Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity.

Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemm's canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity.

Sequencing analysis of the ATOH7 gene in individuals with optic nerve hypoplasia.

BACKGROUND: The Atonal Homolog 7 (ATOH7) gene has been implicated in association studies with optic nerve head diameter size. Hence, we screened optic nerve hypoplasia (ONH) patient DNA samples from Australia, France, and the United States for sequence variants in theATOH7 gene using Sanger sequencing. METHODS: Sanger sequencing of theATOH7 gene was performed on 34 affected individual DNA samples. Sequencing was also carried out in three unaffected family members to confirm segregation of identified single nucleotide variations. RESULTS: Seven sequence variations were identified in ATOH7. No disease-causing sequence changes in the ATOH7 gene was discovered in the ONH patient samples. CONCLUSIONS: Mutations within the ATOH7 gene are not implicated in the pathogenesis of optic nerve hypoplasia in our patient cohort.

Scleral micro-RNA signatures in adult and fetal eyes.

INTRODUCTION: In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. METHODS: Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods. RESULTS: Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10(11)). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples. CONCLUSION: For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.

Mutations in SCO2 are associated with autosomal-dominant high-grade myopia.

Myopia, or near-sightedness, is an ocular refractive error of unfocused image quality in front of the retinal plane. Individuals with high-grade myopia (dioptric power greater than -6.00) are predisposed to ocular morbidities such as glaucoma, retinal detachment, and myopic maculopathy. Nonsyndromic, high-grade myopia is highly heritable, and to date multiple gene loci have been reported. We performed exome sequencing in 4 individuals from an 11-member family of European descent from the United States. Affected individuals had a mean dioptric spherical equivalent of -22.00 sphere. A premature stop codon mutation c.157C>T (p.Gln53*) cosegregating with disease was discovered within SCO2 that maps to chromosome 22q13.33. Subsequent analyses identified three additional mutations in three highly myopic unrelated individuals (c.341G>A, c.418G>A, and c.776C>T). To determine differential gene expression in a developmental mouse model, we induced myopia by applying a -15.00D lens over one eye. Messenger RNA levels of SCO2 were significantly downregulated in myopic mouse retinae. Immunohistochemistry in mouse eyes confirmed SCO2 protein localization in retina, retinal pigment epithelium, and sclera. SCO2 encodes for a copper homeostasis protein influential in mitochondrial cytochrome c oxidase activity. Copper deficiencies have been linked with photoreceptor loss and myopia with increased scleral wall elasticity. Retinal thinning has been reported with an SC02 variant. Human mutation identification with support from an induced myopic animal provides biological insights of myopic development.

Mutation in collagen II alpha 1 isoforms delineates Stickler and Wagner syndrome phenotypes.

PURPOSE: Stickler syndrome is an arthro-ophthalmopathy with phenotypic overlap with Wagner syndrome. The common Stickler syndrome type I is inherited as an autosomal dominant trait, with causal mutations in collagen type II alpha 1 (COL2A1). Wagner syndrome is associated with mutations in versican (VCAN), which encodes for a chondroitin sulfate proteoglycan. A three-generation Caucasian family variably diagnosed with either syndrome was screened for sequence variants in the COL2A1 and VCAN genes. METHODS: Genomic DNA samples derived from saliva were collected from all family members (six affected and four unaffected individuals). Complete sequencing of COL2A1 and VCAN was performed on two affected individuals. Direct sequencing of remaining family members was conducted if the discovered variants followed segregation. RESULTS: A base-pair substitution (c.258C>A) in exon 2 of COL2A1 cosegregated with familial disease status. This known mutation occurs in a highly conserved site that causes a premature stop codon (p.C86X). The mutation was not seen in 1,142 ethnically matched control DNA samples. CONCLUSIONS: Premature stop codons in COL2A1 exon 2 lead to a Stickler syndrome type I ocular-only phenotype with few or no systemic manifestations. Mutation screening of COL2A1 exon 2 in families with autosomal dominant vitreoretinopathy is important for accurate clinical diagnosis.

