Natural history of Becker muscular dystrophy: DMD gene mutations predict clinical severity.

BACKGROUND: Becker muscular dystrophy (BMD) is an X-linked neuromuscular disease due to mutations in the DMD gene, leading to a deficient and less functional dystrophin mainly in skeletal and cardiac muscle. Understanding the natural history of BMD is crucial for optimizing patient care and developing targeted treatments. MATERIALS AND METHODS: Retrospective data were collected from 943 patients diagnosed with BMD based on a combination of clinical, biochemical and genetic criteria followed by 17 Italian neuromuscular centers. Patients' demographics, main signs and symptoms at BMD onset, neuropsychiatric comorbidities, age at loss of ambulation (LoA), cardiac left ventricular ejection fraction (LVEF), pulmonary forced vital capacity (FVC), and DMD mutations were collected. Disease milestones were analysed in specific DMD mutational groups. RESULTS: the median age at the last assessment was 26.0 (16.6-41.9) years, with a median age at diagnosis of 7.5 (4.0-14.0) years. In 55% of patients, the diagnosis was prompted by the incidental finding of hyperCKemia. At the last assessment, 13.5% of patients had lost the ability to walk at a median age estimated by Kaplan-Meier analysis of 69 years. Thirty percent of patients exhibited left ventricular impairment and 2.7% respiratory involvement. Ten percent of patients carried out-of-frame mutations, 4% nonsense mutations, and 86% in-frame deletions/duplications. The subset of in-frame deletions was further classified based on the specific mutations. Patients carrying del45-49 compared to del45-47 were associated with an earlier LoA (P=1×10-4), where patients with del45-55 (P=.005), del48 (P=.02), and del48-49 (P=.02) correlated with a later LoA compared to del45-47. del45-55 (P=.002) and del48 (P=.003) were significantly associated with decreased odds of developing a pathological LVEF% compared to del45-47. CONCLUSION: Our results contribute to the better understanding of the natural history of BMD and capture precious data in the era of the emerging therapies. The knowledge of the specific DMD mutation may help to define a prognosis in a subset of BMD patients and will serve as a model for the design of future therapies.

Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable?

OBJECTIVE: The aim of this study was to provide an overview of the clinical phenotypes associated with 4 SMN2 copies. METHODS: Clinical phenotypes were analyzed in all the patients with 4 SMN2 copies as part of a nationwide effort including all the Italian pediatric and adult reference centers for spinal muscular atrophy (SMA). RESULTS: The cohort includes 169 patients (102 men and 67 women) with confirmed 4 SMN2 copies (mean age at last follow-up = 36.9 ± 19 years). Six of the 169 patients were presymptomatic, 8 were classified as type II, 145 as type III (38 type IIIA and 107 type IIIB), and 8 as type IV. The remaining 2 patients were asymptomatic adults identified because of a familial case. The cross-sectional functional data showed a reduction of scores with increasing age. Over 35% of the type III and 25% of the type IV lost ambulation (mean age = 26.8 years ± 16.3 SD). The risk of loss of ambulation was significantly associated with SMA type (p < 0.0001), with patients with IIIB and IV less likely to lose ambulation compared to type IIIA. There was an overall gender effect with a smaller number of women and a lower risk for women to lose ambulation. This was significant in the adult (p = 0.009) but not in the pediatric cohort (p = 0.43). INTERPRETATION: Our results expand the existing literature on natural history of 4 SMN2 copies confirming the variability of phenotypes in untreated patients, ranging from type II to type IV and an overall reduction of functional scores with increasing age. ANN NEUROL 2023;94:1126-1135.

