne DMD/BMD dijagnoze. Primenjene su metoda lančane reakcije polimerizacije (PCR) i metoda višestrukog umnožavanja vezanih proba (MLPA) za detekciju delecija, odnosno delecija i duplikacija u genu za distrofin. Rezultati. Kod 31-og DMD/BMD probanda utvrđeno je 87,1% delecija i 12,9% duplikacija jednog ili više egzona. Od 29 testiranih majki probanada, mutacije su nađene kod njih 17 (14 delecija i 3 duplikacije). Mutacije su nađene kod 57,9% (11/19) majki probanada sa DMD fenotipom i kod 60% majki probanada sa BMD. Takođe, kod probanada sa delecijom 56% (14/25) majki su potvrđene kao nosioci, a kod probanada sa duplikacijom 3 od 4 majke (75%). Od preostalih 9 ženskih srodnika, mutacije su nađene kod nijh 4. Prenatalnom dijagnostikom utvrđene su delecije kod jednog muškog i jednog ženskog ploda iste majke. Zaključak. Istraživanje je pokazalo da su majke bile nosioci u skoro 60% izolovanih DMD/BMD slučajeva sa delecijama i duplikacijama. Takođe, učestalost majki nosioca kod probanada sa duplikaciom (75%) se pokazala većom nego kod majki probanada sa delecijom (56%). U slučaju majke koja je bila potvrđena kao nosilac, delecija je otkrivena kod njena 2 ploda od 3 ispitana

Uporedna analiza duplikacija i delecija u genu za distrofin u grupi bolesnika sa distrofinopatijom iz Srbije

Background/Aim. Duchenne muscular dystrophy (DMD) and its allelic form Becker muscular dystrophy (BMD) are X-linked diseases that affect males, characterized by progressive muscle and cardiopulmonary weakness, especially in DMD as a severe form of the disease. They result from mutations in the dystrophin gene, and the most common changes are large intragenic deletions and duplications (80%). One third of patients have de novo mutation and 2/3 of the mothers are estimated as carriers. The aim of the study was to analyze the frequency of duplications versus deletions in the dystrophin gene in patients with dystrophinopathies, as well as to analyze the phenotypic effect of large mutations obtained and to determine the carrier status of female relatives in probands with duplications. Methods. We examined 22 DMD and 35 BMD unrelated patients and 6 female relatives of the probands where duplications were found. We used polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) methods, according to the protocol, to detect or confirm mutations in probands and female carriers. Results. In probands, there were 34 (59.6%) large deletions (mostly affected exons 44-60) and 6 (10.5%) large duplications in 4 DMD and 2 BMD patients. Also, duplications were found in 3 out of 4 (75%) tested mothers. The distribution of duplications was heterogeneous, affecting N-terminal and central rod domain, and included more exons, except for one DMD patient who had duplication of exon 2. An exception from the Monaco rule was present in 9.5% of DMD and 15.8% of BMD probands, i.e. in 12.5% of DMD/BMD cases. Conclusion. In 57 DMD/BMD probands, we found 59.6% of large deletions and 10.5% of large duplications. The most affected region of the DMD gene was the central rod domain. An exception to Monaco's rule was present in 12.5% of DMD/BMD cases. Three out of 4 examined proband's mothers were confirmed as carriers.Uvod/Cilj. Dišenova mišićna distrofija (DMD) i njegova alelna forma, Bekerova mišićna distrofija (BMD), su Xvezane nasledne bolesti od kojih obolevaju muškarci, a karakteriše ih progresivna mišićna i kardiopulmonalna slabost, posebno kod DMD kao težeg oblika bolesti. Ove bolesti nastaju kao posledica mutacija u genu za distrofin, a najčešće su prisutne intragenske delecije i duplikacije (80%). Novonastalu mutaciju ima1/3 bolesnika, a procenjeno je da su 2/3 majki nosioci. Cilj rada je bio da se analizira učestalost duplikacija u odnosu na delecije u genu za distrofin kod bolesnika sa distrofinopatijom, kao i da se ispita efekat dobijenih mutacija na fenotip kod probanda i utvrdimo status nosioca kod ženskih srodnika probanda sa duplikacijama. Metode. Studijom je bilo obuhvaćeno 22 DMD i 35 BMD nesrodnih bolesnika i šest ženskih srodnika probanda kod kojih su bile otkrivene duplikacije. Za otkrivanje ili potvrdu mutacije, kod probanda i ženskih nosioca, korišćene su metode: lančana reakcija polimerazom (PCR) i višestruko umnožavanje vezanih sondi (MLPA), prema datom protokolu. Rezultati. Kod probanda je nađeno 34 (59,6%) velikih delecija (najčešće su bili zahvaćeni egzoni 44-60) i 6 velikih duplikacija (10,5%) kod 4 DMD i 2 BMD bolesnika. Takođe, duplikacije su nađene kod 3 od 4 (75%) testirane majke. Distribucija duplikacija je bila heterogena, obuhvatala je N-terminalni i štapićasti region i uključivala je veći broj egzona, osim kod jednog DMD bolesnika koji je imao duplikaciju egzona 2. Odstupanje od Monakovog pravila je bilo prisutno kod 9,5% DMD probanda, odnosno kod 15,8% BMD probanda, to jest kod 12,5% slučajeva. Zaključak. Kod 57 DMD/BMD probanda nađeno je 59,6% velikih delecija i 10,5% velikih duplikacija. Najčešće je bio zahvaćen štapićasti domen u DMD genu. Odstupanje od Monakovog pravila je bilo prisutno u 12,5% DMD/BMD slučajeva. Tri od četiri ispitane majke probanda su bile potvrđene kao nosioci

