Assembly of the ß4-Integrin Interactome Based on Proximal Biotinylation in the Presence and Absence of Heterodimerization.

Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific ß4-integrin that, as α6ß4-heterodimer, forms the core of HDs. The analysis identified ∼150 proteins that were specifically labeled by BirA-tagged integrin-ß4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-ß4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate ß4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of α6ß4-heterodimers, the assembly of ß4-interactome was not strictly dependent on α6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the ß4-integrin.

RAD51B in Familial Breast Cancer.

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11-1.19, P = 8.88 x 10-16) and among familial cases (OR: 1.24, 95% CI: 1.16-1.32, P = 6.19 x 10-11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk.

Rare copy number variants observed in hereditary breast cancer cases disrupt genes in estrogen signaling and TP53 tumor suppression network.

Breast cancer is the most common cancer in women in developed countries, and the contribution of genetic susceptibility to breast cancer development has been well-recognized. However, a great proportion of these hereditary predisposing factors still remain unidentified. To examine the contribution of rare copy number variants (CNVs) in breast cancer predisposition, high-resolution genome-wide scans were performed on genomic DNA of 103 BRCA1, BRCA2, and PALB2 mutation negative familial breast cancer cases and 128 geographically matched healthy female controls; for replication an independent cohort of 75 similarly mutation negative young breast cancer patients was used. All observed rare variants were confirmed by independent methods. The studied breast cancer cases showed a consistent increase in the frequency of rare CNVs when compared to controls. Furthermore, the biological networks of the disrupted genes differed between the two groups. In familial cases the observed mutations disrupted genes, which were significantly overrepresented in cellular functions related to maintenance of genomic integrity, including DNA double-strand break repair (P = 0.0211). Biological network analysis in the two independent breast cancer cohorts showed that the disrupted genes were closely related to estrogen signaling and TP53 centered tumor suppressor network. These results suggest that rare CNVs represent an alternative source of genetic variation influencing hereditary risk for breast cancer.

Mutation screening of the RNF8, UBC13 and MMS2 genes in Northern Finnish breast cancer families.

BACKGROUND: Currently known susceptibility genes such as BRCA1 and BRCA2 explain less than 25% of familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. RNF8, UBC13 and MMS2 are involved in the DNA damage response pathway and play important roles in BRCA1-mediated DNA damage recognition. Based on the evidence that several players in the ubiquitin-mediated BRCA1-dependent DDR seem to contribute to breast cancer predisposition, RNF8, UBC13 and MMS2 were considered plausible candidate genes for susceptibility to breast cancer. METHODS: The entire coding region and splice junctions of RNF8, UBC13 and MMS2 genes were screened for mutations in affected index cases from 123 Northern Finnish breast cancer families by using conformation sensitive gel electrophoresis, high resolution melting (HRM) analysis and direct sequencing. RESULTS: Mutation analysis revealed several changes in RNF8 and UBC13, whereas no aberrations were observed in MMS2. None of the found sequence changes appeared to associate with breast cancer susceptibility. CONCLUSIONS: The present data suggest that mutations in RNF8, UBC13 and MMS2 genes unlikely make any sizeable contribution to breast cancer predisposition in Northern Finland.

Further evidence for the contribution of the RAD51C gene in hereditary breast and ovarian cancer susceptibility.

RAD51C, a RAD51 paralogue involved in homologous recombination, is a recently established Fanconi anemia and breast cancer predisposing factor. In the initial report, RAD51C mutations were shown to confer a high risk for both breast and ovarian tumors, but most of the replication studies published so far have failed to identify any additional susceptibility alleles. Here, we report a full mutation screening of the RAD51C gene in 147 Finnish familial breast cancer cases and in 232 unselected ovarian cancer cases originating from Finland and Sweden. In addition, in order to resolve whether common RAD51C SNPs are risk factors for breast cancer, we genotyped five tagging single nucleotide polymorphisms, rs12946522, rs304270, rs304283, rs17222691, and rs28363312, all located within the gene, from 993 Finnish breast cancer cases and 871 controls for cancer associated variants. Whereas, none of the studied common SNPs associated with breast cancer susceptibility, mutation analysis revealed two clearly pathogenic alterations. RAD51C c.-13_14del27 was observed in one familial breast cancer case and c.774delT in one unselected ovarian cancer case, thus confirming that RAD51C mutations are implicated in breast and ovarian cancer predisposition, although their overall frequency seems to be low. Independent identification of the very recently reported RAD51C c.774delT mutation in yet another patient originating from Sweden suggests that it might be a recurrent mutation in that population and should be studied further. The reliable estimation of the clinical implications of carrying a defective RAD51C allele still requires the identification of additional mutation positive families.

Mutation analysis of the gene encoding the PALB2-binding protein MRG15 in BRCA1/2-negative breast cancer families.

MRG15 is a recently identified member of the BRCA multiprotein complex, essential for the maintenance of the genome integrity and DNA repair. The functional relationship between PALB2 and MRG15 makes MRG15 a strong candidate breast cancer susceptibility gene. We screened affected probands from 232 BRCA1/2-negative breast cancer families for mutations in MRG15. We identified seven previously unreported variants but in silico analyses revealed that none of these variants appears to modify the function of MRG15. Thus, it seems unlikely that any constitutional changes in MRG15 confer an increased risk for breast cancer.

Development of microbial populations in the anaerobic hydrolysis of grass silage for methane production.

Six batch leach bed (LB) reactors, installed in parallel and connected to a common upflow anaerobic sludge blanket reactor, were fed with grass silage and operated at 35 (+/-1) degrees C. The development and distribution of microorganisms, which firmly and loosely attached to solid materials, and presented in the leachate in the LB reactors, were investigated by 16S rRNA gene-based terminal restriction fragment length polymorphism and clone library analyses. The phylotypes and their relative abundance changed in the respective bacterial community throughout the 49-day run and showed differences between the communities. Large numbers of phylotypes were detected from day 10 onwards. On day 17, the majority of phylotypes in the bacterial community firmly attached to solid residues affiliated to the classes Clostridia and Bacteroidetes. There were high numbers of the phylotypes in the leachate bacterial community. They were closely related to members of classes Clostridia, Bacteroidetes, Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, and OP10. The Clostridium-like species clearly dominated the bacterial community. Archaea were only found in the solid residues on day 17 and in the leachate on days 10 and 17. The majority of the Archaea fell within the hydrogenotrophic genus Methanobacterium. The organism assigned to the aceticlastic genus Methanosarcina was only present in the solid residues.