Epithelial recurrent erosion dystrophy (ERED) from the splice site altering COL17A1 variant c.3156C>T in families of Finnish-Swedish ancestry.

PURPOSE: To describe four Finnish families with epithelial recurrent erosion dystrophy (ERED) caused by the pathogenic variant c.3156C>T in collagen type XVII alpha 1 chain gene (COL17A1). METHODS: Eleven affected and two unaffected individuals underwent clinical ophthalmological examination, anterior segment photography, and corneal topography. Two of them underwent phototherapeutic keratectomy (PTK). Genetic analysis included both next-generation and Sanger sequencing. Specimens from the manual keratectomy of one patient were available for ophthalmic pathologic examination, including immunohistochemistry. RESULTS: The common splice-site altering synonymous variant c.3156C > T, p.(Gly1052=) in COL17A1 was confirmed in 15 individuals with ERED from the four families. Subepithelial corneal scarring grades varied and increased with age, leading to decreased best-corrected visual acuity. PTK improved vision in 58- and 67-year-old individuals without reactivating the disease. The keratectomy specimens showed an uneven epithelium and a spectrum of basement membrane abnormalities, including breaks, fragmentation, multiplication and entrapment within the subepithelial scar, reflecting recurrent erosions. The stromal cells consisted of varying proportions of bland and activated fibroblasts and myofibroblasts, reflecting different ages of scars. The family with the largest number of known affected generations originated from Southern Sweden. CONCLUSION: The phenotype in the Finnish ERED families is consistent with earlier reports of the c.3156C > T variant, although the severity has varied between reports. The phenotype may be modulated by other genes. This study suggests a likely founder effect of the variant in both Finnish and Swedish populations due to their shared population histories. If vision is compromised, PTK can be considered especially in older patients.

Analysis of glaucoma genes in Finnish patients with juvenile open-angle glaucoma.

PURPOSE: To identify germline variants in myocilin (MYOC) and other known monogenic glaucoma genes in Finnish patients with juvenile open-angle glaucoma (JOAG). METHODS: Finnish patients with JOAG treated between 2010 and 2018 at the Department of Ophthalmology, Helsinki University Hospital, Finland, were enrolled. We sequenced all exonic regions and flanking splice sites of MYOC for five patients and one healthy relative using Sanger sequencing. In 48 patients, we performed exome sequencing to identify variants also in 28 other glaucoma-related genes. RESULTS: Fifty-three individuals with JOAG from 50 pedigrees, and one healthy relative, participated. The mean age at diagnosis was 30.8 years [SD 7.6; range 11 to 39]. Five probands had probably pathogenic variants in MYOC: c.1102C>T p.(Gln368Ter), c.1109C>T p.(Pro370Leu), c.1130C>T p.(Thr377Met), c.1132G>A p.(Asp378Asn) and c.1456C>T p.(Leu486Phe). Four of these patients had a family history of dominantly inherited JOAG. The frequency of MYOC variants was 10% (5 of 50 families). One patient and his mother with JOAG had a novel loss-of-function variant in the FOXC1 gene, c.366G>A p.(Trp122Ter). A patient with sporadic JOAG had a homozygous likely pathogenic variant in the LTBP2 gene, c.3938G>A p.(Cys1313Tyr). The genetic variants explained 14% (7 out of 50 families; 95% CI, 6%-23%) of JOAG in our cohort. CONCLUSIONS: The frequency of pathogenic variants in previously known glaucoma-associated genes is low in Finnish patients with JOAG. Because of the distinct genetic background of Finns, it might be possible to identify novel glaucoma genes through our JOAG series in the future.

A novel missense TGFBI variant p.(Ser591Phe) in a Finnish family with variant lattice corneal dystrophy.

INTRODUCTION: We describe the phenotype of a variant lattice corneal dystrophy (LCD) potentially caused by a novel variant c.1772C>T p.(Ser591Phe) in exon 13 of the transforming growth factor beta-induced (TGFBI) gene. CASE REPORT: The proband, a 71-year-old woman referred because of bilateral LCD, first seen at the age of 65 years, with recent progressive symptoms, underwent a clinical ophthalmological examination, anterior segment optical coherence tomography and confocal microscopy. Additionally, three siblings and three children were examined. The identified TGFBI variant was screened in six family members using Sanger sequencing. A corneal dystrophy gene screen was performed for the proband. Translucent subepithelial irregularities and central to midperipheral stubby branching corneal stromal lattice lines, asymmetric between the right and the left eye, were visible and resulted in mild deterioration of vision in one eye. Genetic testing revealed a novel variant c.1772C>T in TGFBI, leading to the amino acid change p.(Ser591Phe). One daughter carried the same variant but had only thick stromal nerve fibres at the age of 49 years. The other family members neither had corneal abnormalities nor carried the variant. No keratoplasty is yet planned for the proband. CONCLUSIONS: We classify the novel variant in TGFBI as possibly pathogenic, potentially causing the late-onset, asymmetric variant LCD. Our findings add to the growing number of TGFBI variants associated with a spectrum of phenotypes of variant LCD.