Diaphragm Involvement in Duchenne Muscular Dystrophy (DMD): An MRI Study.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by progressive weakness and wasting of skeletal, cardiac, and respiratory muscles, with consequent cardiopulmonary failure as the main cause of death. Reliable outcome measures able to demonstrate specific trends over disease progression are essential. PURPOSE: To investigate MRI as a noninvasive imaging modality to assess diaphragm impairment in DMD. In particular, we sought to correlate MRI measurement of diaphragm structure and function with pulmonary function tests and with the abdominal volumes (VAB ) measured by optoelectronic plethysmography, being an index of the action of the diaphragm. STUDY TYPE: Cross-sectional study. POPULATION: Twenty-six DMD patients (17.9 ± 6.2 years) and 12 age-matched controls (17.8 ± 5.9 years). FIELD STRENGTH/SEQUENCE: 3-Point gradient echo Dixon sequence at 3T. ASSESSMENT: Images were acquired in breath-hold at full-expiration (EXP) and full-inspiration (INSP). INSP and EXP lung volumes were segmented and the diaphragm surface was reconstructed as the bottom surface of the left and the right lung. The inspiratory and the expiratory diaphragm surfaces were aligned by a nonrigid iterative closest point algorithm. On MRI we measured: 1) craniocaudal diaphragmatic excursion; 2) diaphragm fatty infiltration. STATISTICAL TESTS: Three-parameter sigmoid regression, one-way analysis of variance (ANOVA), Spearman's correlation. RESULTS: In patients, diaphragm excursion decreased with age (r2 = 0.68, P < 0.0001) and fat fraction increased (r2 = 0.51, P = 0.0002). In healthy subjects, diaphragm excursion and fat fraction had no relationship with age. Diaphragm excursion decreased with decreasing FEV1 %pred (r = 0.78, P < 0.0001) and FVC %pred (r = 0.76, P < 0.0001) and correlated with VAB (r = 0.60, P = 0.0002). Fatty infiltration increased with decreasing FEV1 %pred (r = -0.88, P < 0.0001) and FVC %pred (r = -0.88, P < 0.0001). DATA CONCLUSION: The progressive structural and functional diaphragm impairment is highly related to pulmonary function tests and to VAB . The results suggest that MRI might represent a new and noninvasive tool for the functional and structural assessment of the diaphragm. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2020;51:461-471.

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing.

BACKGROUND: Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements. METHODS: We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in DMD gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis. RESULTS: We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n=10 patients), followed by TAG (n=7) and TAA (n=4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the DMD gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65. CONCLUSION: The analysis of our patients' sample, carrying point mutations or complex rearrangements in DMD gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.

A novel acquisition platform for long-term breathing frequency monitoring based on inertial measurement units.

Continuous monitoring of breathing frequency (fB) could foster early prediction of adverse clinical effects and exacerbation of medical conditions. Current solutions are invasive or obtrusive and thus not suitable for prolonged monitoring outside the clinical setting. Previous studies demonstrated the feasibility of deriving fB by measuring inclination changes due to breathing using accelerometers or inertial measurement units (IMU). Nevertheless, few studies faced the problem of motion artifacts that limit the use of IMU-based systems for continuous monitoring. Moreover, few attempts have been made to move towards real portability and wearability of such devices. This paper proposes a wearable IMU-based device that communicates via Bluetooth with a smartphone, uploading data on a web server to allow remote monitoring. Two IMU units are placed on thorax and abdomen to record breathing-related movements, while a third IMU unit records body/trunk motion and is used as reference. The performance of the proposed system was evaluated in terms of long-acquisition-platform reliability showing good performances in terms of duration and data loss amount. The device was preliminarily tested in terms of accuracy in breathing temporal parameter measurement, in static condition, during postural changes, and during slight indoor activities showing favorable comparison against the reference methods (mean error breathing frequency < 5%). Graphical abstract Proof of concept of a wearable, wireless, modular respiratory Holter based on inertial measurement units (IMUS) for the continuous breathing pattern monitoring through the detection of chest wall breathing-related movements.

Ribcage deformity and the altered breathing pattern in children with osteogenesis imperfecta.

