Characterization of severe COL6-related dystrophy due to the recurrent variant COL6A1 c.930+189C>T

Abstract

Collagen VI-related dystrophies manifest with a spectrum of clinical phenotypes, ranging from Ullrich congenital muscular dystrophy (UCMD), presenting with prominent congenital symptoms and characterized by progressive muscle weakness, joint contractures and respiratory insufficiency, to Bethlem muscular dystrophy, with milder symptoms typically recognized later and at times resembling a limb girdle muscular dystrophy, and intermediate phenotypes falling between UCMD and Bethlem muscular dystrophy. Despite clinical and muscle pathology features highly suggestive of collagen VI-related dystrophy, some patients had remained without an identified causative variant in COL6A1, COL6A2 or COL6A3. With combined muscle RNA sequencing and whole-genome sequencing, we uncovered a recurrent, de novo deep intronic variant in intron 11 of COL6A1 (c.930+189C>T) that leads to a dominantly acting in-frame pseudoexon insertion. We subsequently identified and have characterized an international cohort of 44 patients with this COL6A1 intron 11 causative variant, one of the most common recurrent causative variants in the collagen VI genes. Patients manifest a consistently severe phenotype characterized by a paucity of early symptoms followed by an accelerated progression to a severe form of UCMD, except for one patient with somatic mosaicism for this COL6A1 intron 11 variant who manifests a milder phenotype consistent with Bethlem muscular dystrophy. Partial amelioration of the disease phenotype in this individual provides a strong rationale for the development of our pseudoexon skipping therapy to successfully suppress the pseudoexon insertion, resulting in normal COL6A1 transcripts. We have previously shown that splice-modulating antisense oligomers applied in vitro effectively decreased the abundance of the mutant pseudoexon-containing COL6A1 transcripts to levels comparable to the in vivo scenario of the somatic mosaicism shown here, indicating that this therapeutic approach carries significant translational promise for ameliorating the severe form of UCMD caused by this common recurrent COL6A1 variant.

Keywords: COL6A1 c.930+189C>T; COL6A1 intron 11; collagen VI-related dystrophy; pseudoexon; splice-modulating; translational promise.

Figure 1

Clinical phenotype of patients heterozygous for the COL6A1 c.930+189C>T variant versus patients with classical UCMD (UCMD patients who do not have the COL6A1 c.930+189C>T variant). (A) Bar graph demonstrating symptoms at birth in patients with the classical UCMD phenotype (blue) versus the COL6A1 intron 11 phenotype (green). (B) Box plot demonstrating distribution of ages at the time of major motor function and pulmonary function milestones. Onset of independent ambulation: (classical UCMD phenotype, n = 17; blue): box range 1.3–2.0 years; whiskers: 1.0–3.0 years. Onset of independent ambulation (COL6A1 intron 11 phenotype, n = 40; green): box range: 1.2–1.5 years; whiskers: 0.9–2.0 years. Loss of ambulation (classical UCMD phenotype, n = 15; blue): box range: 5.3–10.0 years; whiskers: 4.0–11.0 years. Loss of ambulation (COL6A1 intron 11 phenotype, n = 34; green): box range: 6–10.3 years; whiskers: 3.5–14 years. Onset of non-invasive ventilation (classical UCMD phenotype, n = 12; blue): box range: 7.3–11.0 years; whiskers: 5.0–13.0 years. Onset of non-invasive ventilation (COL6A1 intron 11 phenotype, n = 31; green): box range: 9.0–14.0 years; whiskers: 5–21 years. Asterisks indicate significance at the 0.05 level for Mann–Whitney U-tests. (C) Kaplan–Meier curve depicting independent ambulation in patients with classical UCMD (blue) and patients with COL6A1 intron 11 (green) (P = 0.04). (D) Kaplan–Meier curve depicting ventilation-free status in patients with classical UCMD (blue) and patients with COL6A1 intron 11 (green) (P = 0.003). NIV = non-invasive ventilation; UCMD = Ullrich congenital muscular dystrophy.

Figure 2

Joint contractures in heterozygous patients versus a patient with somatic mosaicism for COL6A1 c.930+189C>T. (A) Mild elbow contractures already noticeable at 3.5 years of age in Patient US1. (B) Severe long finger flexor contractures at age 10 years in Patient IR1. (C) Severe wrist flexion and long finger flexor contractures at age 15 years in Patient US9. (D) Severe elbow, wrist flexion and long finger flexor contractures at age 24 years in Patient US5. (E) Severe elbow contracture (>90° in elbow flexion) at age 29 years in Patient US2. (F) Joint hyperlaxity of the elbow at age 9 years in Patient US16, who has somatic mosaicism for COL6A1 c.930+189C>T.

