Myotonic dystrophy type 1 (DM1) (OMIM #160900), also referred to as myotonia atrophica, dystrophia myotonica (DM), and myotonic muscular dystrophy, is an autosomal dominant, multisystemic disorder affecting skeletal and smooth muscle. It can also affect the eye, the heart, endocrine glands, and the central nervous system. DM is the most common form of adult muscular dystrophy, with an average incidence of one in 8000 (Beers and Robert, 2004). It is characterized by myotonia, progressive muscle weakness and atrophy, cardiac conduction disturbances, cataracts, and abnormal glucose intolerance. The age of onset of symptoms ranges from birth to old age (Day and Ranum, 2005).
DM1 is classified according to the age of onset into three main different forms: mild, congenital, and classic DM (Machuca-Tzili et al., 2005).
The mild form of DM is the least severe form of the disease, with symptoms appearing in middle to old age. The congenital form of DM1 is the most severe form of the disease, with symptoms present at birth. The classic form of DM1 is when the disease first appears in adolescence and early adult life (Tiscornia and Mahadevan, 2000).
The mutation underlying DM has been characterized as an expanded CTG trinucleotide repeat sequence in the 3′ untranslated region of a dystrophia myotonica protein kinase (DMPK) gene on chromosome 19q13.3 whose size correlates with the clinical severity of the disorder. A relationship between the size of the CTG repeat and disease severity has been reported in several studies, although there is an overlap between clinical groups (Passos-Bueno et al., 1995).
The number of CTG repeats varies considerably among individuals, but the average number in a healthy individual is from 5 to 34; patients with DM1 have over 50 and can have as many as 2000 repeats. In asymptomatic individuals at a premutation stage, CTG repeat varies from 35 to 50 (Kaliman and Liagostera, 2008). DM1 is subjected to anticipation; that is, the earlier onset of more severe symptoms in individuals belonging to successive generations of the same family. However, there are fewer reported examples of the opposite phenomenon; that is, a contraction in CTG repeat length in successive generations (Meola, 2000).
The aim of our study was to implement a molecular diagnostic method for DM1 in a sample of Egyptian patients.
Patients and methods
The participants of this study were 13 DM1 probands and seven of their family members as well as 20 apparently healthy controls of the same ethnicity.
All participants were subjected to full assessment of clinical and genetic history and a complete clinical examination. For DM1 patients, pedigree analysis, electromyographic examination, and assessment of serum creatine kinase level were performed.
Genomic DNA was extracted from peripheral blood leukocytes of all participants using the salting-out procedure (Miller et al., 1988).
Expand long PCR (Fermentas, Germany) was used.
The CTG repeat located within the 3′ untranslated region of the DMPK gene was amplified using the following primer set: primer 101: 5′-CTTCCCAGGCCTGCAGTTTGCCCATC-3′; primer 102: 5′-GAACGGGGCTCGAAGGGTCTTGTAGC-3′ (Thornton et al., 1994).
The expanded PCR had a total volume of 15 μl containing 150 ng of DNA, 300 nmol/l of each primer, 1× expand long PCR buffer, 5% glycerol, 350 mmol/l of each dNTP, and 0.1 μl expand enzyme mix (3.5 U/μl). For amplification of premutated and fully mutated CTG alleles, dGTP in dNTPs mixture was substituted with 7-deaza-dGTP in a ratio dGTP : 7-deaza-dGTP=1 : 3 to destabilize secondary structures that were usually formed in these CG-rich DNA sequences. Without 7-deaza-dGTP in the dNTPs mixture, only normal alleles of CTG repeat sequences were amplified.
Expand long PCR was performed with a hot start.
Samples were then subjected to 10 cycles of amplification (97°C for 35 s, 65 or 62°C for 35 s, 68°C for 4 min), followed by 20 cycles with the same temperature and time profile, except that the elongation step was extended for 20 s after each cycle. PCR reactions were performed in 2720 Thermal Cycler (Applied Biosystems, Foster City, California, USA). PCR products were electrophoresed on an 8% polyacrylamide gel, and then stained with ethidium bromide and visualized on a UV transilluminator to determine the CTG repeat size.
