SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer

<p>Abstract</p> <p>Background</p> <p>The <it>ERBB3 </it>gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of <it>ERBB3 </it>or the factors that bind them.</p> <p>Results</p> <p>In this study we identified three transcriptional enhancers at the <it>ERBB3 </it>locus and evaluated their regulatory potential <it>in vitro </it>in NC-derived cell types and <it>in vivo </it>in transgenic zebrafish. One enhancer, termed <it>ERBB3</it>_MCS6, which lies within the first intron of <it>ERBB3</it>, directs the highest reporter expression <it>in vitro </it>and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within <it>ERBB3</it>_MCS6 and demonstrate, <it>in vitro</it>, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous <it>Erbb3 </it>locus is dependent on Sox10. Further we demonstrate <it>in vitro </it>that Sox10 physically interacts with that <it>ERBB3</it>_MCS6. Consistent with its <it>in vitro </it>activity, we also show that <it>ERBB3</it>_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages <it>in vivo </it>in zebrafish in a manner consistent with <it>erbb3b </it>expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for <it>ERBB3</it>_MCS6 expression <it>in vivo </it>in zebrafish.</p> <p>Conclusions</p> <p>Taken collectively, our data suggest that <it>ERBB3 </it>may be directly regulated by SOX10, and that this control may in part be facilitated by <it>ERBB3</it>_MCS6.</p

A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement.

BACKGROUND: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. METHODS: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. RESULTS: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. CONCLUSIONS: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. TRIAL REGISTRATION NUMBERS: NCT01746121 and NCT02397824.journal articleresearch support, non-u.s. gov't2016 Feb2015 10 26importe

Visual outcomes and pacient satisfaction follwing implantation of a supplementary multifocal IOL in patients undergoing cataract surgery

Purpose: To assess visual outcomes and patient satisfaction following implantation of the Sulcoflex® multifocal intraocular lens (IOL; Rayner Intraocular Lenses Ltd., Hove, UK) in a procedure combining capsular bag lens implantation with sulcus placement of the Sulcoflex® IOL. Setting: Instituto de Oftalmologia de Assis, Assis, SP, Brazil. Methods: Cataract patients > 45 years, with hyperopia ≥ 1.50 D and potential acuity measurement ≥ 20/30 undergoing Sulcoflex® multifocal IOL implantation were included. Monocular and binocular uncorrected near and distance visual acuity (VA) were evaluated at five days, one month, and three months postoperatively. Contrast sensitivity and refraction were measured in a subset of patients three months postoperatively. Patient satisfaction was assessed one month postoperative. Results: This non-consecutive case series comprised 25 eyes of 13 patients. Eleven eyes (52%) had pre-existing retinal pathologies. Monocular distance VA improved significantly at all follow-up visits. At final follow-up, 88% of eyes had monocular uncorrected distance VA (UDVA) of at least 20/25 and 24% had monocular UDVA of 20/20. All eyes had binocular UDVA of at least 20/25, and 58% had binocular UDVA of 20/20. Monocular uncorrected near vision (UNVA) was J1 in 68% of eyes and all patients had binocular UNVA of J1. Of all eyes studied, 92% and 58% achieved a spherical equivalent within 1 D and −0.5 D, respectively. The majority of patients reported satisfaction with visual outcomes. Complications included a postoperative intraocular pressure spike in four eyes. Conclusion: The Sulcoflex® multifocal IOL improves near and distance VA in cataract patients with retinal abnormalities and good VA potential

A Polymorphic 3’UTR Element in <i>ATP1B1</i> Regulates Alternative Polyadenylation and Is Associated with Blood Pressure

