We have elucidated the mutation profile of collagen VI-related myopathy in Japan (Table 1). Furthermore, we report 37 novel variants in 40 families, comprising 24 missense, six splicing, three small in-frame deletion, three large genomic deletion, and one nonsense. From the genetic information, we have established the mutation profile of the largest cohort at a single center as far as we are aware. The majority of the variants were mono-allelic (86%, 120/140), and 67% (94/140) of them were likely to be de novo because the parents of the patients were not affected, as has previously been described [6, 9, 10, 17, 18]. Therefore, our mutation profile may be useful as a reference for diverse ethnicities. Given that all cases with collagen VI-related myopathy in this cohort were sent to our center from hospitals in Japan, we calculated the occurrence of severe UCMD in Japan as 2.26 cases per year, which is an estimated incidence of 0.19 in 100,000 births, similar to that found for northern England (0.13/100,000) . This is most likely because in both ethnicities the majority of variants are de novo.
Among the mono-allelic variants, 88% (105/120) were located in the THD. Mono-allelic variants in the THD must be primarily associated with the majority SSCD staining pattern (91%, 92/101) and UCMD phenotype (90%, 94/105), whilst mono-allelic variants outside the THD were also associated with SSCD (71%, 10/14) but a BM phenotype (93%, 14/15). In exceptional cases, genotypes cannot be associated with specific phenotypes, with some variants reported to cause both UCMD and BM phenotypes [9-11, 18]. In fact, in our cohort, the families with c.877G>A in COL6A1, c.856-2A>G in COL6A2, or c.943G>A in COL6A2 showed a wide range of phenotypes from milder BM to severer UCMD, while conversely the variation in phenotypes of families with c.956A>G or c.1022G>A in COL6A1 was quite narrow and on the border between UCMD and BM.
In addition, we found four heterozygous large deletions in families with UCMD phenotype. All the deletions were located in the N-terminal side of the cysteine residue important for the assembly of the collagen VI tetramer. This is in accordance with all the reported multiple exon deletions [12, 14, 19-22]. Intriguingly, the deletion in the region containing the cysteine residue caused relatively mild phenotypes in our cohort and in those of previous reports [6, 23]. Thus, collagen VI proteins with large genomic deletions, which have the deletions no more than amino acid residues may act in a dominant-negative fashion and show a UCMD phenotype.
In this study, we identified ten families having biallelic variants and five each of them showed CD and SSCD collagen VI staining patterns in muscles, respectively. We can presume that families with truncated variants in both alleles will be associated with CD and severe UCMD phenotypes, whilst those with missense variants or in-frame deletions at least in one allele will be associated with SSCD and milder BM phenotypes. In fact, three families with truncated variants in both alleles (CD) and five families with missense or in-frame deletion at least in one allele (SSCD) displayed compatible patterns with the aforementioned presumption, regardless of causative genes. Interestingly, the other two biallelic families had in-frame deletion(s) in one and in two alleles, but they showed CD and severe UCMD phenotypes. To explore the mechanism causing the loss of collagen VI in muscles in these families, we observed the trace of collagen VI remaining in their biopsied muscles. In muscles from patients with truncated variants in both alleles, collagen VI formed small deposits in the extracellular space, while in patients with an in-frame deletion in at least one allele, the collagen VI was retained within mesenchymal cells. Thus, in those cases with extracellular deposits visible, the truncated collagen VI molecules could form tetramers and be secreted, but the secreted collagen VI was unstable and degraded extracellularly. On the other hand, in the cases with a retained trace, the in-frame deleted molecules failed to make a tetramer and be secreted. Additional detailed molecular analyses are required to understand the precise mechanism.
Our results provide comprehensive information on mutation type, incidence, and their consequent effect, performing the role of a ‘mutation catalog’ that thereby replaces the multiple analyses. In the cases with splicing-site variants, intronic pseudoexon-creating variants, and genomic large deletions, which account for about one third of the total, cDNA analysis contributed to successfully identifying the variant. cDNA analysis was especially powerful in identifying intronic pathogenic variants (8%: 11/140) located outside of exon-intron borders and which led to cryptic splicing. Using a combination of genomic and transcript analyses with the collagen VI pathology observed in muscles, we were able to make a conclusive genetic diagnosis in 130/132 cases with suspected collagen VI myopathy clinicopathologically. However, multiple steps of analyses were required to reach the final genetic diagnosis. Because there are a large number of sporadic cases with a de novo collagen VI variant in this disease, our comprehensive mutation catalog together with mutation reports from other published cases may help genetic diagnosis in diverse ethnicities.