Duchenne and Becker muscular dystrophy (DMD/BMD) are both caused by mutations in the DMD gene. Out-of-frame mutations in DMD lead to absence of the dystrophin protein, while in-frame BMD mutations cause production of internally deleted dystrophin. Clinically, patients with DMD loose ambulance around the age of 12, need ventilatory support at their late teens and die in their third or fourth decade due to pulmonary or cardiac failure. BMD has a more variable disease course. The disease course of patients with BMD with specific mutations could be very informative to predict the outcome of the exon-skipping therapy, aiming to restore the reading-frame in patients with DMD. Patients with BMD with a mutation equalling a DMD mutation after successful exon skipping were selected from the Dutch Dystrophinopathy Database. Information about disease course was gathered through a standardised questionnaire. Cardiac data were collected from medical correspondence and a previous study on cardiac function in BMD. Forty-eight patients were included, representing 11 different mutations. Median age of patients was 43 years (range 6-67). Nine patients were wheelchair users (26-56 years). Dilated cardiomyopathy was present in 7/36 patients. Only one patient used ventilatory support. Three patients had died at the age of 45, 50 and 76 years, respectively. This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a 'skipped' DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.
Becker muscular dystrophy (BMD) is characterised by broad clinical variability. Ongoing studies exploring dystrophin restoration in Duchenne muscular dystrophy ask for better understanding of the relation between dystrophin levels and disease severity. We studied this relation in BMD patients with varying mutations, including a large subset with an exon 45–47 deletion.
Methods
Dystrophin was quantified by western blot analyses in a fresh muscle biopsy of the anterior tibial muscle. Disease severity was assessed using quantitative muscle strength measurements and functional disability scoring. MRI of the leg was performed in a subgroup to detect fatty infiltration.
Results
33 BMD patients participated. No linear relation was found between dystrophin levels (range 3%–78%) and muscle strength or age at different disease milestones, in both the whole group and the subgroup of exon 45–47 deleted patients. However, patients with less than 10% dystrophin all showed a severe disease course. No relation was found between disease severity and age when analysing the whole group. By contrast, in the exon 45–47 deleted subgroup, muscle strength and levels of fatty infiltration were significantly correlated with patients' age.
Conclusions
Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45–47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.
Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials.
Methods
Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates.
Results
We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss.
Conclusions
This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.
To the Editor: In response to the case report by Catts et al,1 we present a second patient with myelodysplastic syndrome (MDS) and the same de novo germline RRAS variant. The female patient was born with pulmonary subvalvular and valvular stenosis, which was surgically corrected at age 3. She had normal growth and development and attended regular education. She presented with frequent nosebleeds and menorrhagia at age 15 without probable cause. Two years later she presented with fatigue and persistent menorrhagia, she had low platelets and was diagnosed with MDS with monosomy 7. The patient was treated with an allogeneic stem cell transplantation (matched unrelated donor). During treatment she presented with pain and paralysis of the lower extremities making her confined to a wheelchair, diagnosed as chronic inflammatory demyelinating polyneuropathy (CIDP). Revision of a chest computed tomography made before the stem cell transplantation showed thickened nerves, indicating pretreatment myelinization abnormalities. After treatment with intravenous immunoglobulins, the patient could walk independent, but paresis was not completely resolved. Because of the congenital heart disease, unclassified bleeding disorder and MDS, the patient was referred to a clinical geneticist who suspected Noonan syndrome (NS). Physical examination revealed no typical physical features. NS gene panel testing was negative, and SNP array analysis was normal. Trio whole exome sequencing revealed a de novo heterozygous germline RRAS variant, c.116_118dup, p.Gly39dup. This variant was confirmed by Sanger sequencing in DNA derived from a blood sample taken at age 1, excluding an MDS-related somatic mutation. A highly similar case was reported by Au et al.2 They described a 23-year-old male with subvalvular pulmonary stenosis and concurrent acute demyelinating neuropathy and MDS with monosomy 7. Genetic testing was declined by the family. The patient reported by Catts et al1 was born with craniosynostosis, mild dysmorphic features and developed MDS with monosomy 7 at age 7. A third patient with this de novo variant is a 16-year-old girl with dysmorphic facial features resembling NS and short stature. Her medical history revealed a pulmonary stenosis, delayed motor development, feeding difficulties, and acute myeloid leukemia suspected to represent a blast crisis of juvenile myelomonocytic leukemic at age 13.3 This author also reported a 51-year-old woman with an RRAS c.163G>A, p.Val55Met variant. Physical examination showed dysmorphic facial features, suggesting NS, and she developed an unspecified bone tumor during childhood. These cases show a diversity of symptoms in patients with germline RRAS variants partially overlapping with clinical symptoms of NS. In the patient reported here, the demyelinating neuropathy was striking. RRAS plays an essential role in the differentiation, proliferation, and survival of oligodendrocytes responsible for axon myelination, which might suggest a causal relation.4 Four patients developed a childhood malignancy, including three patients with a hematologic malignancy. This may indicate a higher penetrance for cancer in patients with germline RRAS variants compared with other NS (like) genes. We support the recommendation by Catts et al to add RRAS to the list of genes tested when a RASopathy is suspected. Additionally, we recommend including RRAS in pediatric cancer predisposition gene panels. Nienke van Engelen, MD* Illja Diets, PhD† Dorine Bresters, PhD* Janneke C. van den Bergen, PhD* Alexander F.J.E. Vrancken, PhD‡ Roland P. Kuiper, PhD* Marjolijn C.J. Jongmans, PhD*§ *Princess Máxima Center for Pediatric Oncology ‡Department of Neurology, Brain Centre Rudolf Magnus University Medical Centre Utrecht §Department of Genetics, University Medical Center Utrecht, Utrecht †Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands
In this thesis research is presented about the progressive muscle diseases Duchenne and Becker muscular dystrophy, two hereditary muscle diseases caused by a mutation in the gene coding for dystrophin, a protein involved in muscle membrane stability. The first part describes the disease course of both diseases. It evaluates the effect of developments in care of Duchenne patients, improving age at wheelchair dependence and survival. Data are presented about the relatively mild disease course of selected Becker patients with a mutation that would be the result of exon skipping in a Duchenne patient, illustrating the possible result of this therapeutic approach. The second part of this thesis focusses on research into factors involved in disease variability. Data are presented regarding the role of dystrophin quantity in disease severity in Becker patients, showing no linear relationship. Expression of several dystrophin associated proteins is shown not to influence disease course either. Contrarily, a single nucleotide polymorphism in the LTBP4 gene involved in fibrosis and muscle regeneration is shown to influence disease severity. Lastly, a disease severity scale for Becker patients is presented in this thesis, enabling a better comparison of individual patients for the purpose of scientific research.
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