Analysis of the role of ZEB1 in the pathogenesis of posterior polymorphous corneal dystrophy.

PURPOSE: To determine how nonsense mutations in the transcription factor ZEB1 lead to the development of posterior polymorphous corneal dystrophy type 3 (PPCD3). METHODS: Whole-cell extracts were obtained from cultured human corneal epithelial cells (HCEpCs) as a source of ZEB1 protein. DNA-binding assays were performed using the whole-cell extract and oligonucleotide probes consisting of the two conserved E2-box motifs and surrounding nucleotides upstream of COL4A3. ZEB1 and COL4A3 mRNA expression in primary human corneal endothelial cells (HCEnCs) was assayed in both PPCD3 and control corneas by RT-PCR. Immunohistochemistry was used to localize ZEB1 and COL4A3 expression in normal human cornea. RESULTS: Electromobility shift assays (EMSAs) and competition EMSAs demonstrated binding of protein(s) in the cultured HCEpCs to the E2-box motifs in the probes. The supershift EMSA confirmed that ZEB1, demonstrated to be present in the whole-cell extracts, binds to both the proximal and distal E2-box motifs in the COL4A3 promoter region. Both COL4A3 and ZEB1 are expressed in normal HCEnCs, although in PPCD3, ZEB1 expression is decreased and COL4A3 expression is increased compared with levels of both genes in healthy control corneas. CONCLUSIONS: Inversely related HCEnC expression levels of ZEB1 and COL4A3 in PPCD3 indicate that ZEB1-mediated alterations in COL4A3 expression are most likely associated with the pathogenesis of this corneal endothelial dystrophy. However, the demonstration of COL4A3 expression in healthy adult primary HCEnCs suggests that PPCD3 is more likely to involve an alteration in the timing and/or degree of COL4A3 expression than to result from the dichotomous change implied by the previously proposed ectopic expression model.

TGFB1-induced extracellular expression of TGFBIp and inhibition of TGFBIp expression by RNA interference in a human corneal epithelial cell line.

PURPOSE: To report the increased production of extracellular transforming growth factor ß-induced protein (TGFBIp) by human corneal epithelial cells (HCECs) after induction by TGFB1 and the inhibition of TGFBIp production in induced and noninduced HCECs by RNA interference (RNAi). METHODS: HCECs were cultured in serum-free medium and treated with 0 or 10 ng/mL TGFB1 over a period of 72 hours. Commercially available siRNAs targeting TGFBI mRNA were mixed with a transfection reagent and used to reverse transfect TGFB1-induced and noninduced HCECs. Extracellular and intracellular concentrations of TGFBIp were measured by ELISA and Western blot analysis, respectively, and TGFBI RNA was assayed using semiquantitative RT-PCR. RESULTS: HCECs constitutively express TGFBIp, and treatment with TGFB1 results in up to a fourfold increase in the amount of extracellular TGFBIp. Four commercially available siRNAs targeting TGFBI mRNA produced a >70% decrease in extracellular TGFBIp within 48 hours after transfection of noninduced HCECs but a <25% decrease in extracellular TGFBIp by 48 hours after transfection of TGFB1-induced HCECs. The suppression of extracellular TGFBIp production correlated with a decrease in intracellular TGFBIp production and TGFBI mRNA expression after transfection. CONCLUSIONS: Extracellular TGFBIp expression by HCECs is increased several fold after exposure to TGFB1. Both HCEC-constitutive and HCEC-induced TGFBIp production can be inhibited with RNA interference, though the effect was greater and lasted longer for constitutive than induced TGFBIp production. Given that the corneal deposits in the TGFBI dystrophies consist of TGFBIp derived from HCECs, RNAi represents a potential means to inhibit primary dystrophic deposit formation and recurrence after surgical intervention.

Linkage of posterior amorphous corneal dystrophy to chromosome 12q21.33 and exclusion of coding region mutations in KERA, LUM, DCN, and EPYC.

