Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes

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TL;DR

This study of 15 COL6-related myopathy patients identified 14 pathogenic variants, including three novel ones, and found that combining genetic analysis with muscle biopsy histology, immunohistochemistry, and electron microscopy is essential for diagnosis. Patients showed a wide clinical spectrum from severe Ullrich congenital muscular dystrophy to milder Bethlem myopathy, with ultrastructural findings of sarcolemmal microvilli-like projections and basal lamina alterations. The results highlight the importance of a multimodal diagnostic approach to validate genetic variants and understand the pathogenesis of these disorders.

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**Background:** Collagen VI is a heterotrimeric protein expressed in the extracellular matrix (ECM) of various tissues, including skeletal muscle, where it links the cytoskeleton to the ECM and maintains cell integrity. Mutations in COL6A1, COL6A2, and COL6A3 cause COL6-related myopathies (COL6-RMs), primarily Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), with a phenotypic continuum. Diagnosis is challenging due to clinical heterogeneity and uncertain genetic variants. This study aimed to analyze clinical, genetic, and pathological features in a cohort of COL6-RM patients to improve diagnostic accuracy.

**Methods:** The study included 15 COL6-mutated patients (5 isolated, 10 from 4 families) from a cohort of 53 genetically undiagnosed subjects with progressive muscle weakness. Clinical data, including age of onset, severity, and respiratory/cardiac function, were collected. Genetic analysis was performed using next-generation sequencing (NGS) and whole-exome sequencing (WES), with variants validated by Sanger sequencing and in silico prediction tools. Muscle biopsies from 9 patients were analyzed by histology (H&E), immunohistochemistry (collagen VI and perlecan double staining), and electron microscopy (EM). Skin fibroblasts from 8 patients were studied by immunofluorescence for collagen VI deposition. Western blotting was performed on muscle homogenates from 4 patients.

**Key Results:** The cohort had a mean age at last evaluation of 46 ± 16 years. Eight patients had severe childhood-onset (mean age 4.8 ± 2.9 years) with early tendon retractions and proximal weakness; two lost ambulation at 5 and 10 years. Seven patients had milder adult-onset presentations. Additional features included rigid spine (4 patients), myopathic face with ptosis (3), and respiratory involvement (6 patients, severe in 2). CPK levels were mildly elevated (2-10×) in most, but normal in 3 and up to 60× in one. Cardiac function was normal in all.

Genetic analysis identified 14 different heterozygous variants: 3 in COL6A1, 7 in COL6A2, and 4 in COL6A3. Four variants were novel: COL6A1 c.628C>G (p.Arg210Gly), COL6A2 c.1806C>G (p.Cys602Trp), c.2145C>G (p.Ile715Met), and c.2738_2740del (p.Ser913del). Variants included 10 missense, 1 in-frame deletion, and 3 splice-site alterations. Two variants in the triple-helix domain (COL6A1 c.958-2A>G and c.930+189C>T) were associated with severe phenotypes. Most variants (8/14) were in von Willebrand Factor A domains.

Muscle biopsy histology showed marked fiber size variability, centronuclear fibers, and variable fibrosis. Pediatric patients had significantly increased fibrotic tissue compared to age-matched controls: Pt 1 (27.72 ± 6.52 vs. 9.43 ± 2.14, p<0.0001), Pt 2.1 (39.73 ± 4.08, p<0.0001), Pt 4 (39.92 ± 8.12, p<0.0001), Pt 6 (18.14 ± 5.28, p<0.0001). Adult patients also showed increased fibrosis: Pt 5 (12.91 ± 0.61 vs. 9.11 ± 1.69, p=0.0003), Pt 7.1 (14.31 ± 3.92, p<0.0002). No patient had complete loss of collagen VI; immunohistochemistry showed irregular membrane staining with subtle signal reduction in some, and perimysial signal increase in others. Electron microscopy revealed sarcolemmal microvilli-like projections (4/6 patients), basal lamina splitting or replication, increased collagen fibrils, and subsarcolemmal vacuoles (3 patients). Fibroblast immunofluorescence showed variable collagen VI retention (Pt 1) or altered extracellular deposition (Pt 2 with globular speckled pattern). Western blot showed reduced collagen VI bands in Pt 1 and Pt 2, normal in Pt 3 and Pt 4.1.

**Clinical Implications:** The study underscores the diagnostic complexity of COL6-RMs, where genetic variants alone may be insufficient due to uncertain pathogenicity. Combining NGS with morphological techniques—histology, immunohistochemistry, and electron microscopy on muscle biopsy and skin fibroblasts—is crucial for validating variants and understanding disease mechanisms. The ultrastructural findings of sarcolemmal alterations and ECM disorganization provide insights into pathogenesis, suggesting that mutated collagen VI disrupts ECM integrity, leading to membrane instability and compensatory changes. This multimodal approach improves diagnostic accuracy and may guide future therapeutic strategies targeting ECM stabilization.