Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis.

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels.

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

Management of Full-Thickness Macular Hole in A Genetically Confirmed Case with Usher Syndrome.

INTRODUCTION: Full-thickness macular hole (FTMH) formation is rarely seen in patients with retinitis pigmentosa (RP) and can have an adverse impact on their residual visual function. The underlying mechanisms are unknown, and clinical experience is limited regarding surgical outcomes. Here, we describe the surgical management of FTMH in a young patient with genetically confirmed Usher syndrome, the most common form of syndromic RP. CASE REPORT: A 28-year-old woman presented with blurred vision in her right eye (RE). She had a history of RP and bilateral hearing impairment since childhood. Fundoscopy and spectral-domain optical coherence tomography revealed a FTMH in the RE along with typical RP features bilaterally. After pars plana vitrectomy (PPV) with internal limiting membrane peel and gas tamponade, the FTMH closed. Six months after PPV the patient underwent cataract surgery in the affected eye, and the visual acuity remained stable compared to baseline. The clinical diagnosis of Usher syndrome was genetically confirmed by whole exome sequencing (WES), which revealed the presence of two pathogenic nucleotide variants in trans (compound heterozygosity) in the gene USH2A. CONCLUSION: We report a rare case of successful closure of a FTMH in a patient with Usher syndrome. Surgical treatment of FTMH can help preserve the central vision in RP patients, whose peripheral vision is severely affected.

Functional characterization of the first missense variant in CEP78, a founder allele associated with cone-rod dystrophy, hearing loss, and reduced male fertility.

Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities.

A frequent variant in the Japanese population determines quasi-Mendelian inheritance of rare retinal ciliopathy.

Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e., a 3.5-fold enrichment; p-value = 9.2 × 10-5). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation in trans with the Alu insertion, but might also associate with disease in combination with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.

A novel missense variant in IDH3A causes autosomal recessive retinitis pigmentosa.

BACKGROUND: Inherited retinal degenerations (IRDs) encompass a wide spectrum of genetic ocular diseases characterized by considerable genetic and clinical heterogeneity. METHODS: Complete ophthalmic examination and next-generation sequencing. RESULTS: We describe a patient with no family history of vision loss, who at the age of 28 years developed visual impairment consistent with a severe form of retinitis pigmentosa. Genetic testing by means of whole exome sequencing identified a homozygous variant in the gene IDH3A. To date, only three papers have reported mutations in IDH3A, in families with early-onset retinal degeneration with or without the presence of macular pseudocoloboma. CONCLUSION: This study highlights the importance of including this rarely-mutated gene in the molecular diagnostic set-ups for IRDs, and further delineates the phenotypic spectrum elicited by mutations in IDH3A.

Macular Dystrophy and Cone-Rod Dystrophy Caused by Mutations in the RP1 Gene: Extending the RP1 Disease Spectrum.

Purpose: To describe the clinical and genetic spectrum of RP1-associated retinal dystrophies. Methods: In this multicenter case series, we included 22 patients with RP1-associated retinal dystrophies from 19 families from The Netherlands and Japan. Data on clinical characteristics, visual acuity, visual field, ERG, and retinal imaging were extracted from medical records over a mean follow-up of 8.1 years. Results: Eleven patients were diagnosed with autosomal recessive macular dystrophy (arMD) or autosomal recessive cone-rod dystrophy (arCRD), five with autosomal recessive retinitis pigmentosa (arRP), and six with autosomal dominant RP (adRP). The mean age of onset was 40.3 years (range 14-56) in the patients with arMD/arCRD, 26.2 years (range 18-40) in adRP, and 8.8 years (range 5-12) in arRP patients. All patients with arMD/arCRD carried either the hypomorphic p.Arg1933* variant positioned close to the C-terminus (8 of 11 patients) or a missense variant in exon 2 (3 of 11 patients), compound heterozygous with a likely deleterious frameshift or nonsense mutation, or the p.Gln1916* variant. In contrast, all mutations identified in adRP and arRP patients were frameshift and/or nonsense variants located far from the C-terminus. Conclusions: Mutations in the RP1 gene are associated with a broad spectrum of progressive retinal dystrophies. In addition to adRP and arRP, our study provides further evidence that arCRD and arMD are RP1-associated phenotypes as well. The macular involvement in patients with the hypomorphic RP1 variant suggests that macular function may remain compromised if expression levels of RP1 do not reach adequate levels after gene augmentation therapy.

Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia Defects.

Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa.

Isolated and Syndromic Retinal Dystrophy Caused by Biallelic Mutations in RCBTB1, a Gene Implicated in Ubiquitination.

Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.His325Tyr), in RCBTB1. In affected individuals, it was found to segregate with retinitis pigmentosa (RP), goiter, primary ovarian insufficiency, and mild intellectual disability. Subsequent analysis of WES data in different cohorts uncovered four additional homozygous missense mutations in five unrelated families in whom iRD segregates with or without syndromic features. Ocular phenotypes ranged from typical RP starting in the second decade to chorioretinal dystrophy with a later age of onset. The five missense mutations affect highly conserved residues either in the sixth repeat of the RCC1 domain or in the BTB1 domain. A founder haplotype was identified for mutation c.919G>A (p.Val307Met), occurring in two families of Mediterranean origin. We showed ubiquitous mRNA expression of RCBTB1 and demonstrated predominant RCBTB1 localization in human inner retina. RCBTB1 was very recently shown to be involved in ubiquitination, more specifically as a CUL3 substrate adaptor. Therefore, the effect on different components of the CUL3 and NFE2L2 (NRF2) pathway was assessed in affected individuals' lymphocytes, revealing decreased mRNA expression of NFE2L2 and several NFE2L2 target genes. In conclusion, our study puts forward mutations in RCBTB1 as a cause of autosomal-recessive non-syndromic and syndromic iRD. Finally, our data support a role for impaired ubiquitination in the pathogenetic mechanism of RCBTB1 mutations.

Mutations in the polyglutamylase gene TTLL5, expressed in photoreceptor cells and spermatozoa, are associated with cone-rod degeneration and reduced male fertility.

Hereditary retinal degenerations encompass a group of genetic diseases characterized by extreme clinical variability. Following next-generation sequencing and autozygome-based screening of patients presenting with a peculiar, recessive form of cone-dominated retinopathy, we identified five homozygous variants [p.(Asp594fs), p.(Gln117*), p.(Met712fs), p.(Ile756Phe), and p.(Glu543Lys)] in the polyglutamylase-encoding gene TTLL5, in eight patients from six families. The two male patients carrying truncating TTLL5 variants also displayed a substantial reduction in sperm motility and infertility, whereas those carrying missense changes were fertile. Defects in this polyglutamylase in humans have recently been associated with cone photoreceptor dystrophy, while mouse models carrying truncating mutations in the same gene also display reduced fertility in male animals. We examined the expression levels of TTLL5 in various human tissues and determined that this gene has multiple viable isoforms, being highly expressed in testis and retina. In addition, antibodies against TTLL5 stained the basal body of photoreceptor cells in rat and the centrosome of the spermatozoon flagellum in humans, suggesting a common mechanism of action in these two cell types. Taken together, our data indicate that mutations in TTLL5 delineate a novel, allele-specific syndrome causing defects in two as yet pathogenically unrelated functions, reproduction and vision.

Identification of two novel mutations in CDHR1 in consanguineous Spanish families with autosomal recessive retinal dystrophy.

