Pilot Program of Newborn Screening for 5q Spinal Muscular Atrophy in the Russian Federation.

5q spinal muscular atrophy (5q SMA) is one of the most common autosomal recessive disorders in the Russian Federation. The first medication to treat 5q SMA was registered in the Russian Federation for treatment of all 5q SMA types in 2019, and the last of the three currently available in December 2021. We launched the pilot newborn screening (NBS) program for 5q SMA in Moscow, the Russian Federation, starting in 2019. During the pilot program, 23,405 neonates were tested for the deletion of exon 7 of the SMN1 gene, the most common cause of 5q SMA. We used the SALSA® MC002 SMA Newborn Screen Kit (MRC Holland) to specifically detect homozygous deletions of SMN1 exon 7. We used the restriction fragment length polymorphism (RFLP) approach to validate detected homozygous deletions and the SALSA MLPA Probemix P060 SMA Carrier Kit (MRC Holland) to determine the SMN2 exon 7 copy number to prescribe gene therapy for 5q SMA. Three newborns with a homozygous deletion of the SMN1 gene were detected. The calculated birth prevalence of 1:7801 appears to be similar to the results in other European countries. The children did not show any signs of respiratory involvement or bulbar weakness immediately after birth. Until now, no 5q SMA case missed by NBS has been detected.

Safety and efficacy of risdiplam in patients with type 1 spinal muscular atrophy (FIREFISH part 2): secondary analyses from an open-label trial.

BACKGROUND: Risdiplam is an orally administered therapy that modifies pre-mRNA splicing of the survival of motor neuron 2 (SMN2) gene and is approved for the treatment of spinal muscular atrophy. The FIREFISH study is investigating the safety and efficacy of risdiplam in treated infants with type 1 spinal muscular atrophy versus historical controls. The primary endpoint of part 2 of the FIREFISH study showed that infants with type 1 spinal muscular atrophy attained the ability to sit without support for at least 5 s after 12 months of treatment. Here, we report on the safety and efficacy of risdiplam in FIREFISH part 2 over 24 months of treatment. METHODS: FIREFISH is an ongoing, multicentre, open-label, two-part study. In FIREFISH part 2, eligible infants (aged 1-7 months at enrolment, with a genetically confirmed diagnosis of spinal muscular atrophy, and two SMN2 gene copies) were enrolled in 14 hospitals in ten countries across Europe, North America, South America, and Asia. Risdiplam was orally administered once daily at 0·2 mg/kg for infants between 5 months and 2 years of age; once an infant reached 2 years of age, the dose was increased to 0·25 mg/kg. Infants younger than 5 months started at 0·04 mg/kg (infants between 1 month and 3 months old) or 0·08 mg/kg (infants between 3 months and 5 months old), and this starting dose was adjusted to 0·2 mg/kg once pharmacokinetic data were available for each infant. The primary and secondary endpoints included in the statistical hierarchy and assessed at month 12 have been reported previously. Here we present the remainder of the secondary efficacy endpoints that were included in the statistical hierarchy at month 24: the ability to sit without support for at least 30 s, to stand alone, and to walk alone, as assessed by the Bayley Scales of Infant and Toddler Development, third edition gross motor subscale. These three endpoints were compared with a performance criterion of 5% that was defined based on the natural history of type 1 spinal muscular atrophy; the results were considered statistically significant if the lower limit of the two-sided 90% CI was above the 5% threshold. FIREFISH is registered with ClinicalTrials.gov, NCT02913482. Recruitment is closed; the 36-month extension period of the study is ongoing. FINDINGS: Between March 13 and Nov 19, 2018, 41 infants were enrolled in FIREFISH part 2. After 24 months of treatment, 38 infants were ongoing in the study and 18 infants (44% [90% CI 31-58]) were able to sit without support for at least 30 s (p<0·0001 compared with the performance criterion derived from the natural history of untreated infants with type 1 spinal muscular atrophy). No infants could stand alone (0 [90% CI 0-7]) or walk alone (0 [0-7]) after 24 months of treatment. The most frequently reported adverse event was upper respiratory tract infection, in 22 infants (54%); the most common serious adverse events were pneumonia in 16 infants (39%) and respiratory distress in three infants (7%). INTERPRETATION: Treatment with risdiplam over 24 months resulted in continual improvements in motor function and achievement of developmental motor milestones. The FIREFISH open-label extension phase will provide additional evidence regarding long-term safety and efficacy of risdiplam. FUNDING: F Hoffmann-La Roche.

