Impact of the OncotypeDX score and HER2 RNA PCR levels on HER2-low IHC levels in primary and metastasized tumors.

PURPOSE: One-half of hormone receptor-positive (HR +) breast cancer (BC) patients have low expression of HER2 (HER2-low) and may benefit from trastuzumab deruxtecan (TDXd). This study aimed to identify parameters associated with HER2-low levels in primary and metastatic tumors. We specifically sought to determine whether OncotypeDX and HER2 mRNA levels could identify patients who would otherwise be considered HER2-negative by immunohistochemistry (IHC). METHODS: This retrospective analysis of all consecutive HR + patients who underwent OncotypeDX from January 2004 to December 2020 was conducted in a single medical center (n = 1429). We divided HER2-negative cases into HER2-low (IHC = 1 + or 2 + and non-amplified fluorescent situ hybridization) and HER2-0 (IHC = 0). HER2 RT-PCR was evaluated from the OncotypeDX results. RESULTS: HER2-low cases exhibited significantly higher HER2 RT-PCR scores (p = 2.1e-9), elevated estrogen receptor (ER) levels (p = 0.0114), and larger tumor sizes compared to HER2-0 cases (> 2 cm; 36.6% vs. 22.1%, respectively, p < 0.00001). Primary tumors > 2 cm were more likely to be HER2-low (OR = 2.07, 95% CI: 1.6317 to 2.6475, p < 0.0001). Metastatic BCs expressed higher HER2 IHC scores compared with primary BCs (Wilcoxon signed-rank, p = 0.046). HER2 IHC scores were higher for low-risk vs. medium-risk OncotypeDX (p = 0.0067). No other clinical or pathological parameters were associated with the increase in HER2 levels in the metastatic samples. CONCLUSION: It might be beneficial to use clinical data from the primary tumor, including the HER2 RT-PCR score, to determine a HER2-low status.

CTCF supports preferentially short lamina-associated domains.

More than one third of the mammalian genome is in a close association with the nuclear lamina, thus these genomic regions were termed lamina-associated domains (LADs). This association is fundamental for many aspects of chromatin biology including transcription, replication, and DNA damage repair. LADs association with the nuclear envelope is thought to be dependent on two major mechanisms: The first mechanism is the interaction between nuclear membrane proteins such as LBR with heterochromatin modifications that are enriched in LADs chromatin. The second mechanism is based on proteins that bind the borders of the LADs and support the association of the LADs with the nuclear envelope. Two factors were suggested to support the second mechanism: CCCTC-binding factor (CTCF) and YY1 based on their enriched binding to LADs borders. However, this mechanism has not been proven yet at a whole genome level. Here, to test if CTCF supports the LADs landscape, we generated melanoma cells with a partial loss of function (pLoF) of CTCF by the CRISPR-Cas9 system and determined the LADs landscape by lamin B ChIP-seq analysis. We found that under regular growth conditions, CTCF pLoF led to modest changes in the LADs landscape that included an increase in the signal of 2% of the LADs and a decrease in the signal of 8% of the LADs. However, CTCF importance for the LADs landscape was much higher upon induction of a chromatin stress. We induced chromatin stress by inhibiting RNA polymerase II, an intervention that is known to alter chromatin compaction and supercoiling. Notably, only in CTCF pLoF cells, the chromatin stress led to the dissociation of 7% of the LADs from the lamina. The CTCF-dependent LADs had almost three times shorter median length than the non-affected LADs, were enriched in CTCF binding at their borders, and were higher in their facultative-status (cell-type specific). Thus, it appears that CTCF is a key factor in facilitating the association of short facultative LADs with the nuclear lamina upon chromatin stress.

Evolution of Resistance to Irinotecan in Cancer Cells Involves Generation of Topoisomerase-Guided Mutations in Non-Coding Genome That Reduce the Chances of DNA Breaks.

