Mutation of Rubie, a Novel Long Non-Coding RNA Located Upstream of Bmp4, Causes Vestibular Malformation in Mice

Background: The vestibular apparatus of the vertebrate inner ear uses three fluid-filled semicircular canals to sense angular acceleration of the head. Malformation of these canals disrupts the sense of balance and frequently causes circling behavior in mice. The Epistatic circler (Ecl) is a complex mutant derived from wildtype SWR/J and C57L/J mice. Ecl circling has been shown to result from the epistatic interaction of an SWR-derived locus on chromosome 14 and a C57L-derived locus on chromosome 4, but the causative genes have not been previously identified. Methodology/Principal Findings: We developed a mouse chromosome substitution strain (CSS-14) that carries an SWR/J chromosome 14 on a C57BL/10J genetic background and, like Ecl, exhibits circling behavior due to lateral semicircular canal malformation. We utilized CSS-14 to identify the chromosome 14 Ecl gene by positional cloning. Our candidate interval is located upstream of bone morphogenetic protein 4 (Bmp4) and contains an inner ear-specific, long non-coding RNA that we have designated Rubie (RNA upstream of Bmp4 expressed in inner ear). Rubie is spliced and polyadenylated, and is expressed in developing semicircular canals. However, we discovered that the SWR/J allele of Rubie is disrupted by an intronic endogenous retrovirus that causes aberrant splicing and premature polyadenylation of the transcript. Rubie lies in the conserved gene desert upstream of Bmp4, within a region previously shown to be important for inner ear expression of Bmp4. We found that the expression patterns of Bmp4 and Rubie are nearly identical in developing inner ears

Challenges in Whole Exome Sequencing: An Example from Hereditary Deafness

Whole exome sequencing provides unprecedented opportunities to identify causative DNA variants in rare Mendelian disorders. Finding the responsible mutation via traditional methods in families with hearing loss is difficult due to a high degree of genetic heterogeneity. In this study we combined autozygosity mapping and whole exome sequencing in a family with 3 affected children having nonsyndromic hearing loss born to consanguineous parents. Two novel missense homozygous variants, c.508C>A (p.H170N) in GIPC3 and c.1328C>T (p.T443M) in ZNF57, were identified in the same ∼6 Mb autozygous region on chromosome 19 in affected members of the family. Both variants co-segregated with the phenotype and were absent in 335 ethnicity-matched controls. Biallelic GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss. Thus we conclude that the hearing loss in the family described in this report is caused by a novel missense mutation in GIPC3. Identified variant in GIPC3 had a low read depth, which was initially filtered out during the analysis leaving ZNF57 as the only potential causative gene. This study highlights some of the challenges in the analyses of whole exome data in the bid to establish the true causative variant in Mendelian disease

Toll-Like Receptor 2 Induced Angiogenesis and Invasion Is Mediated through the Tie2 Signalling Pathway in Rheumatoid Arthritis

BACKGROUND: Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo. METHODS: Microvascular endothelial cells (HMVEC) and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml). Angiopoietin 2 (Ang2), Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml) and matched IgG isotype control Ab (10 ug/ml). RESULTS: Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenic tube formation (p<0.05), and upregulated Ang2 production in HMVEC (p<0.05) and RA synovial explants (p<0.05). Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI) to 617±103 (p<0.01). TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05). Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05). CONCLUSION: TLR2 activation promotes angiogenesis, cell adhesion and invasion, effects that are in part mediated through the Tie2 signalling pathway, key mechanisms involved in the pathogenesis of RA

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells.

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule 'programmed death-ligand 1', whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis

Ovarian stimulation for IVF and risk of primary breast cancer in BRCA1/2 mutation carriers

BACKGROUND: The effect of in vitro fertilisation (IVF) on breast cancer risk for BRCA1/2 mutation carriers is rarely examined. As carriers may increasingly undergo IVF as part of preimplantation genetic diagnosis (PGD), we examined the impact of ovarian stimulation for IVF on breast cancer risk in BRCA1/2 mutation carriers. METHODS: The study population consisted of 1550 BRCA1 and 964 BRCA2 mutation carriers, derived from the nationwide HEBON study and the nationwide PGD registry. Questionnaires, clinical records and linkages with the Netherlands Cancer Registry were used to collect data on IVF exposure, risk-reducing surgeries and cancer diagnosis, respectively. Time-dependent Cox regression analyses were conducted, stratified for birth cohort and adjusted for subfertility. RESULTS: Of the 2514 BRCA1/2 mutation carriers, 3% (n = 76) were exposed to ovarian stimulation for IVF. In total, 938 BRCA1/2 mutation carriers (37.3%) were diagnosed with breast cancer. IVF exposure was not associated with risk of breast cancer (HR: 0.79, 95% CI: 0.46-1.36). Similar results were found for the subgroups of subfertile women (n = 232; HR: 0.73, 95% CI: 0.39-1.37) and BRCA1 mutation carriers (HR: 1.12, 95% CI: 0.60-2.09). In addition, age at and recency of first IVF treatment were not associated with breast cancer risk. CONCLUSION: No evidence was found for an association between ovarian stimulation for IVF and breast cancer risk in BRCA1/2 mutation carriers