AIM: Osteogenesis Imperfecta (OI) is a genetic disease characterized by bones fragility and progressive deformity. Life expectancy is reduced in the non-lethal most severe type III form before the age of 10 years. The main cause of death in OI is respiratory insufficiency resulting from impaired thoracic function worsened by ribcage deformity and scoliosis. METHODS: We used opto-electronic plethysmography to study chest geometry, the ventilatory, and the thoraco-abdominal pattern at rest in supine position in children younger than 10 years. Radiographic measurements were used to describe spinal deformity. RESULTS: Eight severe OI (sOI), seven affected by other moderate forms (mOI), and nine healthy controls (CTR) were analyzed. sOI were characterized by Pectus carinatum (sternal angle: 165.2°, CTR: 183.1°; P < 0.01), rapid and shallow breathing (RSBi: 267.4 L-1 min-1 , CTR: 150.7 L-1 min-1 ; P < 0.05) and reduced pulmonary rib cage contribution to tidal volume (5.1%, CTR: 14.6%; P < 0.001) that evolved with age approaching the paradoxical inspiratory inward movement previously found in adults. mOI showed almost normal ventilatory pattern (RSBi: 189.2-1 min-1 ) and absence of sternal deformity (sternal angle: 176.8°). Platyspondyly and kyphosis were common features in all OI children. CONCLUSION: An altered breathing pattern in severe OI is present since childhood and it worsens with age. This is caused by the combination of pectus carinatum, brittle ribs and spinal deformity that put the ribcage muscles in mechanical disadvantage. These results suggest that in severe OI the assessment of the respiratory function should start in early childhood in order to try to reduce the incidence of premature death.

Acute Effects of Mechanical Insufflation-Exsufflation on the Breathing Pattern in Stable Subjects With Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by progressive degeneration, wasting, and weakness of skeletal musculature, including respiratory muscles. Cough is also compromised with disease progression. Among cough-augmentation techniques, mechanical insufflation-exsufflation (MI-E) has demonstrated several clinical benefits in patients with chronic airway secretion obstruction and muscular weakness. In clinical practice, the use of MI-E in DMD patients is also suggested when they are stable with no airway infections. However, there is a paucity of studies that consider the effect of MI-E specifically on stable DMD patients who have adapted to the use of MI-E. METHODS: Twenty subjects with DMD with no active upper airway or lung infections, who used MI-E device regularly at home, were enrolled. They received a single MI-E treatment consisting of 5 cycles of 5 insufflations-exsufflations with their customary settings. Volume variations during quiet breathing, vital capacity, and cough before and after treatment were measured with optoelectronic plethysmography (OEP). RESULTS: A decrease in breathing frequency (P = .001) and the rapid shallow breathing index emerged (P = .007), while cough peak flow (Spirometer P = .86, OEP P = .58), vital capacity (Spirometer P = .78, OEP total chest wall P = .57), and end-expiratory volumes (Total chest wall P = .97, Ribcage P = .14, Abdomen P = .10) were not affected by the treatment. An increment of the chest wall volume variation during the expiratory cough phase was identified (P = .001), particularly due to an increase in abdominal expansion (P = .005). CONCLUSIONS: A single treatment of MI-E in subjects with stable DMD already adapted to the device can provide beneficial changes in breathing pattern through a significant decrease in breathing frequency and rapid shallow breathing. These findings suggest an improvement in short-term dyspnea, although there were no changes in lung-volume recruitment or unassisted cough peak flow.

Angiogenesis in transgenic models of multistep angiogenesis.

The histopathology and the epidemiology of human cancers, as well as studies of animal models of tumorigenesis, have led to a widely accepted notion that multiple genetic and epigenetic changes have to accumulate for progression to malignancy. Formation of new blood vessels (tumor angiogenesis) has been recognized, in addition to proliferative capabilities and ability to down-modulate cell death (apoptosis), as essential for the progressive growth and expansion of solid tumors. Mice overexpressing activated forms of oncogenes or carrying targeted mutations in tumor suppressor genes have proven extremely useful for linking the function of these genes with specific tumor features such as continuous proliferation, escape from apoptosis, invasion and neo-angiogenesis. The interbreeding of these mice allows for studying the extent of cooperativity between different genetic lesions in disease progression, leading to a greater understanding of multi-stage nature of tumorigenesis.