Resistance to chemotherapy is a leading cause of treatment failure. Drug resistance mechanisms involve mutations in specific proteins or changes in their expression levels. It is commonly understood that resistance mutations happen randomly prior to treatment and are selected during the treatment. However, the selection of drug-resistant mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells and thus cannot result from the selection of pre-existent mutations. Accordingly, adaptation must involve the generation of mutations de novo upon drug treatment. Here we explored the origin of resistance mutations to a widely used Top1 inhibitor, irinotecan, which triggers DNA breaks, causing cytotoxicity. The resistance mechanism involved the gradual accumulation of recurrent mutations in non-coding regions of DNA at Top1-cleavage sites. Surprisingly, cancer cells had a higher number of such sites than the reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repairs of DNA double-strand breaks at these sites following initial drug exposures gradually reverted cleavage-sensitive "cancer" sequences back to cleavage-resistant "normal" sequences. These mutations reduced the generation of DNA breaks upon subsequent exposures, thus gradually increasing drug resistance. Together, large target sizes for mutations and their Top1-guided generation lead to their gradual and rapid accumulation, synergistically accelerating the development of resistance.

The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Uncommon side effects of common drugs in patients with familial dysautonomia.

PURPOSE: Patients with the autosomal recessive disorder of familial dysautonomia typically exhibit exacerbated adverse side effects to many common drugs. We aimed to catalog these adverse effects - with a focus on common drugs that are frequently administered to FD patients and compare their incidences to those within the general population. METHODS: We used data of 595 FD patients from an international database with information on drugs received and adverse effects. To investigate the molecular causes of reported differences in drug responses in FD patients, we used expression microarrays to compare the mRNA expression profiles in peripheral blood leukocytes of FD patients (n = 12) and healthy individuals (n = 10). RESULTS: Several drug classes, including cholinergics, anti-cholinergics, anti-convulsants, methylxanthines, SSRIs, and antibiotics caused either unreported symptoms or elevated rates of adverse events in FD patients. FD patients experienced different or more frequent adverse side effects than the general population in 31/123 drugs. These side effects included blood cell dyscrasias, amenorrhea, gastrointestinal bleeding, and bronchospasm. New findings include enhanced reaction of FD patients to H2 antagonist agents and to serotonin receptor agonists. We also detected eight genes differentially expressed between FD patients and healthy individuals that may underlie the differential drug responses of FD patients. CONCLUSION: We provide evidence that suggests the use of several common drugs should be discontinued or reduced in FD patients.

A study of normal copy number variations in Israeli population.

The population of Israel is ethnically diverse, and individuals from different ethnic groups share specific genetic variations. These variations, which have been passed on from common ancestors, are usually reported in public databases as rare variants. Here, we aimed to identify ethnicity-based benign copy number variants (CNVs) and generate the first Israeli CNV database. We applied a data-mining approach to the results of 10,193 chromosomal microarray tests, of which 2150 tests were from individuals of 13 common ethnic backgrounds (n ≥ 10). We found 165 CNV regions (> 50 kbp) that are unique to specific ethnic groups (uCNVRs). The frequency of more than 19% of these uCNVRs is between 1 and 20% of the common ethnic origin, while their frequency in the overall cohort is between 0.5 and 1.6%. Of these 165 uCNVRs, 98 are reported as variants of unknown significance or as not available in dbVar; we postulate that these uCNVRs should be annotated as either "likely benign" or "benign". The ethnic-specific CNVs extracted in this study will allow geneticists to distinguish between relevant pathogenic genomic aberrations and benign ethnicity-related variations, thus preventing variant misinterpretation that may lead to unnecessary pregnancy terminations.

Hope for male fecundity: clinically insignificant changes in semen parameters over 10 years at a single clinic while assessing an infertility population.

PURPOSE: What is the trend in sperm parameters in a group of men attending a single reproductive center, over a 10-year period? METHODS: A retrospective study was conducted on 12,188 semen samples obtained from unique individuals who attended a university reproductive clinic from 2009 to 2018, inclusively. Semen analysis was done using computer-assisted sperm analysis and verified by an andrologist. Analysis was done after dividing the dataset into two groups: above WHO 2010 lower reference limits (ARL) (N = 6325) and below the reference limits (BRL) (N = 5521). RESULTS: Volume increased slightly (ARL, p = 0.049) before returning to baseline or was stable (BRL, p = 0.59). Sperm concentration and total count of the BRL and ARL group declined initially and then recovered slightly (p < 0.0001, in all cases). Although these changes were statistically significant, this was due to the large study population; clinically, these changes were quite mild and would not have been significant for fertility. Sperm total motility and progressive motility of both the BRL group and the ARL group increased slightly from 2009 until 2015 and then decreased back to baseline (p < 0.0001). This change offset the decrease in count seen in those years. A spurious change was observed with sperm morphology that declined after the first 2 years and remained stable thereafter (p < 0.0001, in both groups). However, this change was attributed to a contemporaneous change in the method of analyzing strict morphology which happened when the change occurred. CONCLUSION: While statistically significant changes were found, clinically, these changes were quite mild and would not have been significant for fertility.

