Arabella Poulson

Stickler syndrome and the vitreous phenotype: mutations in COL2A1 and COL11A1.

Stickler syndrome is a dominantly inherited disorder affecting the fibrillar type II/XI collagen molecules expressed in vitreous and cartilage. Mutations have been found in COL2A1, COL11A1 and COL11A2. It has a highly variable phenotype that can include midline clefting, hearing loss, premature osteoarthritis, congenital high myopia and blindness through retinal detachment. Although the systemic phenotype is highly variable, the vitreous phenotype has been used successfully to differentiate between patients with mutations in these different genes. Mutations in COL2A1 usually result in a congenital membranous vitreous anomaly. In contrast mutations in COL11A1 result in a different vitreous phenotype where the lamellae have an irregular and beaded appearance. However, it is now apparent that a new sub‐group of COL2A1 mutations is emerging that result in a different phenotype with a hypoplastic vitreous that fills the posterior chamber of the eye, and is either optically empty or has sparse irregular lamellae. Here we characterise a further 89 families with Stickler syndrome or a type II collagenopathy, and correlate the mutations with the vitreous phenotype. We have identified 57 novel mutations including missense changes in both COL2A1 and COL11A1 and have also detected two cases of complete COL2A1 gene deletions using MLPA. ©2010 Wiley‐Liss, Inc.

Clinical characterisation and molecular analysis of Wagner syndrome.

Aim: To detail the clinical findings in a British family with molecularly characterised Wagner syndrome. Background: Only in the last year has the specific genetic defect in Wagner syndrome been identified, and the background literature of the molecular genetics is outlined. Clinical and laboratory findings in a second case of Wagner syndrome are included to highlight difficulties that can be encountered when identifying pathogenic mutations for disorders arising in complex genes. Methods: Mutation screening was performed using PCR and RT-PCR. Results: A heterozygous mutation was found converting the donor splice site of exon 8 of the chondroitin sulphate proteoglycan 2 (CSPG2). This is the same mutation that has been reported in the original Wagner pedigree. The main clinical features of Wagner syndrome are vitreous syneresis, thickening and incomplete separation of the posterior hyaloid membrane, chorioretinal changes accompanied by subnormal electroretinographic responses, an ectopic fovea and early-onset cataract. A clinical feature present in this family, but previously undescribed, is anterior uveitis without formation of synechiae. Wagner syndrome has a progressive course, resulting in loss of vision even in the absence of retinal detachment. Conclusion: On a background of considerable confusion regarding the distinction between Wagner syndrome and predominantly ocular Stickler syndrome, it is now apparent the that two conditions are both clinically and genetically distinct. This report summarises the clinical findings in Wagner syndrome and extends the phenotypic characteristics.

Clinical features of type 2 Stickler syndrome

The Stickler syndromes1–8 (hereditary arthro-ophthalmopathy; McKusick nos. 108300 and 604841) are one of the more frequently occurring groups of chondrodysplasias and are the commonest inherited cause of rhegmatogenous retinal detachment.9 The majority of patients and pedigrees exhibit the type 1 or “membranous” vitreous phenotype10,11 and harbour mutations in the gene for type II collagen ( COL2A1 ).12–21 While not all mutations in type II collagen result in the membranous vitreous anomaly,22,23 when it is exhibited it appears to be congenital and provides a useful basis for mutant locus assignment. This is particularly helpful for sporadic cases where linkage is impossible, and especially in those individuals with mild or minimal systemic involvement where the diagnosis might otherwise be overlooked.19,24–26 Other pedigrees exhibit a different “beaded” vitreous phenotype and are linked to a different locus.11,27 We reported the first mutation in the gene encoding the α1 chain of type XI collagen ( COL11A1 ) in one of these pedigrees28 and this locus was confirmed in other pedigrees,29,30 and is now known as type 2 Stickler syndrome (McKusick no. 604841). The intimate post-translational molecular associations between types II and XI collagen form the foundation of the close clinical overlap between these two sub-groups of Stickler syndrome, but the extent of this clinical overlap and variation remains to be defined. Whilst type 1 Stickler syndrome pedigrees have a particularly high risk of blindness through giant retinal tear and retinal detachment, Annunen et al suggest that patients with COL11A1 mutations are at a low risk of retinal detachment and have a higher incidence of midfacial hypoplasia.29 This would be an important prognostic difference if confirmed. Here we wish to report the first description of the ophthalmic, oro-facial, audiologial, skeletal, and …

Prevention of retinal detachment in Stickler syndrome: the Cambridge prophylactic cryotherapy protocol.

