Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD)
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 <...
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MDPI AG
2024-02-01
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| Online Access: | https://www.mdpi.com/2073-4409/13/4/329 |
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| author | Sara Aguti Gian Nicola Gallus Silvia Bianchi Simona Salvatore Anna Rubegni Gianna Berti Patrizia Formichi Nicola De Stefano Alessandro Malandrini Diego Lopergolo |
| author_facet | Sara Aguti Gian Nicola Gallus Silvia Bianchi Simona Salvatore Anna Rubegni Gianna Berti Patrizia Formichi Nicola De Stefano Alessandro Malandrini Diego Lopergolo |
| author_sort | Sara Aguti |
| collection | DOAJ |
| description | 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 <i>CAPN3</i> 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 <i>CAPN3</i> isoform. Methods: We analyzed <i>CAPN3</i> 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 <i>CAPN3</i> was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL. Results: Among our patients, seven different <i>CAPN3</i> mutations were detected, of which two were novel. After sequencing <i>CAPN3</i> transcripts from fibroblast and urine, we detected different <i>CAPN3</i> isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel <i>CAPN3</i> 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 <i>CAPN3</i> 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. |
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| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-07T22:37:24Z |
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| spelling | doaj.art-2885ad79e6d1491eb0ea11c7ce31409b2024-02-23T15:11:54ZengMDPI AGCells2073-44092024-02-0113432910.3390/cells13040329Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD)Sara Aguti0Gian Nicola Gallus1Silvia Bianchi2Simona Salvatore3Anna Rubegni4Gianna Berti5Patrizia Formichi6Nicola De Stefano7Alessandro Malandrini8Diego Lopergolo9Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyMolecular Medicine for Neurodegenerative and Neuromuscular Disease Unit, IRCCS Stella Maris Foundation, 56128 Pisa, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyDepartment of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, ItalyObjective: 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 <i>CAPN3</i> 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 <i>CAPN3</i> isoform. Methods: We analyzed <i>CAPN3</i> 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 <i>CAPN3</i> was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL. Results: Among our patients, seven different <i>CAPN3</i> mutations were detected, of which two were novel. After sequencing <i>CAPN3</i> transcripts from fibroblast and urine, we detected different <i>CAPN3</i> isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel <i>CAPN3</i> 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 <i>CAPN3</i> 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.https://www.mdpi.com/2073-4409/13/4/329<i>CAPN3</i>limb girdle muscular dystrophywestern blotting analysisRNA |
| spellingShingle | Sara Aguti Gian Nicola Gallus Silvia Bianchi Simona Salvatore Anna Rubegni Gianna Berti Patrizia Formichi Nicola De Stefano Alessandro Malandrini Diego Lopergolo Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD) Cells <i>CAPN3</i> limb girdle muscular dystrophy western blotting analysis RNA |
| title | Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD) |
| title_full | Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD) |
| title_fullStr | Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD) |
| title_full_unstemmed | Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD) |
| title_short | Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD) |
| title_sort | novel biomarkers for limb girdle muscular dystrophy lgmd |
| topic | <i>CAPN3</i> limb girdle muscular dystrophy western blotting analysis RNA |
| url | https://www.mdpi.com/2073-4409/13/4/329 |
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