Non-Coding RNAs in Normal B-Cell Development and in Mantle Cell Lymphoma: From Molecular Mechanism to Biomarker and Therapeutic Agent Potential.

B-lymphocytes are essential for an efficient immune response against a variety of pathogens. A large fraction of hematologic malignancies are of B-cell origin, suggesting that the development and activation of B cells must be tightly regulated. In recent years, differentially expressed non-coding RNAs have been identified in mantle cell lymphoma (MCL) tumor samples as opposed to their naive, normal B-cell compartment. These aberrantly expressed molecules, specifically microRNAs (miRNAs), circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), have a role in cellular growth and survival pathways in various biological models. Here, we provide an overview of current knowledge on the role of non-coding RNAs and their relevant targets in B-cell development, activation and malignant transformation, summarizing the current understanding of the role of aberrant expression of non-coding RNAs in MCL pathobiology with perspectives for clinical use.

Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer.

We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival. Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N = 209) were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (hazard ratio [HR] = 1.27 p interaction = 0.014 [DCN], HR = 1.58, p interaction = 0.027 [PLAU], HR = 1.71, p interaction = 0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR = 0.73 p interaction = 0.47 [DCN], HR = 0.71, p interaction = 0.73 [PLAU], HR = 0.84; p interaction = 0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). In conclusion, reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy.

Transcriptomic analysis of smooth versus rough Brucella melitensis Rev.1 vaccine strains reveals insights into virulence attenuation.

Brucella melitensis Rev.1 is the live attenuated Elberg-originated vaccine strain of the facultative intracellular Brucella species, and is widely used to control brucellosis in small ruminants. However, Rev.1 may cause abortions in small ruminants that have been vaccinated during the last trimester of gestation, it is pathogenic to humans, and it induces antibodies directed at the O-polysaccharide (O-PS) of the smooth lipopolysaccharide, thus making it difficult to distinguish between vaccinated and infected animals. Rough Brucella strains, which lack O-PS and are considered less pathogenic, have been introduced to address these drawbacks; however, as Rev.1 confers a much better immunity than the rough mutants, it is still considered the reference vaccine for the prophylaxis of brucellosis in small ruminants. Therefore, developing an improved vaccine strain, which lacks the Rev.1 drawbacks, is a highly evaluated task, which requires a better understanding of the molecular mechanisms underlying the virulence attenuation of Rev.1 smooth strains and of natural Rev.1 rough strains, which are currently only partly understood. As the acidification of the Brucella-containing vacuole during the initial stages of infection is crucial for their survival, identifying the genes that contribute to their survival in an acidic environment versus a normal environment will greatly assist our understanding of the molecular pathogenic mechanisms and the attenuated virulence of the Rev.1 strain. Here, we compared the transcriptomes of the smooth and natural rough Rev.1 strains, each grown under either normal or acidic conditions. We found 12 key genes that are significantly downregulated in the Rev.1 rough strains under normal pH, as compared with Rev.1 smooth strains, and six highly important genes that are significantly upregulated in the smooth strains under acidic conditions, as compared with Rev.1 rough strains. All 18 differentially expressed genes are associated with bacterial virulence and survival and may explain the attenuated virulence of the rough Rev.1 strains versus smooth Rev.1 strains, thus providing new insights into the virulence attenuation mechanisms of Brucella. These highly important candidate genes may facilitate the design of new and improved brucellosis vaccines.

An age-based sperm nomogram: the McGill reference guide.

