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Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes (Luminal A, Luminal B, HER2 and triple-negative (TNBC)) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC.

A human transcriptome array on ERα-positive breast cancer continuum of risk identified Secreted Frizzled-Related Protein 1 ( SFRP1 ) as decreased during breast cancer progression. In addition, SFRP1 was inversely associated with breast tissue age-related lobular involution, and differentially regulated in women with regard to their parity status and the presence of microcalcifications. The causal role of SFRP1 in breast carcinogenesis remains, nevertheless, not well understood. In this study, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, in the presence of estradiol (E2) and/or hydroxyapatite microcalcifications (HA). Furthermore, we have modulated SFRP1 expression in breast cancer cell lines, including the MCF10A series, and investigated their tumoral properties. We observed that organoids obtained from multiparous mice were resistant to E2 treatment, while organoids obtained from nulliparous mice developed the luminal phenotype associated with a lower ratio between Sfrp1 and Esr1 expression. The decrease in SFRP1 expression in MCF10A and MCF10AT1 cell lines increased their tumorigenic properties in vitro. On the other hand, the overexpression of SFRP1 in MCF10DCIS, MCF10CA1a, and MCF7 reduced their aggressiveness. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis.

Triple-negative breast cancer (TNBC) is a major concern among the different subtypes of breast cancer (BC) due to the lack of effective treatment. In a previous study by our group aimed at understanding the difference between TNBC and non-TNBC tumors, we identified the gene TBC1 domain family member 9 ( TBC1D9 ), the expression of which was lower in TNBC as compared to non-TNBC tumors. In the present study, analysis of TBC1D9 expression in TNBC ( n = 58) and non-TNBC ( n = 25) patient tumor samples validated that TBC1D9 expression can differentiate TNBC (low) from non-TNBC (high) samples and that expression of TBC1D9 was inversely correlated with grade and proliferative index. Moreover, we found that downregulation of the TBC1D9 gene decreases the proliferation marginally in non-TNBC and was associated with increased migratory and tumorigenic potential in both TNBC and luminal BC cell lines. This increase was mediated by the upregulation of ARL8A , ARL8B , PLK1 , HIF1α , STAT3 , and SPP1 expression in TBC1D9 knockdown cells. Our results suggest that TBC1D9 expression might limit tumor aggressiveness and that it has a differential expression in TNBC vs. non-TNBC tumors.

Ductal carcinoma in situ (DCIS) is considered a non-obligatory precursor for invasive ductal carcinoma (IDC). Around 70% of women with atypical ductal hyperplasia (ADH) undergo unnecessary surgery due to the difficulty in differentiating ADH from low-grade DCIS. If untreated, 14-60% of DCIS progress to IDC, highlighting the importance of identifying a DCIS gene signature. Human transcriptome data of breast tissue samples representing each step of BC progression were analyzed and high expression of carboxypeptidase B1 ( CPB1 ) expression strongly correlated with DCIS. This was confirmed by quantitative PCR in breast tissue samples and cell lines model. High CPB1 expression correlated with better survival outcome, and mRNA level was highest in DCIS than DCIS adjacent to IDC and IDC. Moreover, loss of CPB1 in a DCIS cell line led to invasive properties associated with activation of HIF1α , FN1, STAT3 and SPP1 and downregulation of SFRP1 and OS9 . The expression of CPB1 could predict 90.1% of DCIS in a cohort consisting of DCIS and IDC. We identified CPB1, a biomarker that helps differentiate DCIS from ADH or IDC and in predicting if a DCIS is likely to progress to IDC, thereby helping clinicians in their decisions.

