Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the ...genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.
Objective
Strategic Targeting of Registries and International Database of Excellence (STRIDE) is an ongoing, international, multicenter registry of real-world ataluren use in individuals with ...nonsense mutation Duchenne muscular dystrophy (nmDMD) in clinical practice. This updated interim report (data cut-off: January 31, 2022), describes STRIDE patient characteristics and ataluren safety data, as well as the effectiveness of ataluren plus standard of care (SoC) in STRIDE versus SoC alone in the Cooperative International Neuromuscular Research Group (CINRG) Duchenne Natural History Study (DNHS).
Methods
Patients are followed up from enrollment for at least 5 years or until study withdrawal. Propensity score matching was performed to identify STRIDE and CINRG DNHS patients who were comparable in established predictors of disease progression.
Results
As of January 31, 2022, 307 patients were enrolled from 14 countries. Mean (standard deviation SD) ages at first symptoms and at genetic diagnosis were 2.9 (1.7) years and 4.5 (3.7) years, respectively. Mean (SD) duration of ataluren exposure was 1671 (56.8) days. Ataluren had a favorable safety profile; most treatment-emergent adverse events were mild or moderate and unrelated to ataluren. Kaplan–Meier analyses demonstrated that ataluren plus SoC significantly delayed age at loss of ambulation by 4 years (
p
< 0.0001) and age at decline to %-predicted forced vital capacity of < 60% and < 50% by 1.8 years (
p
= 0.0021) and 2.3 years (
p
= 0.0207), respectively, compared with SoC alone.
Conclusion
Long-term, real-world treatment with ataluren plus SoC delays several disease progression milestones in individuals with nmDMD. NCT02369731; registration date: February 24, 2015.
OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of ...human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild‐type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA‐encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.
Synopsis
This work describes the first confirmed pathogenic variants in OXA1L in a mitochondrial disease patient with tissue‐specific combined OXPHOS deficiencies. OXA1L was shown to be important for complex IV assembly in human cell lines with targeted depletion of OXA1L and in patient tissues.
Bi‐allelic variants in OXA1L (NM_005015.3; c.500_507dup, p.(Ser170Glnfs*18); c.620G>T, p.(Cys207Phe) were identified by whole exome sequencing in a patient with hypotonia, developmental delay and severe encephalopathy who died following cardiorespiratory arrest.
Patient fibroblasts showed decreased protein levels of OXA1L and subunits of OXPHOS complexes I, IV and V.
Introduction of wild‐type OXA1L into patient fibroblasts by retroviral transduction rescued the OXPHOS defects, confirming pathogenicity of the OXA1L variants.
Depletion of OXA1L by siRNA in human cell lines and Drosophila caused combined OXPHOS deficiencies including severe effects on complex IV.
An OXA1L CRISPR/Cas9 knockout cell line with inducible expression of FLAG‐tagged OXA1L was used for OXA1L immunoprecipitation and identified an enrichment of mtDNA‐encoded subunits of complexes I, IV and V.
This work describes the first confirmed pathogenic variants in OXA1L in a mitochondrial disease patient with tissue specific combined OXPHOS deficiencies. OXA1L was shown to be important for complex IV assembly in human cell lines with targeted depletion of OXA1L and in patient tissues.
Several clinical trials are working on drug development for Duchenne and Becker muscular dystrophy (DMD and BMD) treatment, and, since the expected increase in dystrophin is relatively subtle, ...high-sensitivity quantification methods are necessary. There is also a need to quantify dystrophin to reach a definitive diagnosis in individuals with mild BMD, and in female carriers. We developed a method for the quantification of dystrophin in DMD and BMD patients using spectral confocal microscopy. It offers the possibility to capture the whole emission spectrum for any antibody, ensuring the selection of the emission peak and allowing the detection of fluorescent emissions of very low intensities. Fluorescence was evaluated first on manually selected regions of interest (ROIs), proving the usefulness of the methodology. Later, ROI selection was automated to make it operator-independent. The proposed methodology correctly classified patients according to their diagnosis, detected even minimal traces of dystrophin, and the results obtained automatically were statistically comparable to the manual ones. Thus, spectral imaging could be implemented to measure dystrophin expression and it could pave the way for detailed analysis of how its expression relates to the clinical course. Studies could be further expanded to better understand the expression of dystrophin-associated protein complexes (DAPCs).
Highlights • Digital PCR is a powerful technique to quantify miRNAs in sera of DMD/BMD patients. • MiR-30c and miR-181a are valuable serum biomarkers for diagnosis of DMD and BMD. • Steroid treatment ...has not effect on miR-30c and miR-181a levels in DMD patients. • MiR-30c may serve to monitor motor function in DMD.
Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of ...paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.