Figure 3

Muscle MRI and ultrasound in a heterozygous patient versus a patient with somatic mosaicism for COL6A1 c.930+189C>T. (A) Axial TI-weighted MRI of the upper leg in Patient US10 (heterozygous for COL6A1 c.930+189C>T) at age 8 years demonstrating abnormal T1-weighted signal, consistent with a ‘central cloud’ pattern in the rectus femoris muscle (black arrows) and an ‘outside-in’ pattern in the vastus lateralis muscle (white arrowheads). (B) Ultrasound of the rectus femoris muscle in Patient US10 at age 8 years, demonstrating increased echogenicity, consistent with a ‘central cloud’ pattern (white arrow) with a loss of bone echogenicity. (C) Ultrasound of the vastus lateralis muscle in Patient US10 at age 8 years, demonstrating increased echogenicity with a loss of bone echogenicity. (D) Axial TI-weighted MRI of the upper leg in Patient US16 (with somatic mosaicism for COL6A1 c.930+189C>T) at age 9 years, demonstrating mildly abnormal signal in the vastus lateralis muscle suggestive of a subtle ‘outside-in’ pattern (black arrowheads). (E) Ultrasound of the rectus femoris muscle in Patient US16 at 9 years of age, demonstrating an increase in echogenicity of the rectus femoris muscle consistent with a ‘central cloud’ pattern (white arrow) with bone echogenicity (asterisk) preserved. (F) Ultrasound of the vastus lateralis muscle in Patient US16 at 9 years of age, demonstrating an increase in echogenicity of the vastus lateralis with bone echogenicity (asterisk) preserved. RF = rectus femoris; VL = vastus lateralis.

Figure 4

Muscle immunofluorescence. Confocal imaging of muscle co-stained with collagen VI (red) and basement membrane marker laminin (green) along with the nuclear stain DAPI (blue). (A) In control muscle, collagen VI is co-localized with laminin at the basement membrane. (B) In the muscle of Patient US1 from a biopsy performed at age 2 years, collagen VI signal is observed in the interstitial space, indicative of collagen VI mislocalization relative to the basement membrane. (Magnification = ×63; scale bars = 75 µm).

Figure 5

Somatic mosaicism for COL6A1 c.930+189C>T. (A) Genomic DNA sequencing chromatograms at the COL6A1 c.930+189C>T locus, showing comparable peak heights for the cytosine and thymine alleles in a patient heterozygous for COL6A1 c.930+189C>T (Patient CA1), but a higher cytosine peak height compared with thymine in the patient mosaic for COL6A1 c.930+189C>T (Patient US16), in various tissue samples (skin fibroblasts, skin biopsy and blood). (B) Determination of the degree of mosaicism was achieved by droplet digital PCR quantification using a genotyping probe assay and genomic DNA as input. The graph shows the fractional abundance of the thymine (‘T’) allele, calculated as the ratio of ‘T’ concentration (in copies per microlitre) over total (‘T’ + ‘C’) concentration (in copies per microlitre). Error bars represent the Poisson confidence interval. [The heterozygous patients were Patient HK1 (blood) and Patient US12 (skin fibroblasts).] (C) RNA isolated from skin biopsies or from skin-derived primary fibroblasts was reverse transcribed and amplified with primers spanning COL6A1 exons 10–20. In Patient US16 (mosaic for COL6A1 c.930+189C>T), the upper band (transcripts with pseudoexon) appears fainter than in Patient US12 and Patient US5 (heterozygous for COL6A1 c.930+189C>T). (D) Relative expression of COL6A1 with pseudoexon transcripts normalized to total COL6A1 levels in skin fibroblasts and skin biopsy of the patient mosaic for COL6A1 c.930+189C>T (Patient US16), compared with two patients heterozygous for COL6A1 c.930+189C>T (Patient US5 and Patient US12). A fresh skin biopsy was not available for Patient US12.

Figure 6

Comparison of the COL6A1 intron 11 phenotype and the classical UCMD phenotype. (A) Schematic diagram of the natural history of the motor and pulmonary function in patients with the classical UCMD phenotype in the comparison cohort (n = 17). Natural history of pulmonary function data from the largest published international natural history study of patients with UCMD (n = 75) is included (dotted line). (B) Schematic diagram of the natural history of motor and pulmonary function in this cohort of patients who are heterozygous for COL6A1 c.930+189C>T (n = 43). UCMD = Ullrich congenital muscular dystrophy. Created in BioRender. Or Bach, R. (2025) https://BioRender.com/r9a5g60.