PURPOSE The Stickler syndromes are the most common causes of inherited and childhood retinal detachment; however, no consensus exists regarding the effectiveness of prophylactic intervention. We evaluate the long-term safety and efficacy of the Cambridge prophylactic cryotherapy protocol, a standardized retinal prophylactic treatment developed to prevent retinal detachment arising from giant retinal tears in type 1 Stickler syndrome. DESIGN Retrospective comparative case series. PARTICIPANTS Four hundred eighty seven patients with type 1 Stickler syndrome. METHODS Time to retinal detachment was compared between patients who received bilateral prophylaxis and untreated controls, with and without individual patient matching. Patients receiving unilateral prophylaxis (after fellow eye retinal detachment) were similarly compared with an appropriate control subgroup. Individual patient matching ensured equal age and follow-up between groups and that an appropriate control (who had not suffered a retinal detachment before the age at which their individually matched treatment patient underwent prophylactic treatment) was selected. Matching was blinded to outcome events. Individual patient matching protocols purposely weighted bias against the effectiveness of treatment. All treatment side effects are reported. MAIN OUTCOME MEASURES Time to retinal detachment and side effects occurring after prophylactic treatment. RESULTS The bilateral control group (n = 194) had a 7.4-fold increased risk of retinal detachment compared to the bilateral prophylaxis group (n = 229) (hazard ratio [HR], 7.40; 95% confidence interval [CI], 4.53-12.08; P<0.001); the matched bilateral control group (n = 165) had a 5.0-fold increased risk compared to the matched bilateral prophylaxis group (n = 165) (HR, 4.97; 95% CI, 2.82-8.78; P<0.001). The unilateral control group (n = 104) had a 10.3-fold increased risk of retinal detachment compared to the unilateral prophylaxis group (n = 64) (HR, 10.29; 95% CI, 4.96-21.36; P<0.001); the matched unilateral control group (n = 39) had a 8.4-fold increased risk compared to the matched unilateral prophylaxis group (n = 39) (HR, 8.36; 95% CI, 3.24-21.57; P<0.001). No significant long-term side effects occurred. CONCLUSIONS In the largest global cohort of type 1 Stickler syndrome patients published, all analyses indicate that the Cambridge prophylactic cryotherapy protocol is safe and markedly reduces the risk of retinal detachment.

Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations

UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5′ and 3′ of each exon, typically 30 bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patients cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype.

Vitreous phenotype: A key diagnostic sign in Stickler syndrome types 1 and 2 complicated by double heterozygosity

We describe the clinical findings in two patients with double heterozygosity, both involving Stickler syndrome. In case 1, the proposita had Albright hereditary osteodystrophy which was inherited from her mother and type 1 Stickler syndrome which was a new mutation. The combination of manifestations from the two syndromes had resulted in initial diagnostic confusion. Diagnosis of the latter syndrome was made only following ophthalmic examination which documented the presence of a membranous vitreous anomaly characteristic of type 1 Stickler syndrome. Subsequent confirmation was achieved by mutation analysis of the COL2A1 gene. The propositus in case 2 inherited Treacher Collins syndrome paternally and type 2 Stickler syndrome maternally. The overlap of facial anomalies may have resulted in a more severe phenotype for the patient. The diagnosis of Stickler syndrome in the propositus was confirmed initially by vitreous assessment and later by demonstration of mutation in the COL11A1 gene. These two patients highlight the key role of vitreous examination and vitreoretinal phenotyping in the differential diagnosis of Stickler syndrome and its subtypes in cases where the clinical picture is complicated by double heterozygosity.

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

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.

Deep Intronic Sequence Variants in COL2A1 Affect the Alternative Splicing Efficiency of Exon 2, and May Confer a Risk for Rhegmatogenous Retinal Detachment

COL2A1 mutations causing haploinsufficiency of type II collagen cause type 1 Stickler syndrome that has a high risk of retinal detachment and failure of the vitreous to develop normally. Exon 2 of COL2A1 is alternatively spliced, expressed in the eye but not in mature cartilage and encodes a region that binds growth factors TGFβ1 and BMP‐2. We investigated how both an apparently de novo variant and a polymorphism in intron 2 altered the efficiency of COL2A1 exon 2 splicing and how the latter may act as a predisposing risk factor for the occurrence of posterior vitreous detachment (PVD)‐associated rhegmatogenous retinal detachment (RRD) in the general population. Using amplification of illegitimate transcripts and allele‐specific minigenes expressed in cultured cells, we demonstrate variability in exon 2 inclusion not only between different control individuals, but also between different COL2A1 alleles. We identify transacting factors that bind to allele‐specific RNA sequences, and investigate the effect of knockdown and overexpression of these factors on exon 2 splicing efficiency. Finally, using a specific cohort of patients with PVD‐associated RRD and a control population, we demonstrate a significant difference in the frequency of the COL2A1 intronic variant rs1635532 between the two groups.

