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.

Whole genome expression profiling of normal human fetal and adult ocular tissues.

To study growth and development of ocular tissues, gene expression patterns in normal human fetal versus adult eyes were compared. Human retina/retinal pigment epithelium, choroid, sclera, optic nerve* and cornea* tissues were dissected from fetal (24 week gestational age) (N = 9; *N = 6), and adult (N = 6) normal donor eyes. The Illumina(®) whole genome expression microarray platform was used to assess differential expression. Statistical significance for all comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Significant gene expression fold changes > 1.5 were found in adult versus fetal retina/RPE (N = 1185), choroid (N = 6446), sclera (N = 1349), and cornea (N = 3872), but not optic nerve. Genes showing differential expression were assessed using Ingenuity Pathway Analysis (IPA) for enriched functions and canonical pathways. In all tissues, development, cell death/growth, cancer functions, and signaling canonical pathways were enriched. There was also a general trend of down-regulation of collagen genes in adult tissues.

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.

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.