A comprehensive database of Duchenne and Becker muscular dystrophy patients (0–18 years old) in East China

Most of the registrants within the CHFU database were diagnosed during the age of 3–4 (16.6%) and 7–8 (14.8%) years. The high rate of diagnosis at 3–4 year-old group is the result of health screening for children entering Nurseries and Kindergartens. However, the 7–8 year-old children visited the outpatient clinic for clinical symptoms typical for muscular dystrophy such as toe walking and difficulty to climb stairs. Most boys of this age group were from rural areas, indicating the lack of awareness of the disease by both parents and primary care doctors. The data raises the urgent need for health authority and medical institutions to take initiative to educate the public, especially in rural areas to improve early diagnosis and provide preventive care and optimal management.

The large number of DMD/BMD patients in the CHFU database also revealed the specific pattern of mutations in the Chinese population. In this study, we analyzed the distribution of individual exons based on their frequency of deletion or duplication. Our data indicate that exon deletion is the most frequent followed by point mutations, duplications and small insertion/deletion. In DMD patients, exons 45–52 are the most frequently deleted. This is similar to the pattern described in other databases [16,17,20] and those of previously reported in Chinese population [21-23]. Similarly, the most common deletion in BMD patients was exons 45–47 (22.9%) [17]. In general, the severity of the phenotype correlates to the reading-frame rule [24]. Similar to the report by Sylvie Tuffery-Giraud et al. [20], our database shows 95.2% of DMD patients had out of frame or nonsense mutations whereas 85.7% of the BMD mutations were in-frame, consistent with two previous studies in Chinese patients with DMD/BMD [21,22]. The phenotype for in-frame mutations is to some extent determined by the location and the size of the deletion. Large in-frame deletions removing up to 35 exons in the central rod domain have been described for patients with relatively mild BMD [25,26]. Within our database, the muscle biopsy of a patient with a large in-frame deletion of exons 27–57 showed absence of dystrophin expression using antibody NCL-DYS1 (against amino acids 1181–1388), whereas near normal level signals were detected using antibody NCL-DYS2 and NCL-DYS3 (against amino acids 3669–3685 and 321–494 respectively). Another patient with a large in-frame deletion of exons 10–41 showed absence of dystrophin expression using antibody NCL-DYS1 and NCL-DYS3, whereas near normal level signals were detected using antibody NCL-DYS2. Both patients aged 8 years. No detectable muscle weakness was found and the only pathological feature was the elevation of serum creatine kinase (CK) (1926 U/L and 12350 U/L respectively). Most point mutations are nonsense mutations resulting in DMD. However, there were 2 BMD cases with point mutations in our database (Seen in Table 2). One 5-year-old patient with the mutation c.3940C > T had high serum CK levels (11740 U/L) as the only clinical abnormality. Muscle biopsy showed lack of dystrophin using antibody NCL-DYS1 against an epitope encoded by exon 29, but positive staining using antibody NCL-DYS2 and NCL-DYS3, consistent with the previous report of a BMD family [27]. The other 4-year-old boy with the novel point mutation (c.3388G > T) showed the clinical abnormality of bringing the second foot up to join the first rather than going foot over foot when climbing stairs and high serum CK levels (9100 U/L). The muscle biopsy showed lack of dystrophin using antibody NCL-DYS1, NCL-DYS2 and NCL-DYS3. We also found a large BMD pedigree with 4 patients and 10 carriers of a novel frame-shift mutation in exon 4 in this family. The 5-year-old proband showed only mild atrophy of bilateral quadriceps, without symptomatic weakness. Muscle biopsy revealed mild myopathic features including variation in fiber size and a few necrotic and regenerating muscle fibers. Immunohistochemical analysis demonstrated decreased expression with antibody NCL-DYS3 and near normal expression with antibody NCL-DYS1 and NCL-DYS2. Serum CK levels were consistently around 2854 U/L. The oldest patient aged 35 was diagnosed at the age of 9 and lost walking ability in his thirties. The other two patients aged 27 and 23 had the onset of the disorder around 10 years old with difficulty in walking and climbing stairs. All the patients revealed bilateral atrophy limited to quadriceps femoris without hypertrophy of the calves. We considered it as quadriceps myopathy, a clinical variant form of BMD [28-30].

In our study, 23.1% of the probands had a family medical history of the disease and several pedigrees also had a large number of patients and mutation carriers, especially in less developed areas. Through patient registry, we provided information on genetic counseling and prenatal diagnosis for pedigrees and those who had given birth to DMD/BMD babies to prevent this disease. 15 healthy children have been born by prenatal diagnosis in these families in 2013. Genetic counseling is therefore valuable for prenatal diagnosis and vitally important to those big families in less developed areas where literacy level is relatively low and lack of knowledge about genetic diseases.

26.3% of DMD patients in the database were treated with steroids. This rate was relatively low compared to that in the developed countries [16,31,32]. The main reason for this is due to the lack of awareness of physicians specialized in treating muscular dystrophies. The Family Guide for the Diagnosis and Management of Duchenne Muscular Dystrophy [33] became available in our hospital only in January 2013, when DMD Outpatient Clinic was opened. However, this environment is changing with the establishment of the database and the adoption of the TREAT-NMD project guidelines [6,34]. A steady increase in the use of steroid treatment is expected in China. As to the evaluation of cardiac and pulmonary function, we refer to the Family Guide, which indicates that baseline evaluation of cardiac function needs to be performed at the confirmation of the diagnosis or at latest by the age of six years. Evaluation includes an electrocardiogram (ECG) and echocardiogram and occurs at least once every two years until the age of ten. Yearly complete cardiac evaluations begin at approximately ten years of age or at the onset of cardiac signs and symptoms, if earlier. If noninvasive cardiac tests show abnormalities, increased surveillance is required, at least every six months, and drug treatment will be initiated by our cardiologists. In our database, four boys were prescribed for treatment with ACE–inhibitor (1) and the ACE–inhibitor and β-blocker (3). One of them has an abnormal ECG that showed right ventricular hypertrophy and sinus tachycardia and the others have abnormal echocardiograms (ejection fraction ranging from 25-48% of normal levels) [31]. Minimal assessment of pulmonary function (such as measurement of forced vital capacity [FVC] at least annually) allows the child to become familiar with the equipment and the team to assess the maximum respiratory function achieved, while a boy with DMD is still walking. The main emphasis of pulmonary assessment is after the loss of independent walking [33]. However, pulmonary management is far from perfect in China. In our database, none of the patients used ventilator support. No abnormality has been found in the patients who received pulmonary function tests, whereas most wheelchair-bound patients are taken care of by their family members at home and are reluctant to get tested. This is largely due to two factors: lack of awareness by physicians of significance of these measures to the quality of patient’s life and lack of medical insurance to cover the expenses in China. This reinforces the urgency to raise awareness of the disease to the public, physicians and governmental organizations.