PURPOSE: To identify the genetic basis of posterior amorphous corneal dystrophy (PACD) segregating in a large pedigree. METHODS: The authors performed clinical evaluation of a previously unreported pedigree with PACD, light and electron microscopic examination of an excised corneal button, genomewide linkage analysis, fine mapping linkage and haplotype analysis, and screening of four candidate genes (KERA, LUM, DCN, and EPYC). RESULTS: Twenty-one participants were determined to be affected based on the presence of characteristic clinical features of PACD; 15 affected and 39 unaffected individuals from a single pedigree enrolled in the study and provided DNA for analysis. Histopathologic examination of an excised corneal specimen from an affected individual demonstrated disorganized stromal lamellae and stromal staining with colloidal iron. Genomewide analysis demonstrated significant evidence of linkage to chromosome region 12q21.33 and evidence suggestive of linkage to chromosome region 8q22.3. Fine mapping of the chromosome 12 locus confirmed significant linkage; the largest multipoint log odds ratio score was 5.6 at D12S351. The linkage support interval was approximately 3.5 Mb (3.5 cM) in length between flanking markers D12S1812 and D12S95, roughly the entire chromosome band 12q21.33. No coding region mutations were identified in four candidate genes-KERA, LUM, DCN, EPYC-located in the chromosome 12 linkage support interval. CONCLUSIONS: Linkage and haplotype analyses identified 12q21.33 as a locus for PACD. However, no mutations were identified in the candidate genes (KERA, LUM, DCN, EPYC) within this region.

The utility of next-generation sequencing in the evaluation of the posterior polymorphous corneal dystrophy 1 locus.

PURPOSE: To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1) using next-generation sequencing (NGS) of the common PPCD1 support interval, in which Sanger sequencing failed to identify a pathogenic mutation. METHODS: Enrichment of the portion of chromosome 20 containing the common PPCD1 interval was performed on DNA extracted from an affected and an unaffected member of a family previously linked to the PPCD1 locus. NGS using the Roche 454 Titanium platform was performed, followed by computational analysis using NextGENe Software. RESULTS: NGS of the selectively enriched chromosomal 20 region between markers D20S48 and D20S190 produced over 400,000 DNA sequence reads with an average of 350 bases for each of the two DNA samples. Alignment of the DNA sequence reads with the reference sequence from the National Center of Biotechnology Information (NCBI) resulted in over 119 million matched bases per sample. Approximately 68,000 DNA sequence variants were identified in the common PPCD1 support interval in the affected individual, which was approximately twice the number of sequence variants identified in the unaffected individual. In both individuals, approximately 0.5% of the identified variants mapped to the 13 known and 16 predicted genes in the PPCD1 support interval, including 16 of the 17 (94%) variants previously identified by Sanger sequencing in the 13 known genes. In both individuals, the variant not identified by NGS was located in a region of inadequate coverage. CONCLUSIONS: NGS identified all of the exonic sequence variants that were previously identified by Sanger sequencing in known genes in adequately covered regions of the common PPCD1 interval, although the pathogenic variant is yet to be discovered. Given adequate coverage of a selectively enriched chromosomal region of interest, NGS represents a useful technique to screen for sequence variants in candidate gene loci that has multiple advantages over previously employed techniques for mutation discovery.

Exclusion of positional candidate gene coding region mutations in the common posterior polymorphous corneal dystrophy 1 candidate gene interval.

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is an autosomal-dominant disorder of the corneal endothelium associated with visually significant corneal edema and glaucoma. Statistical genetic analysis of 4 families with PPCD has demonstrated linkage to a 2.4 cM common support interval on chromosome 20 bordered by the markers D20S182 and D20S139. We sought to identify the genetic basis of PPCD linked to chromosome 20 (PPCD1) by screening the 26 positional candidate genes between these markers in a family previously mapped to the PPCD1 region. METHODS: The coding regions of the 26 positional candidate genes mapped to the common PPCD1 support interval were amplified and sequenced in affected and unaffected individuals from a family previously linked to the PPCD1 locus. Nine other genes positioned just outside of the common PPCD1 support interval but within the autosomal-dominant congenital hereditary endothelial dystrophy interval were also screened. RESULTS: Four DNA sequence variants in 3 of the positional candidate genes demonstrated complete segregation with the affected phenotype: p.Thr109Thr (rs6111803) in OVOL2, p.Arg56Gln (novel variant-RPSnovel) in RPS19P1, and p.Thr85Thr (rs1053834) and p.Pro99Ser (rs1053839) in C20orf79. Each of these 4 sequence variants demonstrated significant linkage with the affected phenotype in this family (P = 2.5 x 10 for RPSnovel, rs1053834 and rs1053839; P = 8.6 x 10 for rs6111803). However, we also identified each of these 4 sequence variants in > or = affected control individuals. The haplotype on which the disease-causing mutation is segregating was found to have a population frequency of 4.2% in the CEPH HapMap trios. Although a number of other previously described and novel single nucleotide polymorphisms were identified in the 35 positional candidate genes located within the PPCD1 and congenital hereditary endothelial dystrophy intervals, none segregated with the affected phenotype. CONCLUSIONS: We report the absence of a presumed pathogenic coding region mutation in the common PPCD1 support interval. Although minor alleles of 4 single nucleotide polymorphisms were identified that segregated with the affected phenotype, the relatively high frequency of each minor allele in the general population indicates that none is a candidate for the causal variant for PPCD. Instead, the causal variant is most likely a coding region deletion or a variant in a noncoding region of the PPCD1 common support interval.