Association mapping of the high-grade myopia MYP3 locus reveals novel candidates UHRF1BP1L, PTPRR, and PPFIA2.

PURPOSE: Myopia, or nearsightedness, is a common ocular genetic disease for which over 20 candidate genomic loci have been identified. The high-grade myopia locus, MYP3, has been reported on chromosome 12q21-23 by four independent linkage studies. METHODS: We performed a genetic association study of the MYP3 locus in a family-based high-grade myopia cohort (n = 82) by genotyping 768 single-nucleotide polymorphisms (SNPs) within the linkage region. Qualitative testing for high-grade myopia (sphere ≤ -5 D affected, > -0.5 D unaffected) and quantitative testing on the average dioptric sphere were performed. RESULTS: Several genetic markers were nominally significantly associated with high-grade myopia in qualitative testing, including rs3803036, a missense mutation in PTPRR (P = 9.1 × 10(-4)) and rs4764971, an intronic SNP in UHRF1BP1L (P = 6.1 × 10(-4)). Quantitative testing determined statistically significant SNPs rs4764971, also found by qualitative testing (P = 3.1 × 10(-6)); rs7134216, in the 3' untranslated region (UTR) of DEPDC4 (P = 5.4 × 10(-7)); and rs17306116, an intronic SNP within PPFIA2 (P < 9 × 10(-4)). Independently conducted whole genome expression array analyses identified protein tyrosine phosphatase genes PTPRR and PPFIA2, which are in the same gene family, as differentially expressed in normal rapidly growing fetal relative to normal adult ocular tissue (confirmed by RT-qPCR). CONCLUSIONS: In an independent high-grade myopia cohort, an intronic SNP in UHRF1BP1L, rs4764971, was validated for quantitative association, and SNPs within PTPRR (quantitative) and PPFIA2 (qualitative and quantitative) approached significance. Three genes identified by our association study and supported by ocular expression and/or replication, UHRF1BP1L, PTPRR, and PPFIA2, are novel candidates for myopic development within the MYP3 locus that should be further studied.

Whole exome sequencing identifies a mutation for a novel form of corneal intraepithelial dyskeratosis.

BACKGROUND: Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology. METHODS AND RESULTS: A seven member Caucasian French family with two corneal intraepithelial dyskeratosis affected individuals (6-year-old proband and his mother) was ascertained. The proband presented with bilateral complete corneal opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal dyskeratosis were associated with the phenotype. Histopathology studies of cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative PCR ruled out 4q35 duplication, classically described in HBID cases. Next generation sequencing with mean coverage of 50× using the Illumina Hi Seq and whole exome capture processing was performed. Sequence reads were aligned, and screened for single nucleotide variants and insertion/deletion calls. In-house pipeline filtering analyses and comparisons with available databases were performed. A novel missense mutation M77T was discovered for the gene NLRP1 which maps to chromosome 17p13.2. This was a de novo mutation in the proband's mother, following segregation in the family, and not found in 738 control DNA samples. NLRP1 expression was determined in adult corneal epithelium. The amino acid change was found to destabilise significantly the protein structure. CONCLUSIONS: We describe a new corneal intraepithelial dyskeratosis and how we identified its causative gene. The NLRP1 gene product is implicated in inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1 polymorphisms are associated with various diseases.

CYP1B1, MYOC, and LTBP2 mutations in primary congenital glaucoma patients in the United States.

