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

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

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

Rod count and morphological parameters measured from <i>pde6c</i> ^<i>w59</i> retinas with and without SchB treatment.

<p><i>Pde6c</i>^<i>w59</i> retinas were microdissected from 6-dpf larvae. These larvae were either exposed to DMSO carrier (<i>pde6c</i> DMSO) or SchB (<i>pde6c</i> SchB), under the same conditions that induced a positive effect on the Light-On VMR. WT retinas were included as an internal control to compare with the <i>pde6c</i> DMSO, as a previous study indicated a qualitative volume increase in the <i>pde6c</i> ^<i>w59</i> rods [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.ref022" target="_blank">22</a>]. All these retinas were subjected to immunostaining with the rod marker 4c12 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.ref022" target="_blank">22</a>] and imaged by confocal microscopy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.g004" target="_blank">Fig 4A–4C</a>). Then, cell count and morphological analyses of rods were conducted in a central-retinal region on the dorsal side of the optic nerve. This mature region of the retina displayed the most prominent rod degeneration in Stearns and colleagues’ study. Analyzing this region would maximize the chance to detect morphological changes. For cell-count analysis, 4, 6, and 8 retinas were used for WT, <i>pde6c</i> DMSO and <i>pde6c</i> SchB respectively. From these retinas, rods were segmented from the 3D confocal z-stacks using parameters outlined in the methods. The subsequent morphological analyses used only those individual rods that could be successfully segmented from the images and were not distorted by their proximity to the optic nerve. The total rod numbers used in the analyses were 116, 30, and 101 for WT, <i>pde6c</i> DMSO and <i>pde6c</i> SchB respectively. The <i>p-</i>values less than 0.05 are highlighted in boldface. The results not only confirm that <i>pde6c</i> ^<i>w59</i> rods were larger than WT rods as previously observed by Stearns and colleagues, but also reveal that the SchB treatment reduced the abnormal volume of <i>pde6c</i> ^<i>w59</i> rods to a level similar to WT. Furthermore, SchB did not affect other morphological parameters and cell count of the <i>pde6c</i> ^<i>w59</i> rods. The raw data for Table 2 are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.s006" target="_blank">S3 File</a>.</p

The <i>pde6c</i>^<i>w59</i> larvae displayed an attenuated VMR.

<p>(A–H) The activity plots of WT larvae (black traces; N = 34) and <i>pde6c</i> ^<i>w59</i> larvae (red traces; N = 34) from 5 to 8 dpf. Figures A–D show the Light-On VMR, whereas Figures E–H show the Light-Off VMR. The VMR assay consists of three consecutive trials of a Light-On and a Light-Off stimulus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.s001" target="_blank">S1 Fig</a>; also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#sec002" target="_blank">methods</a>). Each stimulus would last for 30 mins. During the experiment, the larval movement was recorded by computer as movement duration per second. Then, the activity of the same type of larvae was averaged across the three Light-On or Light-Off trials and plotted in the figures. The solid traces in each plot show the mean activities from 60 s before light change to 60 s after light change, whereas the ribbons surrounding these activity traces indicate the corresponding standard error of the mean. At the top of the plots, the white and black bars indicate light and dark phases respectively. These plots show that the <i>pde6c</i>^<i>w59</i> larvae displayed a substantially attenuated VMR compared with the WT larvae. The problem was likely caused by the retinal degeneration in the <i>pde6c</i>^<i>w59</i> mutants, which affected their capability to sense light change in the environment. The raw data for Fig 2 are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.s004" target="_blank">S1 File</a>.</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

The survival counts of zebrafish larvae after exposure to various concentrations of SchB from 3 to 8 dpf.

<p>Normal WT embryos were exposed to 1.875, 3.75, 7.5 and 15 μM of SchB or DMSO carrier as controls. The treatment took place from 3 to 8 dpf. The survival of larvae in each condition was counted every day. The DMSO percentage in the controls was 0.025% and 0.2%. These were the corresponding DMSO amounts used as the drug carrier in treatment groups with 1.875 and 15 μM SchB respectively. Both DMSO percentages were lower than the commonly used percentage (< 0.3%) in other zebrafish drug studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.ref060" target="_blank">60</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149663#pone.0149663.ref061" target="_blank">61</a>]. Hence, the use of these specific amounts of DMSO should not affect the survival of the larvae. Indeed, they did not. In each experimental group, 60 embryos were used. Then, the survival was counted every day from 3 to 8 dpf. The 3-dpf count was obtained immediately after the addition of chemicals.</p

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

Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

<div><p>Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in <i>ABCA4</i> and two in <i>PROM1</i> were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in <i>PROM1</i> gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.</p></div