A novel variant of combined granular-lattice corneal dystrophy associated with the Met619Lys mutation in the TGFBI gene.

OBJECTIVE: To report a novel mutation in TGFBI (GenBank NM_000358), p.Met619Lys, associated with a variant of combined granular-lattice corneal dystrophy. METHODS: Slitlamp examination and DNA collection from the proband and affected and unaffected relatives. All 17 exons of TGFBI were amplified and sequenced in the proband. Exon 14 was amplified and sequenced in the proband's family members and in 100 controls. Histopathologic examination of the excised corneal buttons from the proband and 3 family members was also performed. RESULTS: Affected individuals demonstrated an age-dependent phenotype, with the progression from central subepithelial needlelike deposits in younger individuals to polymorphic anterior stromal opacities in older family members. Screening of TGFBI in the proband demonstrated a novel mutation, p.Met619Lys, which was also present in all affected family members. Histopathologic examination revealed stromal deposits that stained with the Congo red and Masson trichrome stains as well as an antibody to the protein product of TGFBI. CONCLUSIONS: We present a unique corneal dystrophy phenotype associated with the novel p.Met619Lys mutation in TGFBI. Clinical Relevance The atypical and variable phenotype and the demonstration of both hyaline and amyloid stromal deposits indicate that neither clinical nor histopathologic features may be relied on to accurately diagnose and classify the corneal dystrophies.

Posterior polymorphous corneal dystrophy is associated with TCF8 gene mutations and abdominal hernia.

Mutations in the two-handed zinc-finger homeodomain transcription factor gene (TCF8) have been associated with posterior polymorphous corneal dystrophy (PPCD) and extraocular developmental abnormalities. We performed screening of TCF8 in 32 affected, unrelated probands, affected and unaffected family members of probands identified with a TCF8 mutation, and in 100 control individuals. Eight different pathogenic mutations were identified in eight probands: four frameshift (c.953_954insA, c.1506dupA, c.1592delA, and c.3012_3013delAG); three nonsense (Gln12X, Gln214X, Arg325X); and one missense (Met1Arg). Screening of TCF8 in affected and unaffected family members in six families demonstrated that each identified mutation segregated with the disease phenotype in each family; two probands did not have additional family members available for analysis. None of the eight TCF8 mutations was identified in 200 control chromosomes. The prevalence of hernias of the abdominal region in affected individuals with PPCD associated with TCF8 mutations was significantly higher than the prevalence in both individuals with PPCD not associated with a TCF8 mutation and in unaffected individuals. Therefore, PPCD is associated with TCF8 mutations in one quarter of affected families in this study, or about one third of all PPCD families that have been screened thus far. In these families, the presence of apparently causative TCF8 mutations is associated with abdominal and inguinal hernias.

Identification of mutations in UBIAD1 following exclusion of coding mutations in the chromosome 1p36 locus for Schnyder crystalline corneal dystrophy.