Natural history of Type 1 spinal muscular atrophy: a retrospective, global, multicenter study.

BACKGROUND: ANCHOVY was a global, multicenter, chart-review study that aimed to describe the natural history of Type 1 spinal muscular atrophy (SMA) from a broad geographical area and provide further contextualization of results from the FIREFISH (NCT02913482) interventional study of risdiplam treatment in Type 1 SMA. METHODS: Data were extracted from medical records of patients with first symptoms attributable to Type 1 SMA between 28 days and 3 months of age, genetic confirmation of SMA, and confirmed survival of motor neuron 2 copy number of two or unknown. The study period started on 1 January 2008 for all sites; study end dates were site-specific due to local treatment availabilities. Primary endpoints were time to death and/or permanent ventilation and proportion of patients achieving motor milestones. Secondary endpoints included time to initiation of respiratory and feeding support. RESULTS: Data for 60 patients from nine countries across Asia, Europe and North and South America were analyzed. The median age (interquartile range [IQR]) for reaching death or permanent ventilation was ~ 7.3 (5.9-10.5) months. The median age (IQR) at permanent ventilation was ~ 12.7 (6.9-16.4) months and at death was ~ 41.2 (7.3-not applicable) months. No patients were able to sit without support or achieved any level of crawling, standing or walking. INTERPRETATION: Findings from ANCHOVY were consistent with published natural history data on Type 1 SMA demonstrating the disease's devastating course, which markedly differed from risdiplam-treated infants (FIREFISH Part 2). The results provide meaningful additions to the literature, including a broader geographical representation.

Risdiplam-Treated Infants with Type 1 Spinal Muscular Atrophy versus Historical Controls.

BACKGROUND: Type 1 spinal muscular atrophy (SMA) is a progressive neuromuscular disease characterized by an onset at 6 months of age or younger, an inability to sit without support, and deficient levels of survival of motor neuron (SMN) protein. Risdiplam is an orally administered small molecule that modifies SMN2 pre-messenger RNA splicing and increases levels of functional SMN protein in blood. METHODS: We conducted an open-label study of risdiplam in infants with type 1 SMA who were 1 to 7 months of age at enrollment. Part 1 of the study (published previously) determined the dose to be used in part 2 (reported here), which assessed the efficacy and safety of daily risdiplam as compared with no treatment in historical controls. The primary end point was the ability to sit without support for at least 5 seconds after 12 months of treatment. Key secondary end points were a score of 40 or higher on the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND; range, 0 to 64, with higher scores indicating better motor function), an increase of at least 4 points from baseline in the CHOP-INTEND score, a motor-milestone response as measured by Section 2 of the Hammersmith Infant Neurological Examination (HINE-2), and survival without permanent ventilation. For the secondary end points, comparisons were made with the upper boundary of 90% confidence intervals for natural-history data from 40 infants with type 1 SMA. RESULTS: A total of 41 infants were enrolled. After 12 months of treatment, 12 infants (29%) were able to sit without support for at least 5 seconds, a milestone not attained in this disorder. The percentages of infants in whom the key secondary end points were met as compared with the upper boundary of confidence intervals from historical controls were 56% as compared with 17% for a CHOP-INTEND score of 40 or higher, 90% as compared with 17% for an increase of at least 4 points from baseline in the CHOP-INTEND score, 78% as compared with 12% for a HINE-2 motor-milestone response, and 85% as compared with 42% for survival without permanent ventilation (P<0.001 for all comparisons). The most common serious adverse events were pneumonia, bronchiolitis, hypotonia, and respiratory failure. CONCLUSIONS: In this study involving infants with type 1 SMA, risdiplam resulted in higher percentages of infants who met motor milestones and who showed improvements in motor function than the percentages observed in historical cohorts. Longer and larger trials are required to determine the long-term safety and efficacy of risdiplam in infants with type 1 SMA. (Funded by F. Hoffmann-La Roche; FIREFISH ClinicalTrials.gov number, NCT02913482.).

Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries.

OBJECTIVE: Genetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries. METHODS: We performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers. RESULTS: We identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations. CONCLUSIONS: Our data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene.

Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.

MRI in sarcoglycanopathies: a large international cohort study.

OBJECTIVES: To characterise the pattern and spectrum of involvement on muscle MRI in a large cohort of patients with sarcoglycanopathies, which are limb-girdle muscular dystrophies (LGMD2C-2F) caused by mutations in one of the four genes coding for muscle sarcoglycans. METHODS: Lower limb MRI scans of patients with LGMD2C-2F, ranging from severe childhood variants to milder adult-onset forms, were collected in 17 neuromuscular referral centres in Europe and USA. Muscle involvement was evaluated semiquantitatively on T1-weighted images according to a visual score, and the global pattern was assessed as well. RESULTS: Scans from 69 patients were examined (38 LGMD2D, 18 LGMD2C, 12 LGMD2E and 1 LGMD2F). A common pattern of involvement was found in all the analysed scans irrespective of the mutated gene. The most and earliest affected muscles were the thigh adductors, glutei and posterior thigh groups, while lower leg muscles were relatively spared even in advanced disease. A proximodistal gradient of involvement of vasti muscles was a consistent finding in these patients, including the most severe ones. CONCLUSIONS: Muscle involvement on MRI is consistent in patients with LGMD2C-F and can be helpful in distinguishing sarcoglycanopathies from other LGMDs or dystrophinopathies, which represent the most common differential diagnoses. Our data provide evidence about selective susceptibility or resistance to degeneration of specific muscles when one of the sarcoglycans is deficient, as well as preliminary information about progressive involvement of the different muscles over time.

Skeletal Muscle Quantitative Nuclear Magnetic Resonance Imaging and Spectroscopy as an Outcome Measure for Clinical Trials.

Recent years have seen tremendous progress towards therapy of many previously incurable neuromuscular diseases. This new context has acted as a driving force for the development of novel non-invasive outcome measures. These can be organized in three main categories: functional tools, fluid biomarkers and imagery. In the latest category, nuclear magnetic resonance imaging (NMRI) offers a considerable range of possibilities for the characterization of skeletal muscle composition, function and metabolism. Nowadays, three NMR outcome measures are frequently integrated in clinical research protocols. They are: 1/ the muscle cross sectional area or volume, 2/ the percentage of intramuscular fat and 3/ the muscle water T2, which quantity muscle trophicity, chronic fatty degenerative changes and oedema (or more broadly, "disease activity"), respectively. A fourth biomarker, the contractile tissue volume is easily derived from the first two ones. The fat fraction maps most often acquired with Dixon sequences have proven their capability to detect small changes in muscle composition and have repeatedly shown superior sensitivity over standard functional evaluation. This outcome measure will more than likely be the first of the series to be validated as an endpoint by regulatory agencies. The versatility of contrast generated by NMR has opened many additional possibilities for characterization of the skeletal muscle and will result in the proposal of more NMR biomarkers. Ultra-short TE (UTE) sequences, late gadolinium enhancement and NMR elastography are being investigated as candidates to evaluate skeletal muscle interstitial fibrosis. Many options exist to measure muscle perfusion and oxygenation by NMR. Diffusion NMR as well as texture analysis algorithms could generate complementary information on muscle organization at microscopic and mesoscopic scales, respectively. 31P NMR spectroscopy is the reference technique to assess muscle energetics non-invasively during and after exercise. In dystrophic muscle, 31P NMR spectrum at rest is profoundly perturbed, and several resonances inform on cell membrane integrity. Considerable efforts are being directed towards acceleration of image acquisitions using a variety of approaches, from the extraction of fat content and water T2 maps from one single acquisition to partial matrices acquisition schemes. Spectacular decreases in examination time are expected in the near future. They will reinforce the attractiveness of NMR outcome measures and will further facilitate their integration in clinical research trials.