STUDY QUESTION: How does age affect various semen parameters? SUMMARY ANSWER: For most semen parameters, the nomogram of the entire population was biphasic, peaking around the fourth decade of life. WHAT IS KNOWN ALREADY: In clinical practice, semen quality is examined by using the WHO 2010 reference limits but these limits do not account for male age. A percentile-based, large-scale nomogram describing how different semen parameters change throughout reproductive life has been lacking. STUDY DESIGN, SIZE, DURATION: A retrospective study was conducted with 12 188 sperm samples, obtained from individuals who attended the McGill University Health Centre reproductive clinic between 2009 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: One sample from each individual who attended the clinic during the study period was analysed by using computer-assisted sperm analysis (CASA). The analysed parameters were human-verified and included sperm concentration, motility, progressive motility, total count, morphology and semen volume. Based on this analysis, the entire dataset (n = 12 188) was further divided into two groups of samples: samples that surpassed the WHO 2010 lower reference limits ('above reference limits' group, ARL; n = 6305), and samples that did not ('below reference limit' group, BRL; n = 5883). Regression quantiles were fitted as a function of age to generate age-dependent nomograms, and these quantiles were divided into 5th, 25th, 50th, 75th and 95th percentiles. MAIN RESULTS AND THE ROLE OF CHANCE: In the entire dataset, age had a significant influence (P < 0.001) on all parameters (except morphology) which demonstrated a biphasic trend peaking in the fourth decade of life. In the ARL group, age had a significant influence (P < 0.01) on all semen parameters except sperm concentration and morphology. However, unlike in the entire dataset, only semen volume demonstrated a biphasic trend in the ARL group (peaking in the fourth decade of life), whereas other parameters either remained unchanged (concentration and morphology) or consistently declined with age (sperm motility, progressive motility and total sperm count). Percentile-based nomograms were generated for individuals between the ages of 20 and 60 years in the entire dataset and in the ARL group. LIMITATIONS, REASONS FOR CAUTION: First, the semen samples were obtained from individuals who were referred to a fertility clinic, such that the entire dataset does not necessarily represent the general population. Second, the cross-sectional sampling design increases variance, and the nomograms are less accurate in the 5th and 95th percentiles and at the extremes of the age distributions. Third, the observed age-dependent changes in semen parameters do not necessarily indicate changes in fertility, as not all factors that affect male fertility were analysed. Fourth, some of our semen analyses employed CASA, which can have variability issues. Finally, our models did not incorporate possible secular trends. WIDER IMPLICATIONS OF THE FINDINGS: We provide the first nomogram that correlates age with semen quality parameters in different population percentiles, thus complementing the current reference limits set by the WHO in 2010. Most examined semen parameters in our study changed non-linearly with age; therefore, age should be regularly employed as a factor in the clinical analysis of semen samples. STUDY FUNDING/COMPETING INTEREST(S): The authors have not received any funding to support this study. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Genomic Analysis of Natural Rough Brucella melitensis Rev.1 Vaccine Strains: Identification and Characterization of Mutations in Key Genes Associated with Bacterial LPS Biosynthesis and Virulence.

Brucella species are facultative intracellular bacteria that cause brucellosis, a zoonotic world-wide disease. The live attenuated B. melitensis Rev.1 vaccine strain is widely used for the control of brucellosis in the small ruminant population. However, Rev.1 induces antibodies against the O-polysaccharide (O-PS) of the smooth lipopolysaccharide thus, it is difficult to differentiate between infected and vaccinated animals. Hence, rough Brucella strains lacking the O-PS have been introduced. In the current study, we conducted a comprehensive comparative analysis of the genome sequence of two natural Rev.1 rough strains, isolated from sheep, against that of 24 Rev.1 smooth strains and the virulent reference strain B. melitensis 16M. We identified and characterized eight vital mutations within highly important genes associated with Brucella lipopolysaccharide (LPS) biosynthesis and virulence, which may explain the mechanisms underlying the formation of the Rev.1 rough phenotype and may be used to determine the mechanism underlying virulence attenuation. Further complementation studies aimed to estimate the specific role of these mutations in affecting Brucella morphology and virulence will serve as a basis for the design of new attenuated vaccines for animal immunization against brucellosis.

The shaping and functional consequences of the microRNA landscape in breast cancer.

MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA-mRNA interactions rather than as on-off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.

[UTILIZATION OF WHOLE EXOME SEQUENCING IN DIAGNOSTICS OF GENETIC DISEASE: RABIN MEDICAL CENTER'S EXPERIENCE].