Background: Obesity fosters worse clinical outcomes in both premenopausal and postmenopausal women with breast cancer. Emerging evidence suggests that an android body fat distribution in particular is deleterious for breast cancer prognosis. The extent of adipose tissue dysfunction, especially how it relates to breast cancer prognostic factors and anthropometric measurements, has not been fully investigated., Objective: Our objective was to examine if markers of adipose tissue dysfunction, such as hypertrophy and macrophage accumulation, are relevant for the pathophysiology of breast cancer and its associated prognostic factors in a well-characterised cohort of women with breast cancer who did not receive treatment before surgery., Methods: A consecutive series of 164 women with breast cancer provided breast adipose tissue sample. Multivariate generalised linear models were used to test associations of anthropometric indices and prognostic factors with markers of adipose tissue dysfunction., Results: We found associations of breast adipocyte size and macrophage infiltration (number of CD68+ cells/100 adipocytes) with adiposity, particularly a strong association between breast adipocyte size and central obesity, independent of total adiposity, age and menopausal status (β adj  = 0.87; p = 0.0001). We also identified relationships of adipocyte hypertrophy and macrophage infiltration with prognostic factors, such as cancer stage and tumour grade (p < 0.05). RNA expression of pro-inflammatory cytokines (IL6, TNF) and leptin was also increased as a function of adipocyte size and CD86+/CD11c+ macrophage number/100 adipocytes (p < 0.05)., Conclusions: Our findings support the model of dysfunctional adipose tissue in obesity-associated breast cancer.

Triple negative breast cancer (TNBC) is one of the most aggressive form of breast cancer (BC) with the highest mortality due to high rate of relapse, resistance, and lack of an effective treatment. Various molecular approaches have been used to target TNBC but with little success. Here, using machine learning algorithms, we analyzed the available BC data from the Cancer Genome Atlas Network (TCGA) and have identified two potential genes, TBC1D9 (TBC1 domain family member 9) and MFGE8 (Milk Fat Globule-EGF Factor 8 Protein), that could successfully differentiate TNBC from non-TNBC, irrespective of their heterogeneity. TBC1D9 is under-expressed in TNBC as compared to non-TNBC patients, while MFGE8 is over-expressed. Overexpression of TBC1D9 has a better prognosis whereas overexpression of MFGE8 correlates with a poor prognosis. Protein-protein interaction analysis by affinity purification mass spectrometry (AP-MS) and proximity biotinylation (BioID) experiments identified a role for TBC1D9 in maintaining cellular integrity, whereas MFGE8 would be involved in various tumor survival processes. These promising genes could serve as biomarkers for TNBC and deserve further investigation as they have the potential to be developed as therapeutic targets for TNBC.

The breast cancer (BC) biomarker HER2 (Human Epidermal Receptor 2) is overexpressed in 25% of BC. Only patients with HER2-positive tumors receive HER2-targeting therapies, like trastuzumab (Herceptin). However, some women with a HER2-negative BC could benefit from trastuzumab. This could be explained by the activation/phosphorylation of HER2 that can be recognized by trastuzumab. The aim of this study is to examine trastuzumab effects on HER2 phosphorylation at tyrosine Y877 (pHER2Y877). HER2 and pHER2Y877 status were evaluated in a cohort of BC patients representative of molecular subtypes distribution (n = 497) and in a series of BC cell lines (n = 7). Immunohistochemistry against pHER2Y877 was performed on tissue micro arrays. Cellular proliferation assays were performed on BC cell lines presenting different combinations of HER2 and pHER2Y877 status and treated with increasing doses of trastuzumab (0-150 μg/ml). The prevalence of pHER2Y877 in this cohort was 6%. Nearly 5% of patients with HER2-negative tumors (n = 406, 82%) overexpressed pHER2Y877. Among triple negative BC patients (n = 39, 8%), 7.7% expressed pHER2Y877. Trastuzumab treatment decreased cell proliferation in HER2-/pHER2Y877+ BC cell lines, to an extent comparable to what occurs in HER2+ cell lines, but did not affect HER2-/pHER2Y877- cell lines. Trastuzumab sensitivity in HER2-/pHER2Y877+ cell line is specific to HER2 tyrosine 877 phosphorylation. Hence, with further confirmation in a bigger cohort, trastuzumab treatment could be envisaged as a treatment option to women presenting with HER2-/pHER2+ tumors, representing more than 1000 BC women in Canada in 2019., Competing Interests: The authors have declared that no competing interests exist.

