26 research outputs found

    Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Significance in Inherited Retinal Dystrophies

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    <div><p>This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in <i>RHO</i>, c.937-2_944del, (ii) one rare homozygous mutation in <i>C2orf71</i>, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in <i>ABCA4,</i> c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of <i>RPGR</i>. The molecular findings for <i>RHO</i> and <i>C2orf71</i> confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two <i>ABCA4</i> mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the <i>RPGR</i> mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.</p></div

    Segregation studies of identified variants in the four analyzed families.

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    <p>Below the individuals, genotypes are presented for each change segregating with RP. Index patients are indicated with a black arrow. [M];[M] represents homozygous mutants; [M];[ = ] indicates heterozygous carriers, [ = ];[ = ] indicates individuals carrying two wild-type alleles, whereas [m] represents hemizygous individuals. NA means non available DNA sample. W means samples processed by WES.</p

    Novel pathogenic variants identified in this study.

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    <p>A) Chromatograms of wild type (WT) <i>RHO</i> DNA sequence (NM_000539.3) and RP subject (MUT) showing the heterozygous mutation c.937-2_944delAGTTCCGGAA. SAS: Splice Acceptor Site. Exon 5 is highlighted in blue. B) The <i>RHO</i> gene structure is composed of 5 exons that are indicated as filled boxes while 5′ and 3′ UTRs are shown as open boxes. The canonical SAS of exon 5 is in blue and the predicted cryptic SAS (CSAS) is in red and indicated with an asterisk. The WT protein product (left) has 348 aminoacids (aa) while the predicted mutant product (MUT) only 326 aa. C) Chromatograms of WT <i>C2orf71</i> (NM_001029883.2) DNA sequence and RP subject (MUT) showing the homozygous mutation c.1795T>C. D) Alignment of C2orf71 orthologous protein sequences showing conservation of the mutated residue p.Cys599Arg among mammals. E) Prediction of disulfide bonds formation for WT and mutant (MUT) C2orf71 proteins. According to the prediction, in the mutant protein structure, two of the disulfide bonds have been disrupted while a new one has been created. The disulfide bonds altered are in red.</p

    Clinical characteristics of affected family members.

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    <p># Consanguineous family</p>†<p>Previously reported by Maugeri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116176#pone.0116176-Maugeri1" target="_blank">[24]</a> in a Stargardt patient in heterozygous state.</p>‡<p>Previously reported by Lewis et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116176#pone.0116176-Lewis1" target="_blank">[25]</a> in a Stargardt patient in heterozygous state.</p>¥<p>Previously reported by Vervoort et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116176#pone.0116176-Vervoort1" target="_blank">[23]</a>.</p><p>N.A.: Not available.</p><p>N.R.: Not recordable.</p><p>Clinical characteristics of affected family members.</p

    <i>PHOX2B</i> deletion detected in exon 2.

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    <p>(A) Schematic representation of <i>PHOX2B</i> showing both nucleotide and amino acid sequence of exon 2; the 18bp corresponding to the deletion are boxed. (B) Sequence chromatogram of <i>PHOX2B</i> exon 2; the arrow indicates the beginning of c.393_410del18bp. (C) Pedigree and agarose gel electrophoresis of the amplification product of exon 2 in the patient and her parents.</p

    Amplification and dHPLC conditions for <i>PHOX2B</i> genomic sequence analysis.

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    <p>Bp: size of the fragment; B: buffer concentration; CM: MgCl2 concentration; DNTPs: dNTPs concentration; P: primers concentration; Taq: units of Taq polymerase; DNA: DNA quantity; D: Denaturing temperature; DT: Denaturing time; H: Hybridization temperature; HT: Hybridization time; E: Extension temperature; ET: Extension time; T dHPLC: Injection temperature in dHPLC. The general PCR protocol was [95°C-5′]→[(D–DT) →(H–HT) → (E–ET)]×35cycles→[72°C–7′]→4°C.</p

    Structure of the homeodomain.

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    <p>(A) Multiple sequence alignment among different species. Aminoacid residues involved in the deletion are included within a red box. (B) Three-dimensional representation of the interaction between the <i>Drosophila sp.</i> homeodomain and the target DNA sequence (orange). Aminoacids encompassed by the deletion are represented in green. C) Prediction of the electrostatic potential for the wild type protein (up) and the mutated one (down). Negative charge is represented in blue and positive charge in red.</p

    Immunostaining of Semaphorin 3D in colon from controls and HSCR patients.

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    <p>The SEMA3D staining illustrated was present at smooth muscle (C, D) and submucous (E, F) layers, as well as in myenteric (G, H) and submucous plexuses (I, J) either in normal colon (A, C, E, G, I) and patients with E198K-3D mutation (B,D, F, H, J). Scale bars: A, B = 200 µm and the rest of pictures = 10 µm.</p

    Immunohistochemical detection of A131T-<i>SEMA3A,</i> S598G-<i>SEMA3A</i> and E198K-<i>SEMA3</i>D mutant proteins in FFPE colon samples.

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    <p>Immunohistochemistry analysis of A131T-3A, S598G-3A and E198K-3D mutant proteins in smooth muscle layer of control samples (3A: A, D; 3D: H, K) and colon tissue from patients with A131T-3A (ganglionic: B, E; aganglionic: C, F), S598G-3A (G, J) and E198K-3D mutations (I, L) was performed. Scale bars: A, B, C, G, H, I = 200 µm and the rest of the pictures = 10 µm.</p
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