Identification of a frameshift mutation in codon 486 of <i>EXT1</i> gene.

<p>(a) Sanger sequencing detected the inserted base in the <i>EXT1</i> gene of all affected subjects. Red arrowhead denotes the mutation position; (b) intron-exon structure of <i>EXT1</i> gene. Mutated exon is indicated by red arrowhead; (c) comparison of the functional domains of EXT1 proteins encoded by mutated and normal <i>EXT1</i> genes; (d) multiple sequence alignment of codon 485 to codon 487. Codon 486 is highly conserved across various vertebrates.</p

Characteristics of study subjects in MO family.

<p>Characteristics of study subjects in MO family.</p

Immunohistochemisty screening of chondrocytes with functional EXT1 in the superficial layers of cartilage caps of MO(a) and extragenetic solitary chondroma(b) (40×).

<p>The chondrocytes with functional EXT1 in MO are less than those in extragenetic solitary chondroma.</p

Advantage of Whole Exome Sequencing over Allele-specific and Targeted Segment Sequencing, in Detection of Novel <i>TULP1</i> Mutation in Leber Congenital Amaurosis

<div><p></p><p><i>Background</i>: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.</p><p><i>Materials and Methods</i>: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.</p><p><i>Results</i>: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C > T, p.Arg361*) in <i>TULP1</i>, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.</p><p><i>Conclusions</i>: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.</p></div