Breast cancer (BC) is a heterogeneous disease where the survival rate of patients decreases with progression of the disease. BC usually has a linear progression, classified into normal/benign, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). This study aimed to identify gene signature for each of these subgroups. We performed human transcriptome array analysis on 5 patient samples from each Normal, ADH, IDC and DCIS and 2 replicates of MCF10A cell line representative of each subgroup. We identified SFRP1 and snoRNAs (especially SNORD115 and SNORD114 ) as the initial regulators of cancer progression, accompanied by significant changes in extracellular matrix organization. Tumor progression to the IDC stage showed upregulation of tumor promoting genes responsible for increased invasion, inflammation, survival in stress environment and metastasis. The gene signatures identified in this study could represent potential biomarkers for each subgroup of breast cancer progression, which could assist in early diagnosis of breast cancer progression as well as treatment interventions. Moreover, these gene signatures could serve in discovery of specific targeted therapies for each subgroup., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Background: Inflammation is a major player in breast cancer (BC) progression. Allograft-inflammatory factor-1 (AIF1) is a crucial mediator in the inflammatory response. AIF1 reportedly plays a role in BC, but the mechanism remains to be elucidated. We identified two AIF1 isoforms, AIF1v1 and AIF1v3, which were differentially expressed between affected and unaffected sisters from families with high risk of BC with no deleterious BRCA1/BRCA2 mutations (BRCAX). We investigated potential functions of AIFv1/v3 in BC of varying severity and breast adipose tissue by evaluating their expression, and association with metabolic and clinical parameters of BC patients., Methods: AIF1v1/v3 expression was determined in BC tissues and cell lines using quantitative real-time PCR. Potential roles and mechanisms were examined in the microenvironment (fibroblasts, adipose tissue, monocytes and macrophages), inflammatory response (cell reaction in BC subgroups), and metabolism [treatment with docosahexaenoic acid (DHA)]. Association of AIF1 transcript expression with clinical factors was determined by Spearman's rank correlation. Bioinformatics analyses were performed to characterize transcripts., Results: AIF1v1/v3 were mostly expressed in the less severe BC samples, and their expression appeared to originate from the tumor microenvironment. AIF1 isoforms had different expression rates and sources in breast adipose tissue; lymphocytes mostly expressed AIF1v1 while activated macrophages mainly expressed AIF1v3 . Bioinformatics analysis revealed major structural differences suggesting distinct functions in BC progression. Lymphocytes were the most infiltrating cells in breast tumors and their number correlated with AIF1v1 adipose expression. Furthermore, DHA supplementation significantly lowered the expression of AIF1 isoforms in BRCAX cell lines. Finally, the expression of AIF1 isoforms in BC and breast adipose tissue correlated with clinical parameters of BC patients., Conclusions: Results strongly suggest that AIF1v1 as much as AIF1v3 play a major role in the crosstalk between BC and infiltrating immune cells mediating tumor progression, implying their high potential as target molecules for BC diagnostic, prognostication and treatment.

Approximately 25% of hereditary breast cancer cases are associated with a strong familial history which can be explained by mutations in BRCA1 or BRCA2 and other lower penetrance genes. The remaining high-risk families could be classified as BRCAX (non- BRCA1/2 ) families. Gene expression involving alternative splicing represents a well-known mechanism regulating the expression of multiple transcripts, which could be involved in cancer development. Thus using RNA-seq methodology, the analysis of transcriptome was undertaken to potentially reveal transcripts implicated in breast cancer susceptibility and development. RNA was extracted from immortalized lymphoblastoid cell lines of 117 women (affected and unaffected) coming from BRCA1 , BRCA2 and BRCAX families. Anova analysis revealed a total of 95 transcripts corresponding to 85 different genes differentially expressed (Bonferroni corrected p-value <0.01) between those groups. Hierarchical clustering allowed distinctive subgrouping of BRCA1/2 subgroups from BRCAX individuals. We found 67 transcripts, which could discriminate BRCAX from BRCA1/BRCA2 individuals while 28 transcripts discriminate affected from unaffected BRCAX individuals. To our knowledge, this represents the first study identifying transcripts differentially expressed in lymphoblastoid cell lines from major classes of mutation-related breast cancer subgroups, namely BRCA1 , BRCA2 and BRCAX. Moreover, some transcripts could discriminate affected from unaffected BRCAX individuals, which could represent potential therapeutic targets for breast cancer treatment., Competing Interests: CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest.