Značaj direktnog genetičkog testiranja za otkrivanje žena prenosioca mutacije kod distrofinopatija

Background/Aim. Duchenne muscular dystrophy (MD) and Becker MD are caused by mutations in the gene for dystrophin (DMD). They are X chromosome-linked reces-sive diseases where males are affected, and females are healthy carriers of the mutation in most cases. It is estimat-ed that 2/3 of mothers of Duchenne MD probands are car-riers, while 1/3 of probands have de novo mutations. The aim of the study was to confirm the carrier status of female members of the families of Duchenne MD/Becker MD probands using direct genetic testing methods. Methods. The study included 38 females from 31 families of Du-chenne MD/Becker MD probands with dele-tion/duplication in the DMD gene. Moreover, 4 cases of prenatal diagnosis of Duchenne MD/Becker MD were in-cluded. The methods of polymerase chain reaction - PCR and the multiplex ligation-dependent probe amplification - MLPA were applied for detecting deletions, i.e., dele-tion/duplication mutations in the DMD gene. Results. In the total of 31 Duchenne MD/Becker MD probands, 87.1% of deletions and 12.9% of duplications of one or more exons in the DMD gene were detected. Of the 29 tested mothers, mutations were found in 17 of them (14 de-letions and 3 duplications). Mutations were detected in 11 (57.9%) out of 19 mothers of probands with the Duchenne MD phenotype and 6 (60%) out of 10 mothers of Becker MD probands. Furthermore, 14 (56%) out of 25 mothers were carriers in probands with deletions, and 3 (75%) out of 4 mothers were carriers in probands with duplications. In the remaining 9 other female relatives of the patients, muta-tions were found in 4. In prenatal diagnosis, we identified a deletion in one male and one female fetus of one single mother who was confirmed as a carrier. Conclusion. The study showed that mothers were carriers in almost 60% of sporadic cases of Duchenne MD/Becker MD with dele-tions and duplications. In addition, the carrier frequency tended to be higher in mothers of the probands with dupli-cations (75%) compared to mothers of probands with dele-tions (56%).Uvod/Cilj. Dišenova mišićna distrofija (MD) i Bekerova MD su uzrokovane mutacijama u genu za distrofin (DMD). To su recesivne bolesti vezane za X hromozom, od kojih obolevaju muškarci, a žene su uglavnom zdravi nosioci mu-tacije. Procenjeno je da su kod probanada obolelih od Dišenove MD 2/3 majki nosioci mutacije, dok 1/3 pro-banada ima de novo mutaciju. Cilj rada bio je da se potvrdi status nosioca mutacije kod ženskih članova porodica pro-banada obolelih od Dišenove MD/Bekerove MD primenom metoda direktnog genetičkog testiranja. Metode. Studija je obuhvatila ukupno 38 žena iz 31 porodice pro-banada obolelih od Dišenove MD/Bekerove MD sa deleci-jom/duplikacijom u DMD genu. Takođe, u studiju su bila uključena i 4 slučaja Dišenove MD/Bekerove MD otkrivena prenatalnom dijagnostikom. Metoda lančane reakcije poli-meraze (polymerase chain reaction – PCR) i metoda višestrukog umnožavanja vezanih proba (multiplex ligation-dependent probe amplification -MLPA) su korišćene za detekciju delecija, od-nosno delecija/duplikacija mutacija u DMD genu. Rezulta-ti. Kod ukupno 31 probanada obolelih od Dišenove MD/Bekerove MD, utvrđeno je 87,1% mutacija tipa deleci-je i 12,9% mutacija tipa duplikacija jednog ili više egzona u DMD genu. Od 29 testiranih majki probanada, mutacije su nađene kod njih 17 (14 delecija i 3 duplikacije). Mutacije su detektovane kod 11 (57,9%) od 19 majki probanada sa feno-tipom Dišenove MD i kod 6 (60%) od 10 majki probanada obolelih od Bekerove MD. Takođe, kod probanada sa de-lecijom, kod 14 (56%) od 25 majki je potvrđeno da su nosioci mutacije, a kod probanada sa duplikacijom, 3 (75%) od 4 majke su bile nosioci mutacije. Od ostalih 9 ženskih srodnika probanada obolelih od Dišenove MD/Bekerove MD, mutacije su nađene kod nijh 4. Prenatalnom dijagnos tikom utvrđene su delecije kod jednog muškog i jednog ženskog fetusa iste majke koja je bila potvrđena kao nosilac mutacije. Zaključak. Istraživanje je pokazalo da su majke bile nosioci mutacija u skoro 60% izolovanih slučajeva ob-olelih od Dišenove MD/Bekerove MD sa delecijama i duplikacijama. Takođe, učestalost majki nosioca mutacije kod probanada sa duplikaciom (75%) se pokazala višom ne-go kod majki probanada sa delecijom (56%)