Inherited retinal dystrophies present extensive phenotypic and genetic heterogeneity, posing a challenge for patients' molecular and clinical diagnoses. In this study, we wanted to clinically characterize and investigate the molecular etiology of an atypical form of autosomal recessive retinal dystrophy in two consanguineous Spanish families. Affected members of the respective families exhibited an array of clinical features including reduced visual acuity, photophobia, defective color vision, reduced or absent ERG responses, macular atrophy and pigmentary deposits in the peripheral retina. Genetic investigation included autozygosity mapping coupled with exome sequencing in the first family, whereas autozygome-guided candidate gene screening was performed by means of Sanger DNA sequencing in the second family. Our approach revealed nucleotide changes in CDHR1; a homozygous missense variant (c.1720C>G, p.P574A) and a homozygous single base transition (c.1485+2T>C) affecting the canonical 5' splice site of intron 13, respectively. Both changes co-segregated with the disease and were absent among cohorts of unrelated control individuals. To date, only five mutations in CDHR1 have been identified, all resulting in premature stop codons leading to mRNA nonsense mediated decay. Our work reports two previously unidentified homozygous mutations in CDHR1 further expanding the mutational spectrum of this gene.

Homozygosity mapping reveals novel and known mutations in Pakistani families with inherited retinal dystrophies.

Inherited retinal dystrophies are phenotypically and genetically heterogeneous. This extensive heterogeneity poses a challenge when performing molecular diagnosis of patients, especially in developing countries. In this study, we applied homozygosity mapping as a tool to reduce the complexity given by genetic heterogeneity and identify disease-causing variants in consanguineous Pakistani pedigrees. DNA samples from eight families with autosomal recessive retinal dystrophies were subjected to genome wide homozygosity mapping (seven by SNP arrays and one by STR markers) and genes comprised within the detected homozygous regions were analyzed by Sanger sequencing. All families displayed consistent autozygous genomic regions. Sequence analysis of candidate genes identified four previously-reported mutations in CNGB3, CNGA3, RHO, and PDE6A, as well as three novel mutations: c.2656C > T (p.L886F) in RPGRIP1, c.991G > C (p.G331R) in CNGA3, and c.413-1G > A (IVS6-1G > A) in CNGB1. This latter mutation impacted pre-mRNA splicing of CNGB1 by creating a -1 frameshift leading to a premature termination codon. In addition to better delineating the genetic landscape of inherited retinal dystrophies in Pakistan, our data confirm that combining homozygosity mapping and candidate gene sequencing is a powerful approach for mutation identification in populations where consanguineous unions are common.

Exome sequencing of index patients with retinal dystrophies as a tool for molecular diagnosis.

BACKGROUND: Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context. METHODOLOGY/PRINCIPAL FINDINGS: We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases. CONCLUSIONS/SIGNIFICANCE: Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.

ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.

Autosomal recessive Stickler syndrome in two families is caused by mutations in the COL9A1 gene.

PURPOSE: To investigate COL9A1 in two families suggestive of autosomal recessive Stickler syndrome and to delineate the associated phenotype. METHODS: The probands of two consanguineous autosomal recessive Stickler families were evaluated for homozygosity using SNP microarray in one and haplotype analysis in the other. Subsequently, the entire COL9A1 open reading frame was analyzed by DNA sequencing in all members of the respective families. Several family members were investigated for dysmorphic features as well as ophthalmic, audiologic, and radiologic abnormalities. RESULTS: A novel homozygous COL9A1 mutation (p.R507X) was identified in two affected Turkish sisters, and the previously published mutation (p.R295X) was found in a Moroccan boy. Ophthalmic assessment revealed myopia, cataracts, distinct vitreous changes, progressive chorioretinal degeneration, and exudative and rhegmatogenous retinal detachments. All three had sensorineural hearing loss and epiphyseal dysplasia. Intervertebral disc bulging was observed in one patient and in two heterozygous carriers of the p.R507X mutation. CONCLUSIONS: A second, novel mutation was identified in COL9A1, causing autosomal recessive Stickler syndrome together with the previously described nucleotide change in two separate families. Although the overall phenotype was comparable to autosomal dominant Stickler, vitreous changes that may enable recognition of patients who are likely to carry mutations in COL9A1 were identified, and exudative retinal detachment was observed as a new finding in Stickler syndrome.

Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP.

