The recurrent deep intronic pseudoexon-inducing variant COL6A1 c.930+189C>T results in a consistently severe phenotype of COL6-related dystrophy: Towards clinical trial readiness for splice-modulating therapy

Abstract

Collagen VI-related dystrophies (COL6-RDs) manifest with a spectrum of clinical phenotypes, ranging from Ullrich congenital muscular dystrophy (UCMD), presenting with prominent congenital symptoms and characterised by progressive muscle weakness, joint contractures and respiratory insufficiency, to Bethlem muscular dystrophy, with milder symptoms typically recognised later and at times resembling a limb girdle muscular dystrophy, and intermediate phenotypes falling between UCMD and Bethlem muscular dystrophy. Despite clinical and immunohistochemical features highly suggestive of COL6-RD, 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 characterised an international cohort of forty-four 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 characterised 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. Characterisation of this individual provides a robust validation for the development of our pseudoexon skipping therapy. 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 causative variant to a Bethlem muscular dystrophy phenotype.

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 (green) phenotype (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; whiskers: 1.0 – 3.0. Onset of independent ambulation (COL6A1 Intron 11 phenotype, n = 40) (green): box range: 1.2 – 1.5; whiskers: 0.9 – 2.0 years. Loss of ambulation (Classical UCMD phenotype, n = 15) (blue): box range: 5.3 – 10.0; whiskers: 4.0 – 11.0. Loss of ambulation (COL6A1 Intron 11 phenotype, n = 28) (green): box range: 6 −10.75; whiskers: 3.5 – 14. Onset on non-invasive ventilation (Classical UCMD phenotype, n = 12) (blue): box range: 7.3 – 11.0; whiskers: 5.0 – 13.0. Onset on non-invasive ventilation (COL6A1 Intron 11 phenotype, n = 24) (green): box range: 9.5 – 14.75; whiskers: 5 – 21. 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 Intron 11 (green) (p=0.07) (D) Kaplan-Meier curve depicting ventilation-free status in patients with Classical UCMD (blue) and patients with Intron 11 (green) (p=0.0009)

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 MRI of the upper leg in patient US10 demonstrating abnormal signaling consistent with a ‘central cloud’ pattern in the rectus femoris (black arrows) and an ‘outside in’ pattern in the vastus lateralis (white arrowheads); (B) Ultrasound of the rectus femoris (RF) muscle in patient US10 demonstrating abnormal signaling consistent with a ‘central cloud’ pattern (white arrow) with a loss of bone echogenicity; (C) Ultrasound of the vastus lateralis (VL) muscle in patient US10 demonstrating increased echogenicity with a loss of bone echogenicity; (D) Axial TI MRI of the upper leg in patient US16 demonstrating mildly abnormal signaling in the vastus lateralis muscle suggestive of a subtle ‘outside in’ pattern (black arrowheads); (E) Ultrasound of the rectus femoris (RF) muscle in patient US16 demonstrating an increase in echogenicity of the rectus femoris (RF) muscle consistent with a ‘central cloud’ pattern (white arrow) with bone echogenicity (asterisk) preserved; (F) Ultrasound of the vastus lateralis (VL) muscle in patient US16 demonstrating an increase in echogenicity of the vastus lateralis (VL) with bone echogenicity (asterisk) preserved.

Figure 4:. Muscle Immunofluorescence

Confocal imaging of muscle co-stained with collagen VI (red) and basement membrane marker laminin (green) along with nuclear stain DAPI (blue). (A) In control muscle, collagen VI is co-localised with laminin at the basement membrane. (B) In the muscle of patient US1 from a biopsy, collagen VI signal is observed in the interstitial space, indicative of collagen VI mislocalisation relative to the basement membrane. (magnification = 63X; scale bar = 75μm)

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) (CA1), but a higher cytosine peak height compared to thymine in the patient mosaic for COL6A1 (c.930+189C>T) (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. Graph shows the fractional abundance of the thymine (“T”) allele, calculated as the ratio of “T” concentration (copies/uL) over total (“T” + “C”) concentration (copies/uL). 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 to 20. In patient US16 [mosaic for COL6A1 (c.930+189C>T)] the upper band (transcripts with pseudoexon) appears fainter than in US12 and US5 [heterozygous for COL6A1 (c.930+189C>T)]. (D) Relative expression of COL6A1 with pseudoexon transcripts normalised to total COL6A1 levels in skin fibroblasts and skin biopsy of the patient mosaic for COL6A1 (c.930+189C>T) (US16), compared to two patients heterozygous for COL6A1 (c.930+189C>T) (US5 and 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 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 included (N=75)² (dotted line). (B) Schematic 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)