PURPOSE: To screen primary congenital glaucoma patients in the United States for sequence variants within the CYP1B1, LTBP2, and MYOC genes using Sanger and whole exome sequencing. DESIGN: Retrospective case-control study. METHODS: Fifty-seven primary congenital glaucoma patients (47 families), 71 unaffected family members of the primary congenital glaucoma probands, and 101 healthy unrelated individuals were recruited from a single institution. Sanger sequencing of the primary congenital glaucoma gene, CYP1B1, was performed on 47 proband deoxyribonucleic acid samples. Simultaneously, whole exome sequencing was conducted on 3 families, each including more than 1 affected individual. Concurrently, 33 of 47 primary congenital glaucoma probands with extended family deoxyribonucleic acid samples were screened for LTBP2 and MYOC gene mutations. Exome-sequenced variations were validated by additional Sanger sequencing to confirm segregation of filtered disease-causing single nucleotide variations. RESULTS: Seven primary congenital glaucoma families (14.9%) manifested disease phenotypes attributable to CYP1B1 mutations. One primary congenital glaucoma family possessed homozygous mutant alleles, whereas 6 families carried compound heterozygous mutations. Five novel combinations of compound heterozygous mutations were identified, of which 2 combinations were found with whole exome sequencing. No disease-causing mutations within the LTBP2 and MYOC genes were discovered. CONCLUSIONS: This study analyzed CYP1B1, LTBP2, and MYOC mutations in a cohort of primary congenital glaucoma patients from the United States, applying whole exome sequencing as a complementary tool to Sanger sequencing. Whole exome sequencing, coupled with Sanger sequencing, may identify novel genes in primary congenital glaucoma patients who have no mutations in known primary congenital glaucoma genes.

Study of a US cohort supports the role of ZNF644 and high-grade myopia susceptibility.

PURPOSE: Myopia, or nearsightedness, is highly prevalent in Asian countries and is considered a serious public health issue globally. High-grade myopia can predispose individuals to myopic maculopathy, premature cataracts, retinal detachment, and glaucoma. A recent study implicated zinc finger protein 644 isoform 1 (ZNF644) variants with non-syndromic high-grade myopia in a Chinese-Asian population. Herein we focused on investigating the role for ZNF644 variants in high-grade myopia in a United States (US) cohort. METHODS: DNA from a case cohort of 131 subject participants diagnosed with high-grade myopia was screened for ZNF644 variants. Spherical refractive error of -≤-6.00 diopters (D) in at least one eye was defined as affected. All coding, intron/exon boundaries were screened using Sanger sequencing. Single nucleotide allele frequencies were determined by screening 672 ethnically matched controls. RESULTS: Sequencing analysis did not detect previously reported mutations. However, our analysis identified 2 novel single nucleotide variants (c.725C>T, c.821A>T) in 2 high-grade myopia individuals- one Caucasian and one African American, respectively. These variants were not found in normal controls. A rare variant - dbsSNP132 (rs12117237→c.2119A>G) - with a minor allele frequency of 0.2% was present in 6 additional cases, but was also present in 5 controls. CONCLUSIONS: Our study has identified two novel variants in ZNF644 associated with high-grade myopia in a US cohort. Our results suggest that ZNF644 may play a role in myopia development.

Genetic association of insulin-like growth factor-1 polymorphisms with high-grade myopia in an international family cohort.

PURPOSE: Evidence from human myopia genetic mapping studies (MYP3 locus), modulated animal models, and observations of glycemic control in humans suggests that insulin-like growth factor (IGF)-1 plays a role in the control of eye growth. This study was conducted to determine whether IGF-1 polymorphisms are associated with myopia in a large, international dataset of Caucasian high-grade myopia pedigrees. METHODS: Two hundred sixty-five multiplex families with 1391 subjects participated in the study. IGF-1 genotyping was performed with 13 selected tag single nucleotide polymorphisms (SNPs) using allelic discrimination assays. A family-based pedigree disequilibrium test (PDT) was performed to test for association. Myopia status was defined using sphere (SPH) or spherical equivalent (SE), and analyses assessed the association of (1) high-grade myopia (

Genome-wide association identifies ATOH7 as a major gene determining human optic disc size.