The age of the DM1 patients ranged from 7 to 60 (27.45±17.27) years. The age of onset of the disease varied from 3 to 46 years. Fourteen men and six women were included in the study. All patients had muscle weakness, as well as muscle wasting of varying degrees affecting distal limb muscles more than the proximal ones. Full ophthalmological assessment indicated that eye abnormalities were detected in 8/20 of cases (40%): two patients had cataract (10%), five had ptosis (25%), and one had optic atrophy (5%). Two patients had nasal tone speech and difficulty in mastication (10%). Two patients had dysarthria (10%). Anthropometric measurements were all within the normal range for age and sex. Echocardiographic and ECG assessment indicated mitral valve prolapse in four patients (20%), whereas the other patients showed no abnormalities. Electromyography and motor conduction studies showed myotonic discharges with mild to moderate dystrophic changes in all the patients studied. Muscle biopsy was performed for only five cases and showed typical sarcoplasmic masses, atrophic small type I fibers, and ringed fibers. Serum CK levels were normal and ranged from 90.7±15.2 U/l, except in one DM1 patient, in whom it was elevated (322 U/l; reference range 38–174 U/l). The descriptive data of the different parameters in the studied groups are shown in Table 1.
The study showed an increase in the number of CTG repeat units in all 13 probands and the seven family members. The expanded alleles ranged from 41 to 299 repeat units. One participant, a relative of a patient, was found to have 41 CTG repeats in the premutation range whereas the rest had the full mutant allele (>50 CTG repeats) (Figs 1 and 2). The CTG repeat numbers in the normal control participants ranged from 5 to 21 and the five repeats was the most frequently detected allele. Anticipation was observed in one of the two families studied, where there was an increase in the length of the CTG repeats than the transmitting parent; the proband had 299 repeats whereas his father had 76 repeats. Another proband of the same family also had an expansion of the father’s upper allele (129 repeats) whereas the father had 96 repeats (Fig. 1). The distribution of CTG expansions in the 20 DM1 patients is shown in Table 2.
Expanded trinucleotide repeats are now recognized as the cause of several neurological diseases; one of them is DM. Identification of the molecular basis in DM1 is important for diagnostic confirmation, genetic counseling, and for understanding the clinical variability among patients (Kim et al., 2008). Molecular diagnosis is the investigation of choice in the diagnosis of individuals with equivocal signs, asymptomatic, and prenatal diagnosis determining whether a fetus has inherited the mutation. This study included 20 patients with DM1, 14 men (70%) and six women (30%), and 20 normal individuals. The significantly higher proportion of affected male patients is in agreement with the reports of Brunner et al. (1993) and Passos-Bueno et al. (1995).
Anticipation is a common genetic phenomenon of DM1 reported in numerous studies (Hunter et al., 1992; Harley et al., 1993; Passos-Bueno et al., 1995; Kim et al., 2008). This anticipation was found in one family. Family 1 included six affected individuals in three generations. An increase in the average of the CTG repeat in successive generations was observed. However, contraction in the size of the trinucleotide repeat was also observed in this study. This phenomenon was reported in other studies of Ashizawa et al. (1994), Passos-Bueno et al. (1995), Amiel et al. (2001), and Kim et al. (2008).
Thus, in conclusion, the molecular diagnosis of DM1 by the expand long PCR technique and PAGE electrophoresis was an efficient method for the diagnosis of DM1. In the group of Egyptian DM1 patients studied, the repeat size varied from 41 to 299 and correlated with the phenotype of the disease, indicating that the CTG repeat number is reliable for confirming diagnosis and providing genetic counseling and is a valuable predictor of the severity of the disease.
Conflicts of interest
There are no conflicts of interest.
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