PURPOSE: To identify the genetic basis of Schnyder crystalline corneal dystrophy (SCCD) through screening positional candidate genes and UBIAD1, in which mutations have been associated with SCCD, in affected families. METHODS: The coding region of each of the 16 positional candidate genes for which mutation screening has not been previously reported was screened with polymerase chain reaction (PCR) amplification and automated sequencing in four affected individuals from two families with SCCD. In addition, the coding region of UBIAD1, located just outside of the originally described SCCD candidate interval on chromosome 1p36, was directly sequenced in affected and unaffected individuals from three families with SCCD. RESULTS: Eighteen novel and 15 previously reported sequence variants were identified in 10 of the 16 positional candidate genes. Only two of the sequence variants segregated with the affected phenotype in either of the families screened. Both were novel single nucleotide polymorphisms (SNPs) predicted to result in synonymous amino acid substitutions in different predicted genes. However, one of these SNPs was also identified in control individuals, and the other SNP was not predicted to alter splicing. Screening of UBIAD1 revealed a different missense mutation in each of the three unrelated probands that was screened: p.Asn102Ser, p.Arg119Gly, and p.Leu121Val. Screening of the affected and unaffected relatives of the probands in whom the p.Asn102Ser and p.Leu121Val mutations were identified demonstrated that each mutation segregated with the affected phenotype. None of the three missense mutations was identified in 110 control individuals. CONCLUSIONS: No presumed pathogenic coding region mutations were identified in the genes mapped to the candidate region for SCCD. However, missense mutations in UBIAD1, located just outside of the originally described SCCD fine mapped region, were identified in each of the three families with SCCD, confirming that mutations in UBIAD1 are associated with SCCD.

An unusual presentation of macular corneal dystrophy associated with uniparental isodisomy and a novel Leu173Pro mutation.

PURPOSE: To report an unusual phenotype of macular corneal dystrophy (MCDC1) associated with a novel CHST6 mutation transmitted via maternal isodisomy. METHODS: Slit lamp examination of the patient and his parents was performed. DNA was collected from each individual for amplification and sequencing of the CHST6 coding region, as well as exons 4 and 12 of TGFBI. Serum antigenic keratan sulfate (AgKS) levels were measured for confirmation of the diagnosis and subtyping of MCDC1. Quantitative real-time PCR (qPCR) was performed to differentiate between homozygous and hemizygous sequence variants. Genotyping at 12 single nucleotide polymorphisms (SNPs) within and surrounding CHST6 was performed to determine the pattern of inheritance of mutations identified in CHST6. RESULTS: Examination of the proband revealed bilateral, discrete, axially distributed, gray-white deposits at the level of Bowman's layer, with diffuse fine corneal stromal haze. Screening of TGFBI exons 4 and 12 in the proband did not reveal any allelic variants. However, screening of CHST6 in the proband demonstrated a novel homozygous missense mutation involving a highly conserved amino acid (c.518T > C; Leu173Pro) and undetectable serum AgKS levels in the proband confirmed the diagnosis of type I MCDC1. Quantitative PCR confirmed that both copies of CHST6 were present in the patient, excluding the possibility that the mutation was present in the hemizygous state. The results of genotyping were consistent with maternal isodisomy, as the patient was homozygous for an allele possessed by his mother at each SNP, two of which were informative and demonstrated nonpaternal inheritance. CONCLUSION: A phenotypically unusual variant of MCDC1 was found to be associated with the novel Leu173Pro mutation in CHST6, transmitted via uniparental isodisomy, a previously unreported pattern of inheritance in the corneal dystrophies.

Keratoconus is not associated with mutations in COL8A1 and COL8A2.

PURPOSE: To evaluate the suggested role of the COL8A1 and COL8A2 genes in the pathogenesis of the corneal ectatic disorders keratoconus and keratoglobus through mutation screening in affected patients. METHODS: DNA extraction, polymerase chain reaction amplification, and sequencing of COL8A1 and COL8A2 were performed in 50 unrelated keratoconus and 2 unrelated keratoglobus patients. RESULTS: No sequence variations were identified in COL8A1 and COL8A2 in the 2 patients with keratoglobus. Screening of COL8A1 in keratoconus patients revealed a previously identified single nucleotide polymorphism (SNP; c.1850C>T; Pro535Pro), in 1 patient. Screening of COL8A2 in keratoconus patients revealed 7 previously described SNPs: c.14G>A (Gly3Arg); c.112G>A (Ala35Ala); c.1012C>G (Leu335Leu); c.1308G>A (Arg434His); c.1492G>A (Gly495Gly); c.1512C>T (Thr502Met); and c.1765C>T (Pro586Pro). Four novel sequence variants were also identified, each in 1 affected patient: c.38_40dupCTG (Leu11dup), also identified in an unaffected relative of the affected proband, c.667G>A (Gly220Gly), c.1588G>A (Pro527Pro), and c.2026C>T (Val673Val). None of the 3 novel synonymous substitutions identified in COL8A2 was predicted to produce a splice acceptor site. CONCLUSIONS: The absence of pathogenic mutations in COL8A1 and COL8A2 in patients with keratoconus indicates that other genetic factors are involved in the pathogenesis of this corneal ectatic disorder.

