Longitudinal evaluation and genotype-phenotype correlation in LGMDR1

INTRODUCTION. Limb–girdle muscular dystrophy R1 (LGMDR1) is a rare autosomal recessive neuromuscular disorder caused by biallelic mutations in the CAPN3 gene, encoding calpain-3, a muscle-specific, calcium-dependent, non-lysosomal protease. The disease presents with proximal muscle weakness, scapular winging, hyperlordosis, and joint contractures. Although muscle biopsy findings and Western Blot (WB) quantification of calpain-3 may support the diagnosis, genetic testing remains the gold standard. No specific therapies are currently available, although several preclinical studies are ongoing. AIMS OF THE STUDY. The aims were: (1) to clinically and molecularly characterize LGMDR1 patients followed at our Neuromuscular Centre; (2) to assess genotype–phenotype correlations in our historical cohort; and (3) to describe 9-year progression of muscle weakness using validated functional tests, in order to identify sensitive and reliable predictors of disease progression for future clinical trials. MATERIALS AND METHODS. Ninety-four patients with molecularly confirmed LGMDR1 (two pathogenic CAPN3 variants) were selected from our database (“retrospective cohort”). Genetic data and WB calpain-3 levels were collected. For 77 patients, age at last neurological evaluation and functional status were also available. Thirty patients underwent longitudinal assessment over 9 years (“cross-sectional” and “longitudinal” cohorts). Natural history data were recorded, including age at symptom onset and age at loss of the ability to rise from the floor, run, or walk. All patients underwent a standardized evaluation protocol comprising Manual Muscle Testing (MMT), North Star Ambulatory Assessment (NSAA), timed functional tests (TFTs: stair climbing, rising from the floor, 10-meter walk), and Performance of Upper Limb (PUL). Assessments were performed at baseline, 2 years, and 9 years. Patients were stratified by WB protein levels, presence of null variants, and variants located in the autolytic domain (exon 11). RESULTS. In the retrospective cohort, an inverse correlation emerged between the number of null variants and WB protein levels: calpain-3 was absent or detectable only in traces in homozygous null patients, whereas levels were variable among carriers of missense variants (p = 0.025). Reduced or absent protein was associated with greater clinical severity (p < 0.05). In the cross-sectional cohort, earlier loss of ambulation significantly correlated with reduced WB protein levels (p = 0.013). MMT revealed a heterogeneous phenotypic spectrum, ranging from asymptomatic or mildly symptomatic presentations to severe limb-girdle weakness, with intermediate forms showing predominant scapulo-brachial involvement. In the longitudinal cohort, patients were analyzed according to age, number of null alleles, exon-11 pathogenic variants, WB protein levels, and sex. Total NSAA and PUL scores declined by 0.30 and 0.50 points per year, respectively (p > 0.05). NSAA declined rapidly until around age 40, then stabilized, whereas PUL declined linearly across all ages. WB protein levels negatively predicted NSAA, PUL, and TFT performance. Exon-11 pathogenic variants were negative predictors of PUL scores. The distal PUL domain remained relatively stable with age. DISCUSSION. Calpain-3 expression was absent or nearly absent in homozygous null patients, whereas expression varied in those with missense variants. Calpain-3 levels represented the strongest predictor of disease severity and were associated with earlier loss of ambulation when reduced or absent. Functional assessments confirmed broad phenotypic variability, encompassing mild to severe forms. Longitudinal data demonstrated progressive decline across functional measures. NSAA was reliable in ambulant patients, while PUL was sensitive across the entire severity range. Preserved calpain-3 expression predicted a slower disease trajectory.