Diego Lopergolo

Objective: To identify novel biomarkers as an alternative diagnostic tool for limb girdle muscular dystrophy (LGMD). Background: LGMD encompasses a group of muscular dystrophies characterized by proximal muscles weakness, elevated CK levels and dystrophic findings on muscle biopsy. Heterozygous CAPN3 mutations are associated with autosomal dominant LGMD-4, while biallelic mutations can cause autosomal recessive LGMD-1. Diagnosis is currently often based on invasive methods requiring muscle biopsy or blood tests. In most cases Western blotting (WB) analysis from muscle biopsy is essential for a diagnosis, as muscle samples are currently the only known tissues to express the full-length CAPN3 isoform. Methods: We analyzed CAPN3 in a cohort including 60 LGMD patients. Selected patients underwent a complete neurological examination, electromyography, muscle biopsy, and skin biopsies for primary fibroblasts isolation. The amount of CAPN3 was evaluated by WB analysis in muscle and skin tissues. The total RNA isolated from muscle, fibroblast and urine was processed, and cDNA was used for qualitative analysis. The expression of CAPN3 was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL. Results: Among our patients, seven different CAPN3 mutations were detected, of which two were novel. After sequencing CAPN3 transcripts from fibroblast and urine, we detected different CAPN3 isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel CAPN3 mutation showed a 30% reduction in protein compared to controls from both tissues. Conclusions: Our findings showed for the first time the presence of the CAPN3 full-length transcript in urine and skin samples. Moreover, we demonstrated surprisingly comparable CAPN3 protein levels between muscle and skin samples, thus allowing us to hypothesize the use of skin biopsy and probably of urine samples as an alternative less invasive method to assess the amount of CAPN3 when molecular diagnosis turns out to be inconclusive.

Pathogenic loss-of-function variants in the IQ motif and SEC7 domain containing protein 2 (IQSEC2) gene cause intellectual disability with Rett syndrome (RTT)-like features. The aim of this study was to obtain systematic information on the natural history and extra-central nervous system (CNS) manifestations for the Italian IQSEC2 population (>90%) by using structured family interviews and semi-quantitative questionnaires. IQSEC2 encephalopathy prevalence estimate was 7.0 to 7.9 × 10−7. Criteria for typical RTT were met in 42.1% of the cases, although psychomotor regression was occasionally evidenced. Genetic diagnosis was occasionally achieved in infancy despite a clinical onset before the first 24 months of life. High severity in both the CNS and extra-CNS manifestations for the IQSEC2 patients was documented and related to a consistently adverse quality of life. Neurodevelopmental delay was diagnosed before the onset of epilepsy by 1.8 to 2.4 years. An earlier age at menarche in IQSEC2 female patients was reported. Sleep disturbance was highly prevalent (60 to 77.8%), with mandatory co-sleeping behavior (50% of the female patients) being related to de novo variant origin, younger age, taller height with underweight, better social interaction, and lower life quality impact for the family and friends area. In conclusion, the IQSEC2 encephalopathy is a rare and likely underdiagnosed developmental encephalopathy leading to an adverse life quality impact.