PMC:4570552 / 4279-7557 JSONTXT

{"project":"2_test","denotations":[{"id":"25865493-8834052-2044006","span":{"begin":186,"end":187},"obj":"8834052"},{"id":"25865493-8867660-2044006","span":{"begin":186,"end":187},"obj":"8867660"},{"id":"25865493-17935251-2044006","span":{"begin":186,"end":187},"obj":"17935251"},{"id":"25865493-12072800-2044006","span":{"begin":186,"end":187},"obj":"12072800"},{"id":"25865493-17394214-2044006","span":{"begin":186,"end":187},"obj":"17394214"},{"id":"25865493-18417983-2044006","span":{"begin":186,"end":187},"obj":"18417983"},{"id":"25865493-25017102-2044007","span":{"begin":1032,"end":1034},"obj":"25017102"},{"id":"25865493-24487276-2044008","span":{"begin":1949,"end":1951},"obj":"24487276"},{"id":"25865493-23843252-2044009","span":{"begin":2063,"end":2065},"obj":"23843252"},{"id":"25865493-11772997-2044010","span":{"begin":2683,"end":2685},"obj":"11772997"},{"id":"25865493-12620964-2044010","span":{"begin":2683,"end":2685},"obj":"12620964"}],"text":"Thirteen subjects were included in this study. All individuals were clinically assessed by experienced clinical geneticists. Among them, nine subjects had previously been reported.1,2,5–8 Clinical features are described in detail in Table S1. Clinical data and biological material collection and storage were attained from the participating families after written informed consent was secured, following procedures in accordance with the ethical standards of the responsible committees on human experimentation (institutional and national). Genomic DNA was isolated from peripheral blood leukocytes, skin fibroblasts, hair bulb cells, and/or buccal mucosal epithelial cells, using standard protocols. We performed WES on genomic DNA extracted from circulating leukocytes of a single affected subject (case 8) (Figure 1A, CaGi_UCSC). Exome capture was performed using NimbleGen SeqCap EZ Exome V. 3.0 (Roche) and sequencing by a HiSeq2000 instrument (Illumina). WES data analysis was performed using an in-house implemented pipeline.10 For sequencing statistics, see Table S2. Data annotation predicted 11,168 high-quality variants having functional impact (i.e., non-synonymous and splice site changes). Among them, 259 private, rare (minor allele frequency \u003c 0.001), or clinically associated changes were retained for further analyses. After excluding the presence of variants compatible with autosomal recessive transmission (Table S3), we reasoned that the clinical symptomatology might be caused by a de novo event. Candidates were stratified through a mixed filtering/prioritization strategy taking into account the predicted impact of each variant and the functional relevance of individual genes on the developmental processes altered in the disorder. Only changes (private, clinically associated, or having unknown frequency or minor allele frequency \u003c 0.001) predicted to be deleterious by the Combined Annotation Dependent Depletion (CADD)11 (score \u003e 15.0) or Database for Nonsynonymous SNPs’ Functional Predictions (dbNSFP) Support Vector Machine (SVM)12 (radial score \u003e 0.0) algorithm were retained and prioritized on the basis of the functional relevance of genes using GeneDistiller. Genes were ranked based on combinations of terms from the OMIM clinical synopsis for MIM 601088 and 601353 (i.e., cataract, deafness, mental retardation, facial dysmorphism, short stature, and seizure) as keywords, using similarity of expression patterns and protein-protein interactions as major weights. We obtained the highest score for MAF, a gene whose mutations had previously been reported to cause autosomal dominant congenital cataracts and lens abnormalities (MIM 610202).13,14 Sanger sequencing confirmed heterozygosity for the c.161C\u003eT (p.Ser54Leu) change in the proband, and sequencing of parental DNAs revealed only the reference allele, evidence for its de novo origin (Figure S1). STR genotyping (AmpFlSTR Identifiler Plus [Life Technologies]) confirmed paternity. The variant was documented in the proband’s skin fibroblasts as well as hair bulb and buccal epithelial cell specimens, strongly arguing against the possibility of a somatic event (Figure S1). All the other candidate variants turned out to be inherited from one of the unaffected parents (Table S4)."}