Autosomal recessive CHED associated with novel compound heterozygous mutations in SLC4A11.

PURPOSE: To determine the genetic basis of autosomal recessive congenital hereditary endothelial dystrophy (CHED2) in an American patient of Chinese ancestry. METHODS: Slit-lamp examination of the proband and his parents, as well as histopathologic examination of excised corneal specimens from the proband, were performed to confirm the diagnosis of autosomal recessive CHED. DNA was collected from the proband and his parents, and all 19 exons of the SLC4A11 gene were amplified and screened. RESULTS: The proband showed diffuse bilateral corneal edema, which was not present in either of his parents. After the performance of bilateral penetrating keratoplasties, histopathologic examination of the excised corneal specimens showed marked corneal stromal edema and an absence of corneal endothelial cells. Screening of SLC4A11 showed 2 heterozygous mutations: c.743G>A (Ser232Asn) and c.1033A>T (Arg329X). The proband's mother was found to be heterozygous for the Ser232Asn missense mutation, and his father was heterozygous for the Arg329X nonsense mutation. No other coding region sequence variants were identified in the proband or his parents, and neither of the identified mutations was identified in 100 control individuals. CONCLUSIONS: CHED2 is associated with mutations in SLC4A11, a member of the SLC4 family of base transporters. Although the majority of affected individuals reported to date have shown homozygous mutations, associated with consanguinity in the Burmese, Indian, and Pakistani populations, we report 2 novel, independently sorting SLC4A11 mutations in an affected individual of Chinese ancestry.

Autosomal dominant cornea plana is not associated with pathogenic mutations in DCN, DSPG3, FOXC1, KERA, LUM, or PITX2.

PURPOSE: To determine the genetic basis of autosomal dominant cornea plana (CNA1) through the performance of a genome-wide linkage analysis and screening of the decorin (DCN), dermatan sulfate proteoglycan 3 (DSPG3), forkhead box C1 (FOXC1), keratocan (KERA), lumican (LUM,) and paired-like homeodomain transcription factor 2 (PITX2) genes in members of an affected multigenerational family. METHODS: Cycloplegic refraction, slit lamp biomicroscopy, corneal pachymetry, and corneal topography were performed to determine each patient's affected status. DNA was obtained from affected and unaffected subjects for the performance of a genome-wide linkage analysis as well as PCR amplification and sequencing of DCN, DSPG3, FOXC1, KERA, LUM, and PITX2. RESULTS: Five affected and three unaffected individuals were examined and provided a peripheral blood sample for DNA isolation. All affected individuals demonstrated an average corneal dioptric power less than 39 D, as well as one or more of the following anomalies: high hyperopia, strabismus, microcornea, posterior embryotoxon, iridocorneal adhesions, iris atrophy, and pupillary irregularities. A genome-wide linkage analysis did not indicate or exclude linkage to the region on chromosome 12 to which CNA1 has been previously mapped, and did not provide a single or multipoint LOD score greater than 2.0 for any of the 400 microsatellite markers. Screening of DCN, DSPG3, FOXC1, KERA, LUM, and PITX2 revealed 12 previously described single nucleotide polymorphisms, 2 previously described duplications, and 1 previously described insertion. None of the mutations previously associated with autosomal recessive cornea plana (CNA2) were identified. Seven novel sequence variants were described, including 5 single nucleotide substitutions, 1 insertion and 1 deletion. None of the identified sequence variants demonstrated complete segregation with the affected phenotype in the pedigree. CONCLUSION: Although missense and nonsense mutations in KERA are associated with CNA2, we did not identify any of the previously described mutations or novel mutations that segregated with the disease phenotype in a family with CNA1. In addition, no pathogenic sequence variations were found in DCN, DSPG3, LUM, PITX2 and FOXC1, which have also been implicated in corneal and anterior segment dysgenesis.

