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. 2013 Nov;50(11):765-71.
doi: 10.1136/jmedgenet-2012-101499. Epub 2013 Aug 6.

Alternative splicing modifies the effect of mutations in COL11A1 and results in recessive type 2 Stickler syndrome with profound hearing loss

Allan J Richards  1 , Gregory S Fincham, Annie McNinch, David Hill, Arabella V Poulson, Bruce Castle, Melissa M Lees, Anthony T Moore, John D Scott, Martin P Snead
Affiliations
Free PMC article

Alternative splicing modifies the effect of mutations in COL11A1 and results in recessive type 2 Stickler syndrome with profound hearing loss

Allan J Richards et al. J Med Genet. 2013 Nov.
Free PMC article

Abstract

Background: Stickler syndromes types 1, 2 and 3 are usually dominant disorders caused by mutations in the genes COL2A1, COL11A1 and COL11A2 that encode the fibrillar collagens types II and XI present in cartilage and vitreous. Rare recessive forms of Stickler syndrome exist that are due to mutations in genes encoding type IX collagen (COL9A1 type 4 Stickler syndrome and COL9A2 type 5 Stickler syndrome). Recently, recessive mutations in the COL11A1 gene have been demonstrated to result in fibrochondrogenesis, a much more severe skeletal dysplasia, which is often lethal. Here we demonstrate that some mutations in COL11A1 are recessive, modified by alternative splicing and result in type 2 Stickler syndrome rather than fibrochondrogenesis.

Methods: Patients referred to the national Stickler syndrome diagnostic service for England, UK were assessed clinically and subsequently sequenced for mutations in COL11A1. Additional in silico and functional studies to assess the effect of sequence variants on pre-mRNA processing and collagen structure were performed.

Results: In three different families, heterozygous COL11A1 biallelic null, null/missense or silent/missense mutations, were found. They resulted in a recessive form of type 2 Stickler syndrome characterised by particularly profound hearing loss and are clinically distinct from the recessive types 4 and 5 variants of Stickler syndrome. One mutant allele in each family is capable of synthesising a normal α1(XI) procollagen molecule, via variable pre-mRNA processing.

Conclusion: This new variant has important implications for molecular diagnosis and counselling families with type 2 Stickler syndrome.

Keywords: Alternative Splicing; Recessive Inheritance; Stickler syndrome.

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Figures

Figure 1
Figure 1
Inheritance patterns of mutations. The pedigrees indicate the inheritance of each mutation in three different families. Solid squares/circles indicate individuals with type 2 Stickler syndrome. Vertical bars are carriers that had minor clinical signs associated with Stickler syndrome or a normal clinical appearance.
Figure 2
Figure 2
Analysis of exon 9 mutations c.1191delT and c.991-24A>G by RT-PCR. RNA from a cell line heterozygous for the c.1191delT mutation was analysed by RT-PCR and sequenced (A). cDNA from cells incubated with emetine to inhibit NMD (−NMD) had the frameshift mutation, whereas cDNA from uninhibited cells (+NMD) had a homozygous sequence from the normal allele only. COL11A1 exon 9, including 682 bp of surrounding intron sequence, was amplified from genomic DNA containing the c.991-24A>G mutation (B). This was cloned into the splicing reporter USR13, between COL2A1 exons 44 and 45. Normal and mutant alleles were expressed in MIO-M1 cells, with the resulting RNA analysed by RT-PCR and sequencing (B). The variant cDNA had an additional 23 bp, which corresponded to the sequence from COL11A1 IVS8 and a de novo acceptor splice site created by the mutation. Green=A, red=T, blue=C, black=G.
Figure 3
Figure 3
Facial photographs of a child with recessive type 2 Stickler syndrome. The child has c.2607A>G, p.Ala869Ala and c.5398G>T, p.Gly1800Cys mutations in COL11A1. The p.Gly1800Cys was inherited from her mother who is also shown.
Figure 4
Figure 4
RT-PCR analysis of the c.2607A>G, p.Ala869Ala mutation. Cultured cells from a heterozygous carrier were incubated with (−NMD) or without (+NMD) emetine to inhibit nonsense mediated decay. NMD was also inhibited in a normal cell line as a control. RNA was analysed by RT-PCR and sequenced in the forward (A) and reverse (B) directions. Text sequence represents the sequence seen in uninhibited cells (upper) and the additional sequence obtained from inhibited cells (lower). In (A) an additional sequence was located between exons 32 and 33 (in bold type), which corresponded to a pseudoexon in intron 32. In the reverse direction, the mutant sequence (arrowed) was present in cDNA from uninhibited (+NMD) cells. When nonsense mediated decay was inhibited an additional transcript was seen which corresponded to the use of the GTGCAC sequence (in bold) as a donor splice site.
Figure 5
Figure 5
Multiple sequence alignment of the C-termini of the fibrillar collagens. Amino acids identical in at least seven of the nine protein sequences are in bold type. The position of the p.Gly1800Cys mutation is indicated.
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