In this study we evaluated whether the sophistication of children's invented spellings in kindergarten was predictive of subsequent reading and spelling in Grade 1, while also considering the influence of well-known precursors. Children in their first year of schooling (mean age = 66 months; N = 171) were assessed on measures of oral vocabulary, alphabetic knowledge, phonological awareness, word reading and invented spelling; approximately 1 year later they were assessed on multiple measures of reading and spelling. Path modeling was pursued to evaluate a hypothesized unique, causal role of invented spelling in subsequent literacy outcomes. Results supported a model in which invented spelling contributed directly to concurrent reading along with alphabetic knowledge and phonological awareness. Longitudinally, invented spelling influenced subsequent reading, along with alphabetic knowledge while mediating the connection between phonological awareness and early reading. Invented spelling also influenced subsequent conventional spelling along with phonological awareness, while mediating the influence of alphabetic knowledge. Invented spelling thus adds explanatory variance to literacy outcomes not entirely captured by well-studied code and language-related skills. (PsycINFO Database Record, ((c) 2016 APA, all rights reserved).)

Aim: The goal of this study is to characterize the specific methylation profile triggered by DNMT3B protein isoforms expressed at different levels in breast cell lines., Materials & Methods: Microarray DNA methylation data were analyzed and associated with functional genome annotation data., Results: A large spectrum of DNMT3B3/DNMT3B2 expression ratio values was observed in parental breast cell lines. According to their methylation profiles, hierarchical clustering of untransfected cell lines revealed clustering based on their ER/PR status. Overexpression of DNMT3B3 triggered methylation changes of thousands of CpG sites in breast cells. Based on the trend of methylation changes, the results suggest an antiproliferative action of the DNMT3B3 isoform through a dominant negative effect on its wild-type counterpart DNMT3B2., Conclusion: This study revealed specific pathways modulated by DNMT3B isoforms, which could regulate cell proliferation and other biological mechanisms. This illustrates the importance of multiple interactions between isoforms in the complexity of methylation processes.

The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Many military personnel returning from deployment experience increases in psychological symptoms, including post-traumatic stress disorder (PTSD), depression, and mood changes. Patient health diaries are commonly used for self-reporting over time away from the clinic. "T2 Mood Tracker" is an application ("app") for smartphones and other mobile devices that enables users to rate their moods, to self-monitor across time, and to report their emotional experiences to health providers. We designed T2 Mood Tracker to track symptoms associated with deployment-related behavioral health issues, including PTSD, Head Injury, Stress, Depression, Anxiety, and General Well-Being. We field-tested T2 Mood Tracker with a small sample of redeployed soldiers under treatment for behavioral health issues at a Warrior Transition Unit. Participants used the app an average of 10 different days over the 2- to 3-week test period. Consensus was that T2 Mood Tracker was easy to use, useful and beneficial. The majority said they would use the app in the future, would recommend it to other service members, and would use the app to share their mood information with a provider. Warrior Transition Unit providers were enthusiastic about the potential of T2 Mood Tracker as a tool for use with their patients., (Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.)