Optic nerve assessment is important for many blinding diseases, with cup-to-disc ratio (CDR) assessments commonly used in both diagnosis and progression monitoring of glaucoma patients. Optic disc, cup, rim area and CDR measurements all show substantial variation between human populations and high heritability estimates within populations. To identify loci underlying these quantitative traits, we performed a genome-wide association study in two Australian twin cohorts and identified rs3858145, P=6.2x10(-10), near the ATOH7 gene as associated with the mean disc area. ATOH7 is known from studies in model organisms to play a key role in retinal ganglion cell formation. The association with rs3858145 was replicated in a cohort of UK twins, with a meta-analysis of the combined data yielding P=3.4x10(-10). Imputation further increased the evidence for association for several SNPs in and around ATOH7 (P=1.3x10(-10) to 4.3x10(-11), top SNP rs1900004). The meta-analysis also provided suggestive evidence for association for the cup area at rs690037, P=1.5x10(-7), in the gene RFTN1. Direct sequencing of ATOH7 in 12 patients with optic nerve hypoplasia, one of the leading causes of blindness in children, revealed two novel non-synonymous mutations (Arg65Gly, Ala47Thr) which were not found in 90 unrelated controls (combined Fisher's exact P=0.0136). Furthermore, the Arg65Gly variant was found to have very low frequency (0.00066) in an additional set of 672 controls.

Mutational hot spot potential of a novel base pair mutation of the CSPG2 gene in a family with Wagner syndrome.

OBJECTIVE: To report a 3-generation white family clinically diagnosed variably with Wagner, Stickler, and Jansen syndromes and screened for sequence variants in the COL2A1 and CSPG2 genes. Wagner syndrome is an autosomal dominant vitreoretinopathy with a predisposition to retinal detachment and cataracts. It has significant phenotypic overlap with allelic Jansen syndrome and ocular Stickler syndrome type 1. Sticker syndrome type 1 maps to chromosome 12q13.11-q13.2, with associated COL2A1 gene mutations. Wagner syndrome maps to chromosome 5q13-q14 and is associated with mutations in CSPG2 encoding versican, a proteoglycan present in human vitreous. METHODS: Genomic DNA samples derived from venous blood were collected from all family members. Complete sequencing of COL2A1 was performed on a proband. Primers for polymerase chain reaction and sequencing were designed to cover all exon and intron-exon boundaries. Direct sequencing of CSPG2 was performed on all family member samples. RESULTS: No detectable COL2A1 mutations were noted, making the diagnosis of ocular Stickler syndrome highly unlikely for this family. A unique base pair substitution (c.9265 + 1G>T) in intron 8 of the CSPG2 gene cosegregating with disease status was identified. This mutation occurred in a highly conserved previously reported splice site with a similar base pair substitution (G>A). Direct sequencing of this splice site mutation in 107 unrelated external controls revealed no variants, supporting the rarity of this base pair change and its causation in Wagner syndrome. This novel base pair substitution is thought to cause the deletion of exon 8 and formation of a truncated protein product. CONCLUSION: Mutation screening of CSPG2 in autosomal dominant vitreoretinopathy families is important for accurate diagnosis. CLINICAL RELEVANCE: This study underscores the importance of obtaining extensive pedigree information and comparative ophthalmologic clinical information, as the phenotypic findings may vary greatly among independent family members. The study also affirms the paradigm shift from diagnosis assignment based on eponyms to that based on gene mutation type.

COL1A1 and COL2A1 genes and myopia susceptibility: evidence of association and suggestive linkage to the COL2A1 locus.

PURPOSE: Collagen involvement in myopia development via scleral remodeling is well-known. Recently, COL1A1 and COL2A1 gene polymorphisms were reported to be associated with high-grade and common myopia, respectively. This study was conducted to investigate whether these collagen genes are associated and/or genetically linked with myopia in large Caucasian family datasets. METHODS: High-grade myopia was defined as

Identification of STRA6 and SKI sequence variants in patients with anophthalmia/microphthalmia.

