Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

PURPOSE: To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS). METHODS: Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing. RESULTS: We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2 years (range 24-63 years) and mean follow-up 6.4 years (range 0-17 years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24 years of age, we documented the progression of corneal disease over the subsequent 17 years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing. CONCLUSION: A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.

The utility of massively parallel sequencing for posterior polymorphous corneal dystrophy type 3 molecular diagnosis.

The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel ∼0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A>G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles.

Schnyder corneal dystrophy and associated phenotypes caused by novel and recurrent mutations in the UBIAD1 gene.

BACKGROUND: The purpose of this study was to identify the genetic cause and describe the clinical phenotype of Schnyder corneal dystrophy (SCD) in six unrelated probands. METHODS: We identified two white Czech, two white British and two South Asian families with a clinical diagnosis of SCD. Ophthalmic assessment included spectral domain optical coherence tomography (SD-OCT) of one individual with advanced disease, and SD-OCT and confocal microscopy of a child with early stages of disease. UBIAD1 coding exons were amplified and Sanger sequenced in each proband. A fasting serum lipid profile was measured in three probands. Paternity testing was performed in one family. RESULTS: A novel heterozygous c.527G>A; p.(Gly176Glu) mutation in UBIAD1 was identified in one Czech proband. In the second Czech proband, aged 6 years when first examined, a previously described de novo heterozygous c.289G>A; p.(Ala97Thr) mutation was found. Two probands of South Asian descent carried a known c.305G>A; p.(Asn102Ser) mutation in the heterozygous state. Previously reported heterozygous c.361C>T; p.(Leu121Phe) and c.308C>T; p.(Thr103Ile) mutations were found in two white British families. Although crystalline deposits were present in all probands the affected area was small in some individuals. Corneal arcus and stromal haze were the most prominent phenotypical feature in two probands. In the Czech probands, SD-OCT confirmed accumulation of reflective material in the anterior stroma. Crystalline deposits were visualized by confocal microscopy. Mild dyslipidemia was found in all three individuals tested. CONCLUSION: Although de novo occurrence of mutations in UBIAD1 is extremely rare, SCD should be considered in the differential diagnosis of bilateral corneal haze and/or crystal deposition, especially in children.

Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4.

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.

Genotype-Phenotype Correlation for TGFBI Corneal Dystrophies Identifies p.(G623D) as a Novel Cause of Epithelial Basement Membrane Dystrophy.

PURPOSE: The majority of anterior corneal dystrophies are caused by dominant mutations in TGFBI (transforming growth factor ß-induced) collectively known as the epithelial-stromal TGFBI dystrophies. Most cases of epithelial basement membrane dystrophy (EBMD) are thought to result from a degenerative (nongenetic) process; however, a minority of cases are associated with specific TGFBI mutations. We evaluated the spectrum of TGFBI mutations and associated phenotypes in a United Kingdom cohort with typical epithelial-stromal TGFBI dystrophies and an EBMD cohort. METHODS: We recruited 68 probands with a clinical diagnosis of epithelial-stromal TGFBI dystrophy and 23 probands with bilateral EBMD. DNA was extracted from peripheral leukocytes, and TGFBI was bi-directly Sanger sequenced. RESULTS: Nine TGFBI mutations were identified. The most common occurred at the mutation hot-spot residues R124 and R555 in 61 probands; these individuals had a genotype-phenotype correlation consistent with prior reports. Four probands with lattice corneal dystrophy carried a mutation in exon 14: p.(A620D), p.(V625D), and p.(H626R). We identified a p.(G623D) mutation in five probands, including two probands from the EBMD cohort. These subjects typically had an onset of severe recurrent corneal epithelial erosion in the fourth decade with mild diffuse or geographic subepithelial corneal opacities and only small anterior stromal lattice structures in older individuals. Symptoms of painful epithelial erosion improved markedly following phototherapeutic keratectomy. CONCLUSIONS: There was a strong correlation between genotype and phenotype for the majority of TGFBI mutations. In this cohort, the p.(G623D) mutation caused a greater proportion of TGFBI-associated disease than anticipated, associated with variable phenotypes including individuals diagnosed with EBMD.

Spectrum of Clinical Signs and Genetic Characterization of Gelatinous Drop-Like Corneal Dystrophy in a Colombian Family.

PURPOSE: To describe the clinical signs of gelatinous drop-like corneal dystrophy (GDLD) in a consanguineous Colombian family and determine the underlying genetic cause. METHODS: We performed ocular examination of available family members and bidirectionally Sanger sequenced the GDLD-associated gene, TACSTD2. In one individual, the presence of subepithelial amyloid was confirmed with biopsy. RESULTS: The parents were consanguineous and 5 of their 10 children had GDLD. Typical mulberry subepithelial deposits with subepithelial vascularization were present in 3 individuals; 2 individuals only had mild polymorphic anterior stromal opacity. We identified a homozygous TACSTD2 missense mutation, c.551A>G, p.(Tyr184Cys), in the affected family members. Both parents were heterozygous for the mutation, and unaffected siblings were either heterozygous or homozygous wild-type for this allele. In the Colombian population, this mutation has a minor allele frequency of 0.53%. CONCLUSION: The clinical presentation of GDLD in this family was variable and does not solely support an age-dependent progression of the phenotype, suggesting that environmental or other genetic factors can modify phenotypic expression. The relatively high prevalence of this mutation in the Colombian population suggests that other individuals may have undiagnosed subclinical disease.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

Heterozygous deletions at the ZEB1 locus verify haploinsufficiency as the mechanism of disease for posterior polymorphous corneal dystrophy type 3.

A substantial proportion of patients with posterior polymorphous corneal dystrophy (PPCD) lack a molecular diagnosis. We evaluated 14 unrelated probands who had a clinical diagnosis of PPCD who were previously determined to be negative for mutations in ZEB1 by direct sequencing. A combination of techniques was used including whole-exome sequencing (WES), single-nucleotide polymorphism (SNP) array copy number variation (CNV) analysis, quantitative real-time PCR, and long-range PCR. Segregation of potentially pathogenic changes with disease was confirmed, where possible, in family members. A putative run of homozygosity on chromosome 10 was identified by WES in a three-generation PPCD family, suggestive of a heterozygous deletion. SNP array genotyping followed by long-range PCR and direct sequencing to define the breakpoints confirmed the presence of a large deletion that encompassed multiple genes, including ZEB1. Identification of a heterozygous deletion spanning ZEB1 prompted us to further investigate potential CNVs at this locus in the remaining probands, leading to detection of two additional heterozygous ZEB1 gene deletions. This study demonstrates that ZEB1 mutations account for a larger proportion of PPCD than previously estimated, and supports the hypothesis that haploinsufficiency of ZEB1 is the underlying molecular mechanism of disease for PPCD3.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.