RAD51 and Breast Cancer Susceptibility: No Evidence for Rare Variant Association in the Breast Cancer Family Registry Study

Background

Although inherited breast cancer has been associated with germline mutations in genes that are functionally involved in the DNA homologous recombination repair (HRR) pathway, including BRCA1, BRCA2, TP53, ATM, BRIP1, CHEK2 and PALB2, about 70% of breast cancer heritability remains unexplained. Because of their critical functions in maintaining genome integrity and already well-established associations with breast cancer susceptibility, it is likely that additional genes involved in the HRR pathway harbor sequence variants associated with increased risk of breast cancer. RAD51 plays a central biological function in DNA repair and despite the fact that rare, likely dysfunctional variants in three of its five paralogs, RAD51C, RAD51D, and XRCC2, have been associated with breast and/or ovarian cancer risk, no population-based case-control mutation screening data are available for the RAD51 gene. We thus postulated that RAD51 could harbor rare germline mutations that confer increased risk of breast cancer.

Methodology/Principal Findings

We screened the coding exons and proximal splice junction regions of the gene for germline sequence variation in 1,330 early-onset breast cancer cases and 1,123 controls from the Breast Cancer Family Registry, using the same population-based sampling and analytical strategy that we developed for assessment of rare sequence variants in ATM and CHEK2. In total, 12 distinct very rare or private variants were characterized in RAD51, with 10 cases (0.75%) and 9 controls (0.80%) carrying such a variant. Variants were either likely neutral missense substitutions (3), silent substitutions (4) or non-coding substitutions (5) that were predicted to have little effect on efficiency of the splicing machinery.

Conclusion

Altogether, our data suggest that RAD51 tolerates so little dysfunctional sequence variation that rare variants in the gene contribute little, if anything, to breast cancer susceptibility.     

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 ¹. Because of the ease of genetic manipulation in mice, these models have also been developed in this species ^2-3.Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume ⁴. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.     

Defining an adequate sample of earlywood vessels for retrospective injury detection in diffuse-porous species

Vessels of broad-leaved trees have been analyzed to study how trees deal with various environmental factors. Cambial injury, in particular, has been reported to induce the formation of narrower conduits. Yet, little or no effort has been devoted to the elaboration of vessel sampling strategies for retrospective injury detection based on vessel lumen size reduction. To fill this methodological gap, four wounded individuals each of grey alder (Alnus incana (L.) Moench) and downy birch (Betula pubescens Ehrh.) were harvested in an avalanche path. Earlywood vessel lumina were measured and compared for each tree between the injury ring built during the growing season following wounding and the control ring laid down the previous year. Measurements were performed along a 10 mm wide radial strip, located directly next to the injury. Specifically, this study aimed at (i) investigating the intra-annual duration and local extension of vessel narrowing close to the wound margin and (ii) identifying an adequate sample of earlywood vessels (number and intra-ring location of cells) attesting to cambial injury. Based on the results of this study, we recommend analyzing at least 30 vessels in each ring. Within the 10 mm wide segment of the injury ring, wound-induced reduction in vessel lumen size did not fade with increasing radial and tangential distances, but we nevertheless advise favoring early earlywood vessels located closest to the injury. These findings, derived from two species widespread across subarctic, mountainous, and temperate regions, will assist retrospective injury detection in Alnus, Betula, and other diffuse-porous species as well as future related research on hydraulic implications after wounding.     

LETM Proteins Play a Role in the Accumulation of Mitochondrially Encoded Proteins in Arabidopsis thaliana and AtLETM2 Displays Parent of Origin Effects