Clinicopathologic Correlation of Lens Epithelial Metaplasia and Late Intraocular Lens Dislocation After Repair of Retinal Detachment.

IMPORTANCE In-the-bag intraocular lens dislocation is an uncommon but serious complication of cataract surgery in patients with previous repair of retinal detachment. The causative mechanism is currently unknown. We report histologic findings from a retrospective case series from 1993 to 2010 and suggest a possible mechanism to explain this association. OBSERVATIONS Clinical characteristics of 8 patients presenting with in-the-bag intraocular lens dislocation after repair of retinal detachment were evaluated. Explanted capsular bags from 3 of these patients were compared with pathologic changes of crystalline lenses associated with retinal detachment. Histologic examination of the explanted capsular bags revealed a paucicellular membrane that covered the concertina-like folded surface of the lens capsule. The lens capsule was devoid of epithelial cell nuclei and showed excessive thickening with the presence of spindle-shaped cells, such as fibroblasts. Collagen fibers were noted in the extracellular matrix. CONCLUSIONS AND RELEVANCE Previous studies of crystalline lens pathologic findings associated with retinal detachment have shown changes in the epithelium with migration and subsequent metaplasia of epithelial cells, resulting in excessive thickening of the anterior capsule with a layer of fibrous tissue. In this retrospective series, similar histologic findings were seen, suggesting that zonular dehiscence and lens dislocation may result from progressive capsular contraction secondary to retinal detachment-induced lens epithelial metaplasia.

Type I membranous anomaly in Stickler syndrome

To the editor: The recent report by Tompson et al. described a single base pair transition in exon 2 ofCOL2A1 in 22 individuals, of whom 15 had features of type 1 Stickler syndrome, while the remaining 7 were described as “clinically unaffected.” Exon 2 of COL2A1 is not expressed in mature cartilage, and the premature termination codon (PTC) mutation described would therefore be expected to result in a predominantly ocular-only phenotype as first described by Richards et al. who highlighted the clinical and genetic differences between Wagner syndrome and a predominantly ocular form of Stickler syndrome. The pathognomonic feature of type 1 Stickler syndrome, due to PTC or frame shift pathogenic changes, is an abnormality in vitreous embryological development, referred to as the type I membranous vitreous anomaly, which consists of vestigial vitreous gel in the retrolental space, bound posteriorly by a convoluted membrane. Subsequent reports of very large pedigrees with predominantly ocular Stickler syndrome have all demonstrated complete penetrance. Tompson et al. report that in their family, mutation positive but apparently unaffected individuals possessed an anterior vitreous that was “clear”; however, there is nomention of the presence or absence of a type 1 membranous anomaly nor indeed in those individuals classified as affected. Lattice degeneration is a late development and is never seen in children, and the use of the term “optically empty vitreous” is not specific to any of the Stickler syndrome subtypes. We agree with the authors that a points system to diagnose Stickler syndrome based on clinical manifestations would not account for subtypes producing ocular-only phenotypes, nor type 3 Stickler syndrome [MIM 184840] which does not exhibit ocular manifestations. The vitreous phenotype can be key to directing laboratory analysis. For instance, for those individuals that present with the membranous anomaly, whole gene (both exon and introns) COL2A1 next generation sequencing (NGS) of long-range PCR products is the most efficient method for detecting pathogenic variants, as it will also detect deep intronic changes which are also known to cause the disorder. For other Stickler syndrome subtypes, NGS using “targeted capture panels” that can simultaneously sequence a number of genes may be more appropriate, and are less expensive than exome sequencing. Early clinical and molecular diagnosis of type 1 Stickler syndrome is important because it allows an opportunity to perform prophylactic cryotherapy, which has been shown to reduce the risk of retinal detachment in these patients. While Stickler syndrome can result in variable expressivity, the suggestion that the disorder is not fully penetrant may lead to affected individuals being wrongly advised as to their risk of retinal detachment.