Neuromuscular disorders. Deep molecular and phenotypic characterization of dystrophinopathies

The overarching aim of this thesis was to provide an integrated and detailed characterisation of the clinical and biochemical spectrum of dystrophinopathies, with four complementary objectives: to define the variability of phenotype in a large Becker muscular dystrophy (BMD) cohort and predictors of clinical severity; to investigate respiratory trajectories and predictors of decline in BMD; to explore the role of dystrophin and utrophin quantification as prognostic biomarkers of disease severity in males with dystrophinopathy; and to delineate the clinical and molecular features of heterozygous females carrying DMD gene variants. The first study retrospectively analysed clinical records of 163 male patients with genetically confirmed BMD followed in Newcastle upon Tyne, United Kingdom, over four decades. The cohort spanned ages 1.4 to 86.3 years, with a mean age at last assessment of 33.2 years. The majority carried large DMD deletions (78%), followed by duplications and small variants (11% each). Initial diagnosis was prompted by skeletal muscle symptoms in just over half of cases. Loss of ambulation (LoA) occurred in 23% of patients, at a mean age of 42.2 years (range 11.2–77.6), and cardiac involvement was identified in 52.3%. Neuropsychiatric issues were noted in 44.2% of patients, though only 18.4% received a formal diagnosis. The second study focused on respiratory outcomes, using 1,360 longitudinal assessments of forced vital capacity (FVC) from 152 patients from the Newcastle BMD cohort (mean follow-up for each patient: 11 years). Respiratory decline was generally slow, with no significant impairment observed in patients younger than 18 years. LoA emerged as a strong predictor of faster decline in FVC percentage of predicted (FVC%) (estimate –0.58%/year, p=0.002). Respiratory support was infrequent (11.2%), always non-invasive, and nocturnal only. The third study examined dystrophin and utrophin levels in skeletal muscle biopsies from 67 male patients with dystrophinopathy followed in Padova, Italy, using targeted mass spectrometry, in collaboration with Binghamton University, United States. All patients with Duchenne muscular dystrophy (DMD) had absent dystrophin, whereas patients with BMD exhibited a wide range (0–110.7%) of dystrophin expression. Individuals carrying in-frame DMD variants at the mildest end of the dystrophinopathy spectrum (i.e., no muscle weakness), showed higher dystrophin expression (up to 166.2%). Higher dystrophin levels were protective against LoA (HR=0.91, p<0.0001), while increased utrophin levels in DMD were associated with earlier LoA (HR=1.003, p=0.038), suggesting utrophin may reflect disease severity without providing functional benefit. Dystrophin quantity correlated closely with prognosis as defined by the underlying DMD gene variant. Specifically, when using patients with the most frequent BMD-associated variants (deletions of exons 45–47 and 45–48) as the reference, higher dystrophin expression was observed in patients with variants previously associated with milder BMD phenotypes, whereas significantly lower levels were detected in BMD patients carrying truncating variants. The fourth study investigated 47 heterozygous females with DMD gene variants followed up in Padova, 43% of whom exhibited muscle weakness and 20% had cardiac involvement. Creatine kinase (CK) levels were normal in a significant proportion of both symptomatic (~20%) and asymptomatic (~46%) carriers, limiting its diagnostic sensitivity. X-chromosome inactivation patterns and common genetic modifiers (SPP1, LTBP4, CD40) did not associate with clinical severity. Taken together, these four studies offer a broad and integrated overview of the spectrum of dystrophinopathies. These findings have direct implications for patient care, support the development of international standards of care and the design and interpretation of current and future clinical trials in dystrophinopathies.