Replication and refinement of linkage of posterior polymorphous corneal dystrophy to the posterior polymorphous corneal dystrophy 1 locus on chromosome 20.

PURPOSE: The study purpose was to identify the genetic basis of posterior polymorphous corneal dystrophy, an autosomal dominant disorder of the corneal endothelium that is associated with the development of corneal edema, necessitating corneal transplantation for visual rehabilitation. Glaucoma also develops in up to 40% of patients with posterior polymorphous corneal dystrophy. METHODS: Linkage analysis, using microsatellite markers previously used to demonstrate linkage of posterior polymorphous corneal dystrophy to the chromosome 20 candidate region known as posterior polymorphous corneal dystrophy 1, was performed in 29 members of a family with posterior polymorphous corneal dystrophy. Thirty-four microsatellite markers were used to refine the posterior polymorphous corneal dystrophy 1 interval. TCF8, located on chromosome 10, was screened in an affected family member to exclude posterior polymorphous corneal dystrophy 3. RESULTS: Significant evidence of linkage to the posterior polymorphous corneal dystrophy 1 interval was obtained with both single-point and multipoint analyses. The largest single-point log odds ratio score obtained was 4.38 (theta=0) at marker D20S471; within 4.7 Mbp (7.2 cM) of D20S471 eight markers provided single-point log odds ratio scores of greater than 3.00 and three markers provided single-point log odds ratio scores greater than 4.00. The largest multipoint log odds ratio score obtained was 4.83, found across the adjacent markers D20S844, D20S191, D20S484, and D20S111. The support interval for posterior polymorphous corneal dystrophy 1 in the family we report is approximately 13.5 Mbp (10 cM) long and lies between the markers D20S182 and D20S195. Eleven markers have multipoint log odds ratio scores greater than 4.0 within this region. No coding region mutations were identified in TCF8 in an affected member of the family, effectively excluding posterior polymorphous corneal dystrophy 3. CONCLUSIONS: The originally described 19.8 cM posterior polymorphous corneal dystrophy 1 candidate disease interval has been refined to a 10 cM interval between markers D20S182 and D20S195. A portion of this refined interval overlaps a more recently reported posterior polymorphous corneal dystrophy 1 interval, with only 20 known and predicted genes mapped to the 2.4 cM common interval.

A clinical and histopathologic examination of accelerated TGFBIp deposition after LASIK in combined granular-lattice corneal dystrophy.

PURPOSE: To report the clinical and histopathologic features of accelerated TGFBI protein (TGFBIp) deposition after lamellar keratorefractive surgery in a patient with combined granular-lattice corneal dystrophy (CGLCD) who underwent bilateral corneal transplantation. DESIGN: Interventional case report. METHODS: A 28-year-old woman with a presumed TGFBI corneal dystrophy, but who retained best corrected visual acuity of 20/20 in each eye, underwent myopic laser-assisted in-situ keratomileusis (LASIK) both eyes (OU). For definitive diagnosis of the corneal dystrophy, buccal epithelial cells were collected as a source of genomic DNA and screening of TGFBI exons 4 and 12 was performed. RESULTS: Four months after the performance of an uncomplicated LASIK procedure, the patient's uncorrected visual acuity was 20/15 OU. Over the following two years, the appearance of confluent white stromal deposits at the LASIK flap interface resulted in disabling glare and a reduced best-corrected visual acuity of 20/40 OU. Corneal transplantation was performed in each eye, and histopathologic examination of the excised corneal buttons was performed. Eosinophilic material that stained positively with the Masson trichrome stain was present in the LASIK flap interface, as well as in the stroma of the flap and the anterior portion of the stromal bed. No amyloid deposits were identified with the Congo red stain. Screening of TGFBI exons 4 and 12 revealed the Arg124His mutation associated with CGLCD. CONCLUSIONS: Accelerated deposition of TGFBIp may occur after lamellar corneal surgery in patients with CGLCD. Therefore, LASIK surgery should be avoided in patients with any of the TGFBI dystrophies, and surgeons should be aware of the potential for rapid interface TGFBIp deposition after lamellar corneal surgery.

No pathogenic mutations identified in the COL8A1 and COL8A2 genes in familial Fuchs corneal dystrophy.