INTRODUCTION: Whole exome sequencing is a diagnostic approach for the identification of molecular etiology in patients with suspected monogenic diseases. In this article we report on our experience with whole-exome sequencing (WES) of DNA samples taken from patients referred for genetic evaluation due to suspected undiagnosed genetic conditions. METHODS: Exome enrichment was achieved by Nextera Rapid Capture Expanded Exome Kit. Whole-exome sequencing was performed on Illumina HiSeq 2500. Potentially damaging rare variants were selected for familial cosegregation analysis. RESULTS: A total of 39 patients presenting a wide range of phenotypes suspected to have a genetic cause were sent to WES. Approximately 80% were children with neurological phenotypes. Variations having a high probability of being causative were identified in 20 families, achieving a 51.3% molecular diagnostic rate. Among these, 7 exhibited autosomal dominant disease, 12 autosomal recessive diseases and one X-linked disease; 28% of the patients (11/39) were found to carry a novel mutation located in previously reported genes. Novel mutations located in genes not known to be associated with genetic disease were identified in 23% of the patients (9/39). CONCLUSIONS: Whole exome sequencing identified the underlying genetic cause in more than half of the patients referred for evaluation in the genetics clinic at the tertiary hospital. These data demonstrate the utility of WES as a powerful tool for effective diagnostics of monogenic genetic diseases.

Elucidating tissue specific genes using the Benford distribution.

BACKGROUND: The RNA-seq technique is applied for the investigation of transcriptional behaviour. The reduction in sequencing costs has led to an unprecedented trove of gene expression data from diverse biological systems. Subsequently, principles from other disciplines such as the Benford law, which can be properly judged only in data-rich systems, can now be examined on this high-throughput transcriptomic information. The Benford law, states that in many count-rich datasets the distribution of the first significant digit is not uniform but rather logarithmic. RESULTS: All tested digital gene expression datasets showed a Benford-like distribution when observing an entire gene set. This phenomenon was conserved in development and does not demonstrate tissue specificity. However, when obedience to the Benford law is calculated for individual expressed genes across thousands of cells, genes that best and least adhere to the Benford law are enriched with tissue specific or cell maintenance descriptors, respectively. Surprisingly, a positive correlation was found between the obedience a gene exhibits to the Benford law and its expression level, despite the former being calculated solely according to first digit frequency while totally ignoring the expression value itself. Nevertheless, genes with low expression that exhibit Benford behavior demonstrate tissue specific associations. These observations were extended to predict the likelihood of tissue specificity based on Benford behaviour in a supervised learning approach. CONCLUSIONS: These results demonstrate the applicability and potential predictability of the Benford law for gleaning biological insight from simple count data.

NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression.

Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.

Utilizing fluorescent life time imaging microscopy technology for identify carriers of BRCA2 mutation.

Worldwide, more than one million women are diagnosed with breast cancer every year, making it the most common female malignancy in the developed world. Germline mutations in BRCA1 and BRCA2 genes are estimated to increase the risk for developing breast cancer by up to 87%. From a clinical point of view, identification of BRCA1 and BRCA2 mutation carriers offers an opportunity to early identify or prevent the development of malignancy; therefore the ability to determine which women are more likely to carry BRCA1 or BRCA2 mutations is of great importance. The available diagnostic tests for mutation analysis of BRCA1 and BRCA2 are time- and labor-intensive, expensive, and do not allow the identification of all the functional mutations. We utilized the Fluorescent lifetime (FLT) imaging microscopy method which allows recognizing different cell populations, in order to distinguish between lymphocytes from BRCA1 and BRCA2 mutation carriers and non-carrier women by using easily obtainable lymphocyte cells from peripheral blood. Our results demonstrate that cells originated from BRCA2-mutation carriers have significantly lower FLT values compared with BRCA1 mutation carriers and control cells. This simple, inexpensive and sensitive method may be utilized in the future to detect BRCA2 mutation carriers, particularly those bearing unknown functional mutations.

VennBLAST­whole transcriptome comparison and visualization tool.

RNA-seq is the method of choice for getting a primary list of genes for non-model organisms. Once this is achieved, one would proceed to annotate the newly discovered genes and consequently strive to position the organism in an evolutionary context. These kinds of studies involving high-throughput sequencing generate large amounts of data, whose analysis might be time consuming for the non-specialist user and merit computational skills. Here we describe VennBLAST, a set of high-performance utilities that combines fast parallelized BLAST filtering with a visualization tool for whole-transcriptomic alignment comparison using Venn diagrams. The software accurately illustrates simple set relationships between numbers of matching sequences and identifies transcriptome conservation among different organisms. The intuitive Venn diagram visualization allows researchers to easily select a desired subset of genes for further inspection, using the DAVID functional annotation tools, for instance, which enables investigators to understand biological meaning behind large lists of genes.