Genomic rearrangements in BRCA1 and BRCA2: A literature review

Women with mutations in the breast cancer genes BRCA1 or BRCA2 have an increased lifetime risk of developing breast, ovarian and other BRCA-associated cancers. However, the number of detected germline mutations in families with hereditary breast and ovarian cancer (HBOC) syndrome is lower than expected based upon genetic linkage data. Undetected deleterious mutations in the BRCA genes in some high-risk families are due to the presence of intragenic rearrangements such as deletions, duplications or insertions that span whole exons. This article reviews the molecular aspects of BRCA1 and BRCA2 rearrangements and their frequency among different populations. An overview of the techniques used to screen for large rearrangements in BRCA1 and BRCA2 is also presented. The detection of rearrangements in BRCA genes, especially BRCA1, offers a promising outlook for mutation screening in clinical practice, particularly in HBOC families that test negative for a germline mutation assessed by traditional methods.     

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.     

Effects of BRCA1 and BRCA2 mutations on female fertility

Women with BRCA1/2 mutations have a significantly higher lifetime risk of developing breast or ovarian cancer. We suggest that female mutation carriers may have improved fitness owing to enhanced fertility relative to non-carriers. Here we show that women who are carriers of BRCA1/2 mutations living in natural fertility conditions have excess fertility as well as excess post-reproductive mortality in relation to controls. Individuals who tested positive for BRCA1/2 mutations who linked into multi-generational pedigrees within the Utah Population Database were used to identify putative obligate carriers. We find that women born before 1930 who are mutation carriers have significantly more children than controls and have excess post-reproductive mortality risks. They also have shorter birth intervals and end child-bearing later than controls. For contemporary women tested directly for BRCA1/2 mutations, an era when modern contraceptives are available, differences in fertility and mortality persist but are attenuated. Our findings suggest the need to re-examine the wider role played by BRCA1/2 mutations. Elevated fertility of female mutation carriers indicates that they are more fecund despite their elevated post-reproductive mortality risks.     

Trypanosoma brucei: Reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic “pulse-chase” analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8 h of initiating transformation. GPI-PLCp is not lost precipitously from newly transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number

Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number.     

Divergent polo box domains underpin the unique kinetoplastid kinetochore

Kinetochores are macromolecular machines that drive eukaryotic chromosome segregation by interacting with centromeric DNA and spindle microtubules. While most eukaryotes possess conventional kinetochore proteins, evolutionarily distant kinetoplastid species have unconventional kinetochore proteins, composed of at least 19 proteins (KKT1–19). Polo-like kinase (PLK) is not a structural kinetochore component in either system. Here, we report the identification of an additional kinetochore protein, KKT20, in Trypanosoma brucei. KKT20 has sequence similarity with KKT2 and KKT3 in the Cys-rich region, and all three proteins have weak but significant similarity to the polo box domain (PBD) of PLK. These divergent PBDs of KKT2 and KKT20 are sufficient for kinetochore localization in vivo. We propose that the ancestral PLK acquired a Cys-rich region and then underwent gene duplication events to give rise to three structural kinetochore proteins in kinetoplastids.     

SAR of 2-amino and 2,4-diamino pyrimidines with in vivo efficacy against Trypanosoma brucei

A series of 2,4-diaminopyrimidines was investigated and compounds were found to have in vivo efficacy against Trypanosoma brucei in an acute mouse model. However, in vitro permeability data suggested the 2,4-diaminopyrimidenes would have poor permeability through the blood brain barrier. Consequently a series of 4-desamino analogs were synthesized and found to have improved in vitro permeability.     

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.     