PURPOSE: Anophthalmia and microphthalmia (A/M) are rare congenital ocular malformations presenting with the absence of eye components or small eyes with or without structural abnormalities. A/M can be isolated or syndromic. The stimulated by retinoic acid gene 6 (STRA6) and Sloan-Kettering viral oncogene homolog (SKI) genes are involved in vitamin A metabolism, and are implicated with A/M developmental abnormalities in human and animal studies. Vitamin A metabolism is vital to normal eye development and growth. This study explores the association of these genes in a cohort of subjects with A/M. METHODS: STRA6 and SKI were screened for sequence variants by direct sequencing of genomic DNA samples from 18 affected subjects with A/M. The DNA samples of 4 external, unrelated controls were initially screened. Eighty-nine additional unrelated controls were screened to confirm that any sequence variants found in the affected subject DNA samples were related to the phenotype. Coding regions, intron-exon boundaries, and untranslated regions were sequenced by standard techniques. Derived DNA sequences were compared to known reference sequences from public genomic databases. RESULTS: For STRA6, a novel coding non-synonymous sequence variant was found in one subject, resulting in an amino acid change from glycine to glutamic acid in residue 217. One novel nonsense sequence variant found in the same subject changed the STRA6 amino acid residue 592 from cytosine to thymine resulting in a premature stop codon. For SKI, a known coding non-synonymous sequence variant (rs28384811) was found in 3 subject DNA samples and 11/89 control DNA samples. Four novel coding-synonymous sequence variants were observed in SKI. CONCLUSIONS: The STRA6 sequence variants reported in this study could play a role in the pathogenesis of A/M by structural changes to the STRA6 protein. We can attribute 4% A/M incidence in this cohort to these sequence variants. Although no SKI sequence variants were found in this cohort, SKI should not be ruled out as a candidate gene for A/M due to the small cohort size.

REEP1 mutation spectrum and genotype/phenotype correlation in hereditary spastic paraplegia type 31.

Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3'-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2-7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.

Common MFRP sequence variants are not associated with moderate to high hyperopia, isolated microphthalmia, and high myopia.

PURPOSE: The membrane-type frizzled-related protein (MFRP) gene is selectively expressed in the retinal pigment epithelium and ciliary body, and mutations of this gene cause nanophthalmos. The MFRP gene may not be essential for retinal function but has been hypothesized to play a role in ocular axial length regulation. The involvement of the MFRP gene in moderate to high hyperopic, isolated microphthalmic/anophthalmic, and high myopic patients was tested in two phases: a mutation screening/sequence variant discovery phase and a genetic association study phase. METHODS: Eleven hyperopic, ten microphthalmic/anophthalmic, and seven non-syndromic high-grade myopic patients of varying ages and 11 control subjects participated in the mutation screening phase. Sixteen primer pairs were designed to amplify the 13 exons of the MFRP gene including intron/exon boundaries. Polymerase chain reactions were performed, and amplified products were sequenced using standard techniques. Normal and affected individual DNA sequences were compared alongside the known reference sequence (UCSC genome browser) for the MFRP gene. The genetic association study included 146 multiplex non-syndromic high-grade myopia families. Seventeen intragenic and flanking single nucleotide polymorphisms (SNPs) were chosen for the MFRP gene and genotyped in the large data set using the Taqman allelic discrimination assay. The family-based association Pedigree Disequilibrium Test (PDT) and GenoPDT were performed. RESULTS: The average spherical refractive error of the hyperopic patient cohort was +4.21 diopters (D; range +2.00 to +9.25 D) and of the myopic patient cohort was -12.36 D (range -8.25 to -14.50 D). A total of 16 SNPs were identified by direct sequencing. No significant association was determined between the 16 MFRP gene SNPs and the moderate to high hyperopia, microphthalmia/anophthalmia affection status, and high myopia. Family based association analysis did not reveal any association between the 17 SNPs genotyped in the larger family data set for any refractive error type. CONCLUSIONS: Sequence variants of the MFRP gene do not appear to be associated with either the less severe forms of hyperopia, extreme forms of limited eye growth and development, or high myopia. These results indicate that the MFRP gene may not play a role in regulating ocular axial length in these phenotypes.

Mutations in the novel mitochondrial protein REEP1 cause hereditary spastic paraplegia type 31.

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. For the novel SPG31 locus on chromosome 2p12, we identified six different mutations in the receptor expression-enhancing protein 1 gene (REEP1). REEP1 mutations occurred in 6.5% of the patients with HSP in our sample, making it the third-most common HSP gene. We show that REEP1 is widely expressed and localizes to mitochondria, which underlines the importance of mitochondrial function in neurodegenerative disease.