Our objective was to investigate the potential of three microRNAs, miR-181a-5p, miR-30c-5p, and miR-206 as prognostic biomarkers for long-term follow up of Duchenne muscular dystrophy (DMD) and ...Becker muscular dystrophy (BMD) patients. We analyzed the expression of three microRNAs in serum of 18 patients (DMD 13, BMD 5) and 13 controls using droplet digital PCR. Over 4 years a minimum of two and a maximum of three measurements were performed at different time points in the same patient. Correlations between microRNA serum levels, age, and functional outcome measures were analyzed. We show the individual evolution of the levels of the three microRNAs in 12 patients and also the effect of corticosteroid treatment on microRNAs expression. We measure the expression of three microRNAs in the muscle of six DMD patients and also the expression of target genes for miR-30c. We found that levels of miR-30c and miR-206 remained significantly elevated in DMD patients relative to controls over the entire study length. The introduction of the corticosteroid treatment did not significantly influence the levels of these microRNAs. We report a trend for microRNA levels to decrease with age. Moreover, miR-206 expression levels are capable to distinguish DMD from BMD patients according to ROC analysis. We found miR-30c expression decreased in the muscle of DMD patients and marked upregulation of the target genes for this microRNA. MiR-30c and miR-206 represent sensitive biomarkers for DMD, while miR-206 may have an additional value to distinguish the DMD and BMD phenotype. This may be particularly relevant to assess the effectiveness of treatments aimed at converting the DMD to the less-severe BMD like phenotype.
Objective
We report natural history data in a large cohort of 199 patients with spinal muscular atrophy (SMA) type III assessed using the Hammersmith Functional Motor Scale Expanded (HFMSE). The aim ...of the study was to establish the annual rate and possible patterns of progression according to a number of variables, such as age of onset, age at assessment, SMN2 copy number, and functional status.
Methods
HFMSE longitudinal changes were assessed using piecewise linear mixed‐effects models. The dependency in the data due to repeated measures was accounted for by a random intercept per individual and an unstructured covariance R matrix was used as correlation structure. An additional descriptive analysis was performed for 123 patients, for a total of 375 12‐month assessments.
Results
A break point at age 7 years was set for the whole cohort and for SMA IIIA and IIIB. Age, SMA type, and ambulatory status were significantly associated with changes in mean HFMSE score, whereas gender and SMN2 copy number were not. The increase in response before the break point of age 7 years is significant only for SMA IIIA (β = 1.79, p < 0.0001). After the break point, the change in the rate of HFMSE score significantly decrease for both SMA IIIA (β = −1.15, p < 0.0001) and IIIB (β = −0.69, p = 0.002).
Interpretation
Our findings contribute to the understanding of the natural history of SMA type III and will be helpful in the interpretation of the real‐world data of patients treated with commercially available drugs. ANN NEUROL 2020;88:1109–1117
•Describe lost or gained activities on the HFMSE in type II SMA over 12 months.•The cohort included 614 12-month assessments from 243 patients.•Data were using with the shift analysis statistical ...method.•The results were analysed by age, functional level and SMN2 copy number.•The shift approach could be used as an additional tool for data analysis.
The advent of clinical trials in spinal muscular atrophy (SMA) has highlighted the need to define patterns of progression using functional scales. It has recently been suggested that the analysis of abilities gained or lost applied to functional scales better reflects meaningful changes. We defined as “gain” a positive change between scores from 0 to either 1 or 2 and as “loss” a negative change from either 2 or 1 to 0. The aim of this study was to describe, over 12 months, which abilities on the Hammersmith Functional Motor Scale Expanded (HFMSE) were more frequently lost or gained in patients with SMA II. The cohort included 614 12-month assessments from 243 patients (age range: 30 months - 63 years; mean 9.94, SD ±7.91). The peak of abilities gained occurred before the age of 5 years while the highest number of lost abilities was found in the group 5–13 years. A correlation between the HFMSE baseline score and the ordinal number of the items was found for both lost (p<0.001) or gained (p<0.001) activities. No correlation was found with SMN2 copy number. These findings will have implications for clinical trial design and for the interpretation of real-world data using new therapeutic approaches.
The term neuromuscular disorder (NMD) includes many genetic and acquired diseases and differential diagnosis can be challenging. Next-generation sequencing (NGS) is especially useful in this setting ...given the large number of possible candidate genes, the clinical, pathological, and genetic heterogeneity, the absence of an established genotype-phenotype correlation, and the exceptionally large size of some causative genes such as
,
and
We evaluated the diagnostic value of a custom targeted next-generation sequencing gene panel to study the mutational spectrum of a subset of NMD patients in Spain. In an NMD cohort of 207 patients with congenital myopathies, distal myopathies, congenital and adult-onset muscular dystrophies, and congenital myasthenic syndromes, we detected causative mutations in 102 patients (49.3%), involving 42 NMD-related genes. The most common causative genes,
accounted for almost 30% of cases. Thirty-two of the 207 patients (15.4%) carried variants of uncertain significance or had an unidentified second mutation to explain the genetic cause of the disease. In the remaining 73 patients (35.3%), no candidate variant was identified. In combination with patients' clinical and myopathological data, the custom gene panel designed in our lab proved to be a powerful tool to diagnose patients with myopathies, muscular dystrophies and congenital myasthenic syndromes. Targeted NGS approaches enable a rapid and cost-effective analysis of NMD- related genes, offering reliable results in a short time and relegating invasive techniques to a second tier.
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