Epidemiology of myotonic dystrophy type 1 in the population of central Serbia

The objective of this epidemiological survey was to estimate the frequency and distribution of Myotonic dystrophy type 1 (MD1) (Steinert's disease) in central Serbia, during the period 1983-2002. The data on the number of diagnosed MD1 patients were obtained using the analysis of hospital records, which were examined in all the relevant neurological institutions in central Serbia in the mentioned period. Incidence rate and prevalence were used for the data analysis. In the study period in central Serbia, 154 patients (78 males and 76 females) with MD1 were identified. The average annual incidence rate of MD1 was 1.3 (95% CI-confidence interval 0.1-7.2) per 1,000,000 population, 1.4/1,000,000 (95% CI 0.1-7.2) for males, 1.3/1,000,000 (95% CI 0.1-7.2) for females. The trend of MD1 incidence rates in the observed period in central Serbia had a tendency of the statistically significant decrease, according to the linear model, in both male (y=0.205-0.0066x, p=0.021) and female populations (y = 0.1788-0.0048×, p = 0.032). The prevalence of MD1 on December 31, 2002 in central Serbia was 3.8/100,000 (95% IP 3.2-4.6), 3.7/100,000 (95% IP 3.3-4.8) for males, 3.3/100,000 (95% IP 3.0-4.4) for females

Cognitive Impairment in Myotonic Dystrophy Type 1 Is Associated with White Matter Damage

<div><p>Objective</p><p>To investigate grey (GM) and white matter (WM) abnormalities and their effects on cognitive and behavioral deficits in a large, phenotypically and genotypically well-characterized cohort of classic adult (aDM1, age at onset ≥20 years) or juvenile (jDM1, age at onset <20 years) patients with myotonic dystrophy type 1 (DM1).</p><p>Methods</p><p>A case-control study including 51 DM1 patients (17 jDM1 and 34 aDM1) and 34 controls was conducted at an academic medical center. Clinical, cognitive and structural MRI evaluations were obtained. Quantitative assessments of regional GM volumes, WM hyperintensities (WMHs), and microstructural WM tract damage were performed. The association between structural brain damage and clinical and cognitive findings was assessed.</p><p>Results</p><p>DM1 patients showed a high prevalence of WMHs, severe regional GM atrophy including the key nodes of the sensorimotor and main cognitive brain networks, and WM microstructural damage of the interhemispheric, corticospinal, limbic and associative pathways. WM tract damage extends well beyond the focal WMHs. While aDM1 patients had severe patterns of GM atrophy and WM tract damage, in jDM1 patients WM abnormalities exceeded GM involvement. In DM1, WMHs and microstructural damage, but not GM atrophy, correlated with cognitive deficits.</p><p>Conclusions</p><p>WM damage, through a disconnection between GM structures, is likely to be the major contributor to cognitive impairment in DM1. Our MRI findings in aDM1 and jDM1 patients support the hypothesis of a degenerative (premature aging) origin of the GM abnormalities and of developmental changes as the principal substrates of microstructural WM alterations in DM1.</p></div

Neuropsychological and behavioural data of DM1 patients.

<p>Values are means ± standard deviations (% of subjects with abnormal test score compared with normative data of reference, see text for further details).</p>#<p>p<0.05 in aDM1 <i>vs</i> jDM1 patients (Poisson model, false-discovery rate adjusted for multiple comparisons;</p><p>*age-adjusted p values). Abbreviations: ACE-R, Addenbrooke’s Cognitive Examination–Revised; aDM1, Myotonic dystrophy type 1 with adult onset (≥20 years); BNT, Boston Naming Test; DM1, Myotonic dystrophy type 1; HDRS, Hamilton Depression Rating Scale; HARS, Hamilton Anxiety Rating Scale; jDM1, Myotonic dystrophy type 1 with early onset (<20 years); ns, not significant; RAVLT, Rey Auditory Verbal Learning test; TMT, Trials Making Test; VOT, Visual Organization Test; WAIS, Wechsler-Adult Intelligence Scale.</p

Tract-based spatial statistics results in patients with myotonic dystrophy 1 compared with age-matched healthy controls and relationship between the Addenbrooke’s Cognitive Examination–Revised (ACE-R) orientation and attention subscores and mean diffusivity values.

<p>Analyses were adjusted for age. A–C: Voxelwise group differences are shown in blue (mean diffusivity) and red (fractional anisotropy). D) Regions where MD values correlated with the ACE-R orientation and attention subscores are shown in blue. Results are overlaid on the sagittal and axial sections of the Montreal Neurological Institute standard brain in radiological convention (right is left), and displayed at p<0.05 corrected for multiple comparisons. The white matter skeleton is green.</p