<div><p>Although variants in many genes have previously been shown to be associated with blood pressure (BP) levels, the molecular mechanism underlying these associations are mostly unknown. We identified a multi-allelic T-rich sequence (TRS) in the 3’UTR of <i>ATP1B1</i> that varies in length and sequence composition (T_22-27 and T₁₂GT ₃GT₆). The 3’UTR of <i>ATP1B1</i> contains 2 functional polyadenylation signals and the TRS is downstream of the proximal polyadenylation site (A2). Therefore, we hypothesized that alleles of this TRS might influence <i>ATP1B1</i> expression by regulating alternative polyadenylation. <i>In vitro</i>, the T₁₂GT ₃GT₆ allele increases polyadenylation at the A2 polyadenylation site as compared to the T₂₃ allele. Consistent with our hypothesis, the relative abundance of the A2-polyadenylated <i>ATP1B1</i> mRNA was higher in human kidneys with at least one copy of the T₁₂GT ₃GT₆ allele than in those lacking this allele. The T₁₂GT ₃GT₆ allele is also associated with higher systolic BP (beta = 3.3 mmHg, <i>p</i> = 0.014) and diastolic BP (beta = 2.4 mmHg, <i>p</i> = 0.003) in a European-American population. Therefore, we have identified a novel multi-allelic TRS in the 3’UTR of <i>ATP1B1</i> that is associated with higher BP and may mediate its effect by regulating the polyadenylation of the <i>ATP1B1</i> mRNA.</p> </div

The T₁₂GT ₃GT₆ allele increases polyadenylation at the A2 polyadenylation site.

<p>A) Schematic of the pRIG vector. MCS: multiple cloning site, where the tested sequences were inserted, pCMV: cytomegalovirus minimal promoter, RFP: red fluorescent protein, IRES: internal ribosomal entry site, eGFP: enhanced green fluorescent protein, SV40 pA: SV40 polyadenylation signal. B) Dot plots from FACS analysis of the expression of RFP and eGFP in HEK293T cells transfected with the T₂₃ and T ₁₂GT ₃GT₆ alleles in the pRIG vector. Each dot represents a cell and the X- and Y-axes are the fluorescence intensity of eGFP and RFP, respectively. C) The ratio of RFP to eGFP expression in pRIG with no insert, T₂₃, and T₁₂GT ₃GT₆.</p

The T₁₂GT ₃GT₆ allele regulates the polyadenylation of <i>ATP1B1</i> mRNA in human tissues

<p>A) Schematic of the primer design used to distinguish between the A2- and A5- polyadenylated mRNAs. The A2-R primer had a 5’ T₁₅ tail to selectively amplify A2-polyadenylated mRNA. Total <i>ATP1B1</i> mRNA was measured using primers that amplified across the exon 5/exon 6 junction. B) Real-time PCR of mRNA from human kidneys. The A2-polyadenylated transcript was quantified relative to total <i>ATP1B1</i> mRNA. 0 and 1 indicate subjects without the T ₁₂GT ₃GT₆ allele and with 1 copy of the T ₁₂GT ₃GT₆ allele, respectively. Error bars represent standard errors. *<i>p</i> < 0.0001. C) Real-time PCR of mRNA from human lymphocytes. Expression of the A2- and A5-polyadenylated mRNAs relative to total <i>ATP1B1</i> mRNA in lymphocytes from subjects with 0 or at least 1 copy of the T ₁₂GT ₃GT₆ allele. *<i>p</i> = 0.0001 between the A5-polyadenylated transcript levels in the 2 groups. Data represent the average across samples from each genotype group with standard error. D) Expression of ATP1B1 protein in lymphocytes from individuals with 0 or at least 1 copy of the T ₁₂GT ₃GT₆ allele. Tubulin was used as a loading control. The graph quantifies the average level of ATP1B1 protein relative to tubulin with standard error. *<i>p</i> = 0.05.</p

Structure and genotyping of the T-rich sequence in <i>ATP1B1</i>.

<p>A) Genomic architecture of the <i>ATP1B1</i> gene. The red box highlights the 3’UTR, which is enlarged and shown below. TGA: translational stop codon, *: G/A: SNP rs12079745, A2: A2 polyadenylation signal, TRS: T-rich element, A5: A5 polyadenylation signal. B) Representative Sanger sequencing chromatograms of the T₂₃ and T ₁₂GT ₃GT₆ alleles. Alleles are named based on the portion of the TRS that is unique to each allele (denoted by a horizontal bar). C) Sequence alignment of the TRS across mammals. The A2 polyadenylation signal and the TRS are highlighted in red and green respectively. D) Aligned are the 2a consensus sequence of the CstF binding site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076290#B38" target="_blank">38</a>] and the sequences of the TRS with the T₂₃ and T₁₂GT ₃GT₆ alleles. Asterisks mark the nucleotides in the TRS that deviate from the consensus CstF binding sequence. Y = C/T, N = A/C/G/T.</p