PURPOSE: To investigate the genetic basis of late-onset, familial Fuchs endothelial corneal dystrophy (FECD) through screening of the COL8A1 and COL8A2 genes, in which mutations have been associated with both early and late-onset, familial and sporadic FECD. METHODS: DNA extraction, PCR amplification, and direct sequencing of the COL8A1 and COL8A2 genes was performed in affected and unaffected members of 15 unrelated families with two or more members with late-onset FECD. RESULTS: Screening of the COL8A1 gene did not reveal sequence variants in any affected individuals from the 15 FECD families. In the COL8A2 gene, the previously identified mutations presumed to play a pathogenic role in cases of familial FECD (Arg155Gln, Leu450Trp, and Gln455Lys) were not discovered in any of the affected patients. A mutation previously considered causative of FECD (Arg434His) was shown not to segregate with the disease in the one family in which it was identified. Two previously identified single-nucleotide polymorphisms (SNPs), Pro575Leu and Pro586Pro, were identified in a single affected individual and three affected individuals (two families), respectively. CONCLUSIONS: The Arg434His mutation in the COL8A2 gene, previously associated with FECD, has been shown not to segregate with the disease phenotype, and thus may not be considered a disease-causing mutation. The absence of pathogenic mutations identified in the COL8A1 or COL8A2 genes in affected members of 15 pedigrees with familial FECD indicates that other genetic factors are involved in the development of this autosomal dominant corneal dystrophy.

No VSX1 gene mutations associated with keratoconus.

PURPOSE: To determine whether mutations of the VSX1 gene play a pathogenetic role in the development of keratoconus (KTCN). METHODS: DNA extraction, PCR amplification, and direct sequencing of the VSX1 gene were performed in 100 unrelated patients with diagnoses of clinical and topographic features of KTCN. RESULTS: Of the four previously identified presumed pathogenic mutations in the VSX1 gene (Leu17Pro, Asp144Glu, Leu159Met, and Arg166Trp), only Asp144Glu was identified in a single affected patient. Two novel single nucleotide polymorphisms (SNPs), both resulting in synonymous substitutions, were identified: c.53G>T (Ser6Ser) in four affected patients and c.209G>T (Pro58Pro) in two affected patients. Two previously reported SNPs were also identified: c.426C>A (Arg131Ser) in one affected patient and c.581A>G (Ala182Ala) in 51 of the 100 affected patients. CONCLUSIONS: Only one of the presumed pathogenic mutations in the VSX1 gene, Asp144Glu, was identified in a single member of the cohort of affected patients. However, as previously demonstrated, Asp144Glu is a non-disease-causing polymorphism. The absence of pathogenic mutations in the VSX1 gene in a large number of unrelated KTCN patients indicates that other genetic factors are involved in the development of this disorder.

No pathogenic mutations identified in the TGFBI gene in polymorphic corneal amyloid deposition.

PURPOSE: To determine whether primary, polymorphic, corneal amyloid deposition is associated with a mutation of the TGFBI gene. METHODS: Interventional case series of 8 patients. Slit lamp examination of all patients and photodocumentation of 5 patients were performed. Genomic DNA was isolated from buccal mucosal swabs obtained from all patients and all 17 exons of the TGFBI gene were amplified and sequenced. RESULTS: Multiple polymorphic, refractile deposits were noted throughout the central corneal stroma in all patients. The deposits appeared gray-white on direct illumination and translucent on retroillumination, characteristic of amyloid. In 2 patients, linear, branching opacities, reminiscent of lattice corneal dystrophy, were identified. Histopathologic examination confirmed the presence of stromal amyloid in the cornea of 1 patient who required corneal transplantation for pseudophakic corneal edema. Screening of the entire coding region of the TGFBI gene revealed 4 previously described synonymous substitutions, Leu217Leu, Val327Val, Leu472Leu, and Phe540Phe. A previously unreported missense change, Asp299Asn, was identified in one affected patient but not in her affected sister. No pathogenic mutations, including the Ala546Asp missense mutation previously associated with polymorphic corneal amyloidosis, were identified in any of the patients. CONCLUSIONS: TGFBI gene mutations were not identified in a series of patients with polymorphic corneal amyloid deposition. As bilateral, discrete stromal amyloid deposits may be dystrophic or degenerative, differentiation between these phenotypically similar conditions is facilitated with the use of molecular genetic analysis.

