Comprehensive Molecular Diagnosis of Bardet-Biedl Syndrome by High-Throughput Targeted Exome Sequencing

<div><p>Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder with significant genetic heterogeneity. BBS is linked to mutations in 17 genes, which contain more than 200 coding exons. Currently, BBS is diagnosed by direct DNA sequencing for mutations in these genes, which because of the large genomic screening region is both time-consuming and expensive. In order to develop a practical method for the clinic diagnosis of BBS, we have developed a high-throughput targeted exome sequencing (TES) for genetic diagnosis. Five typical BBS patients were recruited and screened for mutations in a total of 144 known genes responsible for inherited retinal diseases, a hallmark symptom of BBS. The genomic DNA of these patients and their families were subjected to high-throughput DNA re-sequencing. Deep bioinformatics analysis was carried out to filter the massive sequencing data, which were further confirmed through co-segregation analysis. TES successfully revealed mutations in BBS genes in each patient and family member. Six pathological mutations, including five novel mutations, were revealed in the genes <i>BBS2</i>, <i>MKKS</i>, <i>ARL6</i>, <i>MKS1</i>. This study represents the first report of targeted exome sequencing in BBS patients and demonstrates that high-throughput TES is an accurate and rapid method for the genetic diagnosis of BBS.</p></div

Conservation of Missense Mutations in BBS Genes.

<p>A: Coverage of the reads > 10 and > 4 in each sample; B: Gene and protein structures of <i>BBS2</i>, including p.I188fs200X and p.R480X..The yellow region indicates the coiled coil domain; the blue indicates the flanking peptide chain region; the red box represents the frame shift peptide chain (p.I188fs200X). C: All the missense mutations of BBS genes were located within a highly conserved region compared to different species. D: Seventeen genes were mapped and identified with BBS so far, in which patients with <i>BBS1</i> or <i>BBS10</i> accounts for more than 20% respectively. E: More than 10% reported mutations of <i>BBS2</i> are located at exon 2, 4, 6, while the two novel mutations discovered in this study were located at exon 5 and 12. Asterisk represents the locations of mutations.</p

Identified Mutations in <i>BBS</i> Genes.

<p>Identified Mutations in <i>BBS</i> Genes.</p

Causative Genes of Bardet-Biedl Syndrome.

<p>Seventeen <i>BBS</i> genes have been mapped and identified, including a total of 242 coding exons.</p

Five BBS Pedigrees underwent Comprehensive Mutational Screening.

<p>The pedigrees of five families with Bardet-beidl syndrome (BBS) are shown. A: In pedigree of WZ036, mutation c.563C>T was re-confirmed by restriction fragment length polymorphism method with restrictionenzymeEcoRV. The homozygous sample (WZ036-I:2) was not digested, while the heterozygous sample (WZ036-I:1) was partially digested. B, C, D: <i>BBS</i> genes mutations were detected by TES, and confirmed by direct sequencing with intra-familiar members. Squares indicate males; circles indicate females; solid symbols indicate affected; open symbols indicate unaffected; Bar on the symbol indicates the proband examined by TES; WT, wildtype; M indicates mutation.</p