Anophthalmia/microphthalmia (A/M) is a developmental ocular malformation defined as complete absence or reduction in size of the eye. a normal brain MRI in the proband at 16 months. No pathogenic mutations were identified in 71 known A/M genes. Further analysis identified a shared heterozygous mutation in c.2317G>A p.(Gly773Arg) that was not seen in the unaffected parents and siblings. Analysis of twenty-four unrelated A/M exomes identified a novel c.2122G>A p.(Gly708Arg) mutation in an additional patient with unilateral microphthalmia bilateral microcornea glaucoma and Peters anomaly; the mutation was absent in the unaffected mother and the unaffected father was not available. Mutations in have been linked to a spectrum of human disorders; the most consistent feature is usually cerebrovascular disease with variable ocular anomalies kidney and muscle defects. This study expands the spectrum of phenotypes and indicates screening in patients with A/M regardless of MRI findings or presumed inheritance pattern. and represent the most common genetic factors each accounting for approximately 10-20 4 and 15% of dominant Mupirocin ((11) (12) (13 14 and (15). In some Mupirocin cases whole exome sequencing has allowed for the expansion of phenotypes associated with genes previously reported to play a role in human disease (16). Materials and Methods Human patients This human study was approved by the Institutional Review Board of the Children’s Hospital of Wisconsin with written informed consent obtained from each participant and/or their legal representative as appropriate. DNA was extracted from blood samples using standard protocols and analyzed for quantity/quality using the NanoDrop 1000 spectrophotometer (Thermo Fisher USA). Whole Exome Sequencing and Data Analysis Genomic DNA Rabbit Polyclonal to Cox2. was submitted for whole exome sequencing by Perkin Elmer Inc (Branford CT); exome capture was performed with the Agilent Sure Select v4 + UTR and Mupirocin 100 base pair paired end sequencing was performed using the Illumina HiSeq 2000. The obtained data were aligned using the Burrows-Wheeler Aligner (BWA) and variants were called using the Genome Analysis Toolkit Mupirocin (GATK v2.10 or v2.20) analysis pipeline available through Perkin Elmer. The samples were analyzed for mutations in 71 genes previously associated with A/M or coloboma (Online Resource 1) and other ocular genes (NEIBank list of Human Eye Disease Genes at http://neibank.nei.nih.gov/index.shtml) using the SNP & Variation Suite (Golden Helix Bozeman MT); analysis of variants for their possible effect on protein function was performed within the SVS program by accessing data from dbNSFP which provides scores from multiple functional prediction programs (SIFT Polyphen2 Mutation Taster MutationAssessor and FATHMM) as well as two conservation scores (GERP++ and PhyloP) (17). The observed variants were evaluated for their frequency in the general population using publicly available databases such as dbSNP (http://www.ncbi.nlm.nih.gov/snp) Exome Variant Server (NHLBI Exome Sequencing Project (ESP) Seattle WA (http://evs.gs.washington.edu/EVS/) and 1000 Genomes project (http://www.1000genomes.org/data). Variant Confirmation Primers flanking variant sites were designed and genomic DNA was amplified in probands and all available family members to confirm the variant and determine its inheritance. The following primers and conditions were utilized for the mutations: mutation identified in Family 1 The proband’s genomic DNA was submitted first for whole exome sequencing. Mean coverage of the target region in Patient 1A was 67.74X with 79% of the region showing greater than 10X coverage. Screening in 71 genes associated with A/M and/or coloboma (Online Resource 1) identified 2 novel and 2 rare heterozygous variants in the proband (Table 1). Among the identified variants a nonsense allele (p.Gln20*) predicted to result in an early truncation of the encoded protein and a missense allele (p.Arg25Trp) predicted to be damaging by 4 out of 5 functional effect predictor programs appeared to be the most significant; neither allele has been previously reported in general populations and mutations in both and were reported in association with a dominant pattern of inheritance (18 19 All four variants were confirmed by Sanger sequencing in the proband and analyzed for co-segregation in other family members. None of the alleles co-segregated with the affected phenotype; specifically both and alleles were absent in the affected sibling and present in one of the unaffected parents.