Unilateral lattice corneal dystrophy associated with the novel His572del mutation in the TGFBI gene.

PURPOSE: To report a novel mutation in the TGFBI gene, c.1761_1763del (p.His572del), associated with a unilateral variant of lattice corneal dystrophy (LCD). METHODS: A 63-year-old man presenting with the complaint of decreased vision in one eye was noted to have a unilateral lattice corneal dystrophy. Examination of the patient's wife and two sons, ages 20 and 27 years old, failed to reveal the presence of any corneal opacities. Following the collection of DNA from the patient and his family members, the TGFBI gene was screened for mutations previously associated with lattice corneal dystrophy and any novel coding region changes. RESULTS: In the affected patient, none of the mutations previously associated with the classic and variant forms of LCD were identified. However, a novel mutation, c.1761_1763del (p.His572del), was identified in exon 13 of TGFBI in the patient and his sons. This mutation was not identified in the patient's wife or in 200 control chromosomes. CONCLUSIONS: The novel TGFBI gene mutation (p.His572del) is associated with a unilateral, late-onset variant of lattice corneal dystrophy. This case highlights the utility of molecular genetic analysis in differentiating corneal dystrophies associated with an atypical phenotype from nondystrophic conditions.

Analysis of fifteen positional candidate genes for Schnyder crystalline corneal dystrophy.

PURPOSE: To identify the genetic basis of Schnyder crystalline corneal dystrophy (SCCD) through screening of positional candidate genes in affected patients. METHODS: Mutation screening of fifteen genes (CORT, CLSTN1, CTNNBIP1, DFFA, ENO1, GPR157, H6PD, KIF1B, LOC440559, LZIC, MGC4399, PEX14, PGD, PIK3CD, and SSB1) that lie within the candidate gene region for SCCD was performed in members of two families affected with SCCD. RESULTS: No presumed disease-causing mutations were identified in affected patients. Seventeen previously described single nucleotide polymorphisms (SNPs) were identified in eight of the candidate genes. Novel SNPs were identified in both affected and unaffected individuals in GPR157 (c.795C>T [Arg218Leu]; c.811C>T [Ala223Val]), MGC4399 (c.1024G>C [Leu277Leu]), and H6PD (c.754A>C [Asp151Ala]). CONCLUSIONS: No pathogenic mutations were identified in fifteen positional candidate genes in two families with SCCD. As the candidate gene region in each SCCD family previously examined with haplotype analysis has been mapped to the same chromosomal region, the absence of pathogenic mutations in these positional candidates in the families we examined reduces the number of remaining positional candidate genes by half, and the number of remaining candidate genes with a known gene function by two-thirds. We anticipate that screening of the remaining positional candidate genes will lead to the identification of the genetic basis of SCCD.

A unique corneal dystrophy of Bowman's layer and stroma associated with the Gly623Asp mutation in the transforming growth factor beta-induced (TGFBI) gene.

PURPOSE: To report a unique corneal dystrophy characterized by deposits at Bowman's layer and stromal lattice lines associated with the Gly623Asp missense mutation in the transforming growth factor beta-induced (TGFBI) gene. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: The proband, 3 affected siblings, 4 unaffected relatives, and 100 control individuals. METHODS: Slit-lamp examination, photographic documentation, and isolation of genomic DNA from buccal mucosal swabs obtained from each family member examined. Exons 4 and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members and in control individuals. MAIN OUTCOME MEASURES: Clinical characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene. RESULTS: Significant phenotypic variability, including polymorphic Bowman's layer opacities and stromal lattice lines, was noted in the 4 affected siblings who were examined. Screening of TGFBI exon 14 in the proband, 3 affected siblings, and a 19-year-old unaffected relative revealed a missense change, Gly623Asp, that was absent in the other 3 unaffected relatives screened and in 200 control chromosomes. CONCLUSIONS: We report a novel corneal dystrophy phenotype secondary to the Gly623Asp mutation in the TGFBI gene that is associated with clinical features of both lattice corneal dystrophy and a Bowman's layer dystrophy. The presence of clinical features considered atypical for a TGFBI-associated dystrophy in this pedigree, as well as the wide range of phenotypic expressions of the Gly623Asp mutation in affected members, underscore the clinical utility of molecular genetic analysis in the diagnosis of suspected corneal dystrophies.