Retinal miRNAs variations in a large cohort of inherited retinal disease

<p><b>Background</b>: Although great efforts have been paid on identification of genetic predisposition in the inherited retinal disease (IRD), genetic causes of a large proportion of patients remain a mystery. This dilemma makes us attempt to speculate that genetic components other than coding genes might be an additional pool predisposing IRD. In this study, we aim to perform a mutational screening in a large cohort of IRD patients with a particular focus on retina-specific or abundant microRNAs (miRs).</p> <p><b>Material and methods</b>: A total of 324 unrelated patients with IRD were recruited. Targeted next-generation sequencing (tNGS) was performed to survey genetic mutations in 32 known miRs highly expressed in the retina, followed by validation with Sanger sequencing, co-segregation analysis in each family, and computational assessments.</p> <p><b>Results</b>: Novel genotype-phenotype associations have been uncovered. In total, six different variants in the miRs were identified, including four rare ones, miR-216a (n.56C>A), miR-216b (n.43_44insG), miR-7–2 (n.107C>T), and miR-7–3 (n.95G>A). The other two variants, miR-182 (n.106G>A) and miR-216a (n.105T>A), were considered as polymorphic.</p> <p><b>Conclusions</b>: We for the first time screened candidate retinal miRs in patients with IRD. Although there is no convincing evidence that these variants are responsible for the IRD, the results enhance the current knowledge of the associations between IRD and miRNAs variants.</p

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

Targeted Exome Sequencing Identified Novel <i>USH2A</i> Mutations in Usher Syndrome Families

<div><p>Usher syndrome (USH) is a leading cause of deaf-blindness in autosomal recessive trait. Phenotypic and genetic heterogeneities in USH make molecular diagnosis much difficult. This is a pilot study aiming to develop an approach based on next-generation sequencing to determine the genetic defects in patients with USH or allied diseases precisely and effectively. Eight affected patients and twelve unaffected relatives from five unrelated Chinese USH families, including 2 pseudo-dominant ones, were recruited. A total of 144 known genes of inherited retinal diseases were selected for deep exome resequencing. Through systematic data analysis using established bioinformatics pipeline and segregation analysis, a number of genetic variants were released. Eleven mutations, eight of them were novel, in the <i>USH2A</i> gene were identified. Biparental mutations in <i>USH2A</i> were revealed in 2 families with pseudo-dominant inheritance. A proband was found to have triple mutations, two of them were supposed to locate in the same chromosome. In conclusion, this study revealed the genetic defects in the <i>USH2A</i> gene and demonstrated the robustness of targeted exome sequencing to precisely and rapidly determine genetic defects. The methodology provides a reliable strategy for routine gene diagnosis of USH.</p></div

MOESM1 of Identification of novel mutations by targeted exome sequencing and the genotype-phenotype assessment of patients with achromatopsia

Additional file 1: Table S1. The list of 201 disease-causing genes of capture panel

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

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

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

Genotype-Phenotype Association Study Reveals <i>CFI</i>-Rs13104777 to be a Protective Genetic Marker Against Acute Anterior Uveitis

<p><i>Purpose</i>: To investigate the roles of <i>CFI</i>, genotype-phenotype associations were identified in AAU.</p> <p><i>Methods</i>: A case–control study was conducted in a total of 575 subjects consisting of 279 AAU patients and 296 healthy controls. Genotypic analyses were performed using Sequenom MassARRAY technology. Analyses were stratified to a series of clinical ophthalmic confounding factors.</p> <p><i>Results</i>: A lower frequency of the <i>CFI</i>-rs13104777 C allele was found in the AAU cohort compared with the controls, and, thus, was significantly associated with AAU pathogenesis (<i>p</i> = 0.041, OR = 0.712, 95% CI: 0.513–0.987). Stratified analysis also demonstrated the associations may differ depending on the HLA-B27 status and laterality status.</p> <p><i>Conclusions</i>: This study has revealed a significant genetic role for <i>CFI</i>-rs13104777 in AAU. This influence may be dependent on human leukocyte antigen (HLA)-B27 and disease laterality. Overall, the results provide evidence for a pathogenic role for <i>CFI</i> in AAU and expand our knowledge on the genetic basis of AAU.</p