Identifying haplotypes in recessive inherited retinal dystrophies using whole-genome linked-read sequencing.

Conventional next-generation sequencing methods, used in most gene panels, cannot separate maternally and paternally derived sequence information of distant variants. In recessive diseases, two or more equally plausible causative variants with unsolved phase information prevent accurate molecular diagnosis. In reality, close relatives might be unavailable for segregation analysis. Here, we utilized whole genome linked-read sequencing to assign variants to haplotypes in two patients with inherited retinal dystrophies. Patient 1 with macular dystrophy had variants c.3442T>C, p.(Cys1148Arg), c.4209G>T, p.(Glu1403Asp), and c.1182C>T, p.(Cys394=) in CRB1, and Patient 2 with nonsyndromic retinitis pigmentosa had c.1328T>A, p.(Val443Asp) and c.3032C>G, p.(Ser1011*) in AHI1. The relatives were not available for genotyping. Using whole genome linked-read sequencing we phased the variants to haplotypes providing genetic background for the retinal dystrophies. In future, when the price of sequencing methods that provides long-read data decreases and their read-depth and accuracy increases, they are probably considered the primary or adjunctive sequencing methods in genetic testing, allowing the immediate collection of phase information and thus obviating the need for the carrier testing and segregation analysis.

Germline loss-of-function variants in MBD4 are rare in Finnish patients with uveal melanoma.

Uveal melanoma (UM) is a rare intraocular cancer with the highest incidence in northern latitudes. Metastases develop in approximately 50% of patients, whereafter the median survival is 13 months. Generally, the mutation burden of these tumors is low. Germline variants predisposing to UM have been previously described in BRCA1-associated protein 1 (BAP1). Recently, germline and somatic loss-of-function (LOF) variants in the methyl-CpG-binding domain 4 (MBD4) gene have been found to cause a hypermutated UM, and MBD4 also has been put forward as a gene predisposing to UM. We sequenced for MBD4 germline variants in 440 Finnish patients with UM and identified seven rare exonic missense variants in 16 (3.6%) patients, of which one likely alters MBD4 function. The frequency of likely pathogenic variants in our cohort is 0.23% (1/432; 95% CI, 0.01-1.28). We identified no LOF variants though their frequency in the Finnish population is 0.052%. Thus, our data do not support the suggestion that MBD4 germline variants predispose to UM. Somatic loss of MBD4 might modify the mutational burden in UM and change its response to immune checkpoint inhibitors.

In Vivo Corneal Confocal Microscopy and Histopathology of Keratitis Fugax Hereditaria From a Pathogenic Variant in NLRP3.

PURPOSE: To apply in vivo corneal confocal microscopy (IVCM) to study the pathogenesis of keratitis (keratoendotheliitis) fugax hereditaria, an autosomal dominant cryopyrin-associated periodic keratitis, associated with the c.61G>C pathogenic variant in the NLRP3 gene, in its acute and chronic phase, and to report histopathologic findings after penetrating keratoplasty. DESIGN: This was an observational case series. METHODS: The study population included 6 patients during an acute attack, 18 patients in the chronic phase, and 1 patient who underwent penetrating keratoplasty. Interventions included Sanger sequencing for the NLRP3 variant c.61C>G, a clinical examination, corneal photography, IVCM, light microscopy, and immunohistochemistry. Our primary outcome measures included IVCM and histopathologic findings. RESULTS: During the acute attack, hyperreflective cellular structures consistent with inflammatory cells transiently occupied the anterior to middle layers of the corneal stroma. Other corneal layers were unremarkable. With recurring attacks, central oval stromal opacities accumulated. IVCM revealed that they contained long, hyperreflective, needle-shaped structures in the extracellular matrix. Using light microscopy, the anterior half of the stroma displayed thin and finely vacuolated lamellae, and keratocytes throughout the stroma were immunopositive for syndecan. CONCLUSIONS: The acute attacks and chronic stromal deposits mainly involve the anterior to middle layers of the corneal stroma, and the disease is primarily a keratitis rather than a keratoendotheliitis. IVCM shows that inflammatory cells invade only the stroma during an acute attack. IVCM and light microscopic findings suggest that the central corneal opacities represent gradual deposition of extracellular lipids. The disease could make a good in vivo model to study activation of the NLRP3 inflammasome in cryopyrin-associated periodic syndromes.

Population-based analysis of BAP1 germline variations in patients with uveal melanoma.