The Arabidopsis thaliana genome contains two genes with homology to the mitochondrial protein LETM1 (leucine zipper-EF-hand-containing transmembrane protein). Inactivation of both genes, Atletm1 and Atletm2, together is lethal. Plants that are hemizygous for AtLETM2 and homozygous for Atletm1 (letm1(−/−) LETM2(+/−)) displayed a mild retarded growth phenotype during early seedling growth. It was shown that accumulation of mitochondrial proteins was reduced in hemizygous (letm1(−/−) LETM2(+/−)) plants. Examination of respiratory chain proteins by Western blotting, blue native PAGE, and enzymatic activity assays revealed that the steady state level of ATP synthase was reduced in abundance, whereas the steady state levels of other respiratory chain proteins remained unchanged. The absence of a functional maternal AtLETM2 allele in an Atletm1 mutant background resulted in early seed abortion. Reciprocal crosses revealed that maternally, but not paternally, derived AtLETM2 was absolutely required for seed development. This requirement for a functional maternal allele of AtLETM2 was confirmed using direct sequencing of reciprocal crosses of Col-0 and Ler accessions. Furthermore, AtLETM2 promoter β-glucuronidase constructs displayed exclusive maternal expression patterns.     

The protein phosphatase PP2A/Bα binds to the microtubule-associated proteins Tau and MAP2 at a motif also recognized by the kinase Fyn: implications for tauopathies

The predominant brain microtubule-associated proteins MAP2 and tau play a critical role in microtubule cytoskeletal organization and function. We have previously reported that PP2A/Bα, a major protein phosphatase 2A (PP2A) holoenzyme, binds to and dephosphorylates tau, and regulates microtubule stability. Here, we provide evidence that MAP2 co-purifies with and is dephosphorylated by endogenous PP2A/Bα in bovine gray matter. It co-localizes with PP2A/Bα in immature and mature human neuronal cell bodies. PP2A co-immunoprecipitates with and directly interacts with MAP2. Using in vitro binding assays, we show that PP2A/Bα binds to MAP2c isoforms through a region encompassing the microtubule-binding domain and upstream proline-rich region. Tau and MAP2 compete for binding to and dephosphorylation by PP2A/Bα. Remarkably, the protein-tyrosine kinase Fyn, which binds to the proline-rich RTPPKSP motif conserved in both MAP2 and tau, inhibits the interaction of PP2A/Bα with either tau or MAP2c. The corresponding synthetic RTPPKSP peptide, but not the phosphorylated RpTPPKSP version, competes with Tau and MAP2c for binding to PP2A/Bα. Significantly, down-regulation of PP2A/Bα and deregulation of Fyn-Tau protein interactions have been linked to enhanced tau phosphorylation in Alzheimer disease. Together, our results suggest that PP2A/Bα is part of segregated MAP2 and tau signaling scaffolds that can coordinate the action of key kinases and phosphatases involved in modulating neuronal plasticity. Deregulation of these compartmentalized multifunctional protein complexes is likely to contribute to tau deregulation, microtubule disruption, and altered signaling in tauopathies.     

Bluetongue: a historical and epidemiological perspective with the emphasis on South Africa

ABSTRACT: Bluetongue (BT) is a non-contagious, infectious, arthropod transmitted viral disease of domestic and wild ruminants that is caused by the bluetongue virus (BTV), the prototype member of the Orbivirus genus in the family Reoviridae. Bluetongue was first described in South Africa, where it has probably been endemic in wild ruminants since antiquity. Since its discovery BT has had a major impact on sheep breeders in the country and has therefore been a key focus of research at the Onderstepoort Veterinary Research Institute in Pretoria. Several key discoveries were made at this Institute, including the demonstration that the aetiological agent of BT was a dsRNA virus that is transmitted by Culicoides midges and that multiple BTV serotypes circulate in nature. It is currently recognized that BT is endemic throughout most of South Africa and 22 of the 26 known serotypes have been detected in the region. Multiple serotypes circulate each vector season with the occurrence of different serotypes depending largely on herd -immunity. Indigenous sheep breeds, cattle and wild ruminants are frequently infected but rarely demonstrate clinical signs, whereas improved European sheep breeds are most susceptible. The immunization of susceptible sheep remains the most effective and practical control measure against BT. In order to protect sheep against multiple circulating serotypes, three pentavalent attenuated vaccines have been developed. Despite the proven efficacy of these vaccines in protecting sheep against the disease, several disadvantages are associated with their use in the field.