Wnt signaling is a crucial component of the cell machinery orchestrating a series of physiological processes such as cell survival, proliferation, and migration. Among the plethora of roles that Wnt signaling plays, its canonical branch regulates eye organogenesis and angiogenesis. Mutations in the genes encoding the low density lipoprotein receptor protein 5 (LRP5) and frizzled 4 (FZD4), acting as coreceptors for Wnt ligands, cause familial exudative vitreoretinopathy (FEVR). Moreover, mutations in the gene encoding NDP, a ligand for these Wnt receptors, cause Norrie disease and FEVR. Both FEVR and Norrie disease share similar phenotypic characteristics, including abnormal vascularization of the peripheral retina and formation of fibrovascular masses in the eye that can lead to blindness. In this mutation update, we report 21 novel variants for FZD4, LRP5, and NDP, and discuss the putative functional consequences of missense mutations. In addition, we provide a comprehensive overview of all previously published variants in the aforementioned genes and summarize the phenotypic characteristics in mouse models carrying mutations in the orthologous genes. The increasing molecular understanding of Wnt signaling, related to ocular development and blood supply, offers more tools for accurate disease diagnosis that may be important in the development of therapeutic interventions.

Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

L1 retrotransposition can occur early in human embryonic development.

L1 elements are autonomous retrotransposons that can cause hereditary diseases. We have previously identified a full-length L1 insertion in the CHM (choroideremia) gene of a patient with choroideremia, an X-linked progressive eye disease. Because this L1 element, designated L1(CHM), contains two 3'-transductions, we were able to delineate a retrotransposition path in which a precursor L1 on chromosome 10p15 or 18p11 retrotransposed to chromosome 6p21 and subsequently to the CHM gene on chromosome Xq21. A cell culture retrotransposition assay showed that L1(CHM) is one of the most active L1 elements in the human genome. Most importantly, analysis of genomic DNA from the CHM patient's relatives indicated somatic and germ-line mosaicism for the L1 insertion in his mother. These findings provide evidence that L1 retrotransposition can occur very early in human embryonic development.

Erosive vitreoretinopathy and wagner disease are caused by intronic mutations in CSPG2/Versican that result in an imbalance of splice variants.

PURPOSE: Linkage intervals for erosive vitreoretinopathy (ERVR) and Wagner disease previously were found to overlap at 5q14.3. In a Japanese family with Wagner disease, a CSPG2/Versican splice site mutation (c.4004-2A-->G) was recently reported that resulted in a 39-nucleotide exon 8 in-frame deletion. We investigated whether CSPG2/Versican was mutated in six Dutch families and one Chinese family with Wagner disease and in a family with ERVR. METHODS: In all families, extensive ophthalmic examinations, haplotype analysis of the 5q14.3 region, and sequence analysis of CSPG2/Versican were performed. The effects of splice site mutations were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (QPCR). RESULTS: Three novel intron 7 sequence variants (c.4004-5T-->C, c.4004-5T-->A, c.4004-1G-->A) were identified in seven families. The c.4004-5T-->C variant was identified in four families with Wagner disease and a family with ERVR. The families were shown to carry the same 5q14.3 haplotype, strongly suggesting that this is a common Dutch founder variant. All three changes segregated with the disease in the respective families and were absent in 250 healthy individuals. In patients with the c.4004-5T-->A and c.4004-1G-->A variants, RT-PCR analysis of CSPG2/Versican showed activation of a cryptic splice site resulting in a 39-nt exon 8 in-frame deletion in splice variant V0. QPCR revealed a highly significant (P < 0.0001) and consistent increase of the V2 (>38-fold) and V3 (>12-fold) splice variants in all patients with intron 7 nucleotide changes and in a Chinese Wagner disease family, in which the genetic defect remains to be found. CONCLUSIONS: Wagner disease and ERVR are allelic disorders. Seven of the eight families exhibit a variant in intron 7 of CSPG2/Versican. The conspicuous clustering of sequence variants in the splice acceptor site of intron 7 and the consistent upregulation of the V2 and V3 isoforms strongly suggest that Wagner disease and ERVR may belong to a largely overlooked group of diseases that are caused by mRNA isoform balance shifts, representing a novel disease mechanism.