Pathogenic germline variants in the BRCA1-associated protein 1 (BAP1) gene cause the BAP1 tumor predisposition syndrome (BAP1-TPDS) with increased risk of several cancers, the most frequent of which is uveal melanoma (UM). Pathogenicity of loss-of-function (LOF) BAP1 variants is clear, as opposed to that of missense and regulatory region variants. We sequenced the coding, promoter, untranslated region (UTR) and intronic regions of BAP1 and analyzed copy number variations (CNVs). In this nationwide study, the cohort comprised UM patients diagnosed between 2010 and 2017. These included 432 of 520 consecutive Finnish UM patients, 16 of whom were familial, and one additional patient from a Finnish-Swedish family. Twenty-one different rare variants were found: seven exonic, seven intronic, four 3' UTR and three promoter. We considered five variants likely to be pathogenic by effect on splicing, nuclear localization or deubiquitination activity. Intron 2 (c.67+1G>T) and exon 14 (c.1780_1781insT) LOF variants were presumed founder mutations, occurring in two and four families, respectively; both abolished nuclear localization in vitro. Intron 2, exons 5 (c.281A>G) and 9 (c.680G>A) missense variants markedly reduced deubiquitinating activity. A deep intronic 25 base pair deletion in intron 1 caused aberrant splicing in vitro. On the basis of functional studies and family cancer history, we classified four exon 13 missense variants as benign. No CNVs were found. The prevalence of pathogenic variants was 9/433 (2%) and 4/16 (25%) in Finnish UM families. Family cancer history and functional assays are indispensable when establishing the pathogenicity of BAP1 variants. Deep intronic variants can cause BAP1-TPDS.

Comprehensive Study of the Clinical Phenotype of Germline BAP1 Variant-Carrying Families Worldwide.

Background: The BRCA1-associated protein-1 (BAP1) tumor predisposition syndrome (BAP1-TPDS) is a hereditary tumor syndrome caused by germline pathogenic variants in BAP1 encoding a tumor suppressor associated with uveal melanoma, mesothelioma, cutaneous melanoma, renal cell carcinoma, and cutaneous BAP1-inactivated melanocytic tumors. However, the full spectrum of tumors associated with the syndrome is yet to be determined. Improved understanding of the BAP1-TPDS is crucial for appropriate clinical management of BAP1 germline variant carriers and their families, including genetic counseling and surveillance for new tumors. Methods: We collated germline variant status, tumor diagnoses, and information on BAP1 immunohistochemistry or loss of somatic heterozygosity on 106 published and 75 unpublished BAP1 germline variant-positive families worldwide to better characterize the genotypes and phenotypes associated with the BAP1-TPDS. Tumor spectrum and ages of onset were compared between missense and null variants. All statistical tests were two-sided. Results: The 181 families carried 140 unique BAP1 germline variants. The collated data confirmed the core tumor spectrum associated with the BAP1-TPDS and showed that some families carrying missense variants can exhibit this phenotype. A variety of noncore BAP1-TPDS -associated tumors were found in families of variant carriers. Median ages of onset of core tumor types were lower in null than missense variant carriers for all tumors combined (P < .001), mesothelioma (P < .001), cutaneous melanoma (P < .001), and nonmelanoma skin cancer (P < .001). Conclusions: This analysis substantially increases the number of pathogenic BAP1 germline variants and refines the phenotype. It highlights the need for a curated registry of germline variant carriers for proper assessment of the clinical phenotype of the BAP1-TPDS and pathogenicity of new variants, thus guiding management of patients and informing areas requiring further research.

Keratoendotheliitis Fugax Hereditaria: A Novel Cryopyrin-Associated Periodic Syndrome Caused by a Mutation in the Nucleotide-Binding Domain, Leucine-Rich Repeat Family, Pyrin Domain-Containing 3 (NLRP3) Gene.

PURPOSE: To describe the phenotype and the genetic defect in keratoendotheliitis fugax hereditaria, an autosomal dominant keratitis that periodically affects the corneal endothelium and stroma, leading in some patients to opacities and decreased visual acuity. DESIGN: Cross-sectional, hospital-based study. METHODS: Patient Population: Thirty affected and 7 unaffected subjects from 7 families, and 4 sporadic patients from Finland. OBSERVATION PROCEDURES: Ophthalmic examination and photography, corneal topography, specular microscopy, and optical coherence tomography in 34 patients, whole exome sequencing in 10 patients, and Sanger sequencing in 34 patients. MAIN OUTCOME MEASURES: Clinical phenotype, disease-causing genetic variants. RESULTS: Unilateral attacks of keratoendotheliitis typically occurred 1-6 times a year (median, 2.5), starting at a median age of 11 years (range, 5-28 years), and lasted for 1-2 days. The attacks were characterized by cornea pseudoguttata and haze in the posterior corneal stroma, sometimes with a mild anterior chamber reaction, and got milder and less frequent in middle age. Seventeen (50%) patients had bilateral stromal opacities. The disease was inherited as an autosomal dominant trait. A likely pathogenic variant c.61G>C in the NLRP3 gene, encoding cryopyrin, was detected in all 34 tested patients and segregated with the disease. This variant is present in both Finnish and non-Finnish European populations at a frequency of about 0.02% and 0.01%, respectively. CONCLUSION: Keratoendotheliitis fugax hereditaria is an autoinflammatory cryopyrin-associated periodic syndrome caused by a missense mutation c.61G>C in exon 1 of NLRP3 in Finnish patients. It is additionally expected to occur in other populations of European descent.