Purpose: To explore parental perceptions and experiences regarding the return of genomic incidental research findings in children with rare diseases., Methods: Parents of children affected by various rare diseases were invited to participate in focus groups or individual telephone interviews in Montreal and Ottawa. Fifteen participants were interviewed and transcriptions were analysed using thematic analysis., Results: Four emergent themes underscored parental enthusiasm for receiving incidental findings concerning their child's health: (1) right to information; (2) perceived benefits and risks; (3) communication practicalities: who, when, and how; and (4) service needs to promote the communication of incidental findings. Parents believed they should be made aware of all results pertaining to their child's health status, and that they are responsible for transmitting this information to their child, irrespective of disease severity. Despite potential negative consequences, respondents generally perceived a favourable risk-benefit ratio in receiving all incidental findings., Conclusions: Understanding how parents assess the risks and benefits of returning incidental findings is essential to genomic research applications in paediatric medicine. The authors believe the study findings will contribute to establishing future best practices, although further research is needed to evaluate the impact of parental decisions on themselves and their child., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Prior research has shown superior orthographic learning resulting from spelling practice relative to repeated reading. One mechanism proposed to underlie this advantage of spelling in establishing detailed orthographic representations in memory is the motoric component of the manual movements evoked in printing or writing. This study investigated this contention directly by testing the effects of typing vs. printing on the orthographic learning achieved through spelling practice, and further evaluated whether practice modality interacts with pre-existing individual characteristics. Forty students in grade 2 (mean age 7 years 5 months) were introduced to 10 novel non-words. Some of the students practiced spelling the items by printing, while the others practiced spelling them on a keyboard. Participants were tested for recognition and spelling of these items 1 and 7 days later. Results revealed high rates of orthographic learning with no main effects of practice modality, testing time, or post-test modality. Hierarchical regression analyses revealed an interaction between typing proficiency and practice modality, such that pre-existing keyboarding skills constrained or facilitated learning within the typing-practice group. A similar interaction was not found between printing skills and learning within the printing group. Results are discussed with reference to both prominent reading theory and educational applications.

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

We describe here a strain of Yersinia pestis, G1670A, which exhibits a baseline mutation rate elevated 250-fold over wild-type Y. pestis. The responsible mutation, a C to T substitution in the mutS gene, results in the transition of a highly conserved leucine at position 689 to arginine (mutS(L689R)). When the MutSL 689R protein of G1670A was expressed in a ΔmutS derivative of Y. pestis strain EV76, mutation rates observed were equivalent to those observed in G1670A, consistent with a causal association between the mutS mutation and the mutator phenotype. The observation of a mutator allele in Yersinia pestis has potential implications for the study of evolution of this and other especially dangerous pathogens., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The majority of genes associated with breast cancer susceptibility, including BRCA1 and BRCA2 genes, are involved in DNA repair mechanisms. Moreover, among the genes recently associated with an increased susceptibility to breast cancer, four are Fanconi Anemia (FA) genes: FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/PALB2 and FANCO/RAD51C. FANCA is implicated in DNA repair and has been shown to interact directly with BRCA1. It has been proposed that the formation of FANCA/G (dependent upon the phosphorylation of FANCA) and FANCB/L sub-complexes altogether with FANCM, represent the initial step for DNA repair activation and subsequent formation of other sub-complexes leading to ubiquitination of FANCD2 and FANCI. As only approximately 25% of inherited breast cancers are attributable to BRCA1/2 mutations, FANCA therefore becomes an attractive candidate for breast cancer susceptibility. We thus analyzed FANCA gene in 97 high-risk French Canadian non-BRCA1/2 breast cancer individuals by direct sequencing as well as in 95 healthy control individuals from the same population. Among a total of 85 sequence variants found in either or both series, 28 are coding variants and 19 of them are missense variations leading to amino acid change. Three of the amino acid changes, namely Thr561Met, Cys625Ser and particularly Ser1088Phe, which has been previously reported to be associated with FA, are predicted to be damaging by the SIFT and PolyPhen softwares. cDNA amplification revealed significant expression of 4 alternative splicing events (insertion of an intronic portion of intron 10, and the skipping of exons 11, 30 and 31). In silico analyzes of relevant genomic variants have been performed in order to identify potential variations involved in the expression of these spliced transcripts. Sequence variants in FANCA could therefore be potential spoilers of the Fanconi-BRCA pathway and as a result, they could in turn have an impact in non-BRCA1/2 breast cancer families., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