LIM-only protein FHL3 interacts with CDC25B2 phosphatase

LIM domain proteins are important regulators of the growth, determination, and differentiation of cells. In this report, FHL3 (human four-and-a-half LIM-only protein 3) is shown to interact with human phosphatase CDC25B, a cell cycle regulator involved in the control of G2/M. We found that this interaction was specific to the CDC25B2 isoform. Deletion and point mutation studies indicated that the second LIM domain of FHL3 was essential for this interaction. FRET experiments in C2C12 cells showed that, although both proteins were colocated in the cytoplasm and the nucleus, they interacted only in the nucleus. Finally, we showed that FHL3 binding impaired neither CDC25B2 phosphatase activity nor its localization. Further work is now needed to elucidate the consequences of this interaction on myoblast fate decision and cycle control.     

Interactions of antimicrobial peptides with Leishmania and trypanosomes and their functional role in host parasitism

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection.     

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.     

A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.     

Trypanosoma brucei brucei: biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.     

Identification and characterization of a novel protein ISOC2 that interacts with p16INK4a

p16(INK4a) is a multiple tumor suppressor, playing an important role in proliferation and tumorigenesis. To screen the p16(INK4a)-associated proteins, we performed a yeast two-hybrid assay and identified a novel protein isochorismatase domain containing 2 (ISOC2). ISOC2 conserves in different species, and encodes 205 and 210 amino acids in human and mouse, respectively. The expression of ISOC2 in mouse is universal but predominantly in uterus, stomach, and urinary tract system. Interaction between ISOC2 and p16(INK4a) was verified using in vitro pull-down assays and in vivo co-immunoprecipitation. Confocal microscopy studies using green and cyan fluorescent fusion proteins determined that ISOC2 co-localizes with p16(INK4a). Over-expressed ISOC2 is able to inhibit p16(INK4a) in dose-dependent manner. Our data indicated that ISOC2 is a novel functional protein, which is able to bind and co-localize with a tumor suppressor gene p16(INK4a). Over-expressed ISOC2 inhibits the expression of p16(INK4a), suggesting that this novel gene may play a role during the tumor development by interacting with p16(INK4a).     

Prevalence of 185delAG and 5382insC mutations in BRCA1, and 6174delT in BRCA2 in women of Ashkenazi Jewish origin in southern Brazil

Certain mutations in BRCA1 and BRCA2 genes are frequent in the Ashkenazi Jewish population. Several factors contribute to this increased frequency, including consanguineous marriages and an event known as a “bottleneck”, which occurred in the past and caused a drastic reduction in the genetic variability of this population. Several studies were performed over the years in an attempt to elucidate the role of BRCA1 and BRCA2 genes in susceptibility to breast cancer. The aim of this study was to estimate the carrier frequency of certain common mutations in the BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT) genes in an Ashkenazi Jewish population from Porto Alegre, Brazil. Molecular analyses were done by PCR followed by RFLP (ACRS). The carrier frequencies for BRCA1 185delAG and 5382insC were 0.78 and 0 respectively, and 0.4 for the BRCA2 6174deT mutation. These findings are similar to those of some prior studies but differ from others, possibly due to excluding individuals with a personal or family history of cancer. Our sample was drawn from the community group and included individuals with or without a family or personal history of cancer. Furthermore, increased dispersion among Ashkenazi subpopulations may be the result of strong genetic drift and/or admixture. It is therefore necessary to consider the effects of local admixture on the mismatch distributions of various Jewish populations.     

WCB is a C2 domain protein defining the plasma membrane - sub-pellicular microtubule corset of kinetoplastid parasites

WCB is a protein that locates between the inner face of the plasma membrane and the sub-pellicular corset of microtubules in Trypanosoma brucei. We provide the molecular identity of WCB and bioinformatic analysis suggests that it possesses a C2 domain implicated in membrane/protein interactions and a highly charged region possessing characteristics of a putative tubulin-binding domain. Functional analyses via RNA interference (RNAi) depletion show that WCB is essential for cell morphogenesis. Depletion results in gross abnormalities in cell shape, mainly at the cytoskeletal/plasma membrane dynamic posterior end of the trypanosome. Failures in cytokinesis and zoid production are also evident. Furthermore, electron microscopy reveals that RNAi-induced trypanosomes lose local plasma membrane to microtubule corset integrity.