ZNF350/ZBRK1 is a transcription factor, which associates with BRCA1 to co-repress GADD45A to regulate DNA damage repair, and the expression of ZNF350 is altered in different human carcinomas. In a previous study, we identified ZNF350 genomic variants potentially involved in breast cancer susceptibility in high-risk non-BRCA1/2 breast cancer individuals, which pointed toward a potential association for variants in the 5'-UTR and promoter regions. Therefore, direct sequencing was undertaken and identified 12 promoter variants, whereas haplotype analyses put in evidence four common haplotypes with a frequency>2%. However, based on their frequency observed in breast cancer and unrelated healthy individuals, these are not statistically associated with breast cancer risk. Luciferase promoter assays in two breast cancer cell lines identified two haplotypes (H11 and H12) stimulating significantly the expression of ZNF350 transcript compared with the common haplotype H8. The high expression of the H11 allele was associated with the variant c.-874A. Using MatInspector and Transcription Element Search softwares, in silico analyses predicted that the variant c.-874A created a binding site for the factors c-Myc and myogenin. This study represents the first characterization step of the ZNF350 promoter. Additional studies in larger cohorts and other populations will be needed to further evaluate whether common and/or rare ZNF350 promoter variants and haplotypes could be associated with a modest risk of breast cancer.

Plague disease caused by the gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19(th) century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen.

This study investigated the relevance of type of practice and presence of semantic representation for orthographic learning in learning to spell. A total of 36 students in Grade 2 (mean age = 7 years 10 months) were exposed to 10 novel nonwords, 5 of which were paired with semantic information. Half of the participants practiced reading these new items, whereas the others spelled them. The students were then tested 1 and 7 days later on a dictation task. Results revealed a significant main effect of practice type favoring spelling practice and a main effect of the teaching condition, where taught items paired with semantic information were spelled correctly more often than words presented without semantic support. There was no effect of testing time, with learned words being retained well over the 1-week delay, nor were there any significant interactions. The findings support the contention that spelling offers an excellent milieu for orthographic learning to occur. Furthermore, semantics are proposed as a relevant factor in learning to spell, supporting the view that orthographic learning involves the integration of phonological, orthographic, and semantic representations., (Copyright 2010 Elsevier Inc. All rights reserved.)

Inactivating mutations of the CHEK2 and STK11 genes are responsible for Li-Fraumeni and Peutz-Jeghers syndrome, respectively, both autosomal dominant syndromes associated with an increased risk of breast cancer. The PALB2/FANCN gene encodes a nuclear partner of BRCA2 and acts as a linker between BRCA1 and BRCA2. Monoallelic PALB2 truncating mutations were shown to confer higher risk of breast cancer. To evaluate the proportion of French Canadian non-BRCA1/BRCA2 families with high risk of breast cancer potentially harboring alterations in these three breast cancer susceptibility genes, the whole coding and flanking intronic sequences were analyzed in a series of 96 high-risk breast cancer individuals. Despite no PALB2 deleterious truncating mutations being identified, the c.1100delC breast-cancer-associated CHEK2 mutation and a STK11 mutation reported to be the causative mutation in a Peutz-Jeghers family were identified. This extensive analysis also led to the identification of several variants in these genes. Ascertainment of allele frequency of these variants in a cohort of 96 healthy unrelated women suggests a difference in allele frequency for two STK11 intronic variants. In addition, large genomic rearrangements in both STK11 and PALB2 were also examined. Our analysis led to the conclusion that CHEK2, STK11, and PALB2 mutations or large genomic rearrangements of either STK11 or PALB2 are rare, and do not contribute to a substantial fraction of breast cancer susceptibility in high-risk French Canadian breast cancer families.

This study investigated whether semantic information presented along with novel printed nonwords facilitates orthographic learning and examined predictors of individual differences in this important literacy skill. A sample of 35 fourth graders was tested on a variety of language and literacy tests, and participants were then exposed to 10 target nonwords, 5 of which were presented with semantic information. Children were tested 1 and 4days later on their ability to correctly recognize and spell the target nonwords. Results revealed a significant main effect on the recognition task, where items presented with semantic information were identified correctly more often than were words presented in isolation. No significant effect of training condition was found for the spelling posttests. Furthermore, multiple regression analyses revealed that both phonological and semantic factors were significant predictors of orthographic learning. The findings support the view that orthographic learning, as measured through visual recognition, involves the integration of phonological, orthographic, and semantic representations.

Background: The Nijmegen Breakage Syndrome is a chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, and increased frequency of cancers. Familial studies on relatives of these patients indicated that they also appear to be at increased risk of cancer., Methods: In a candidate gene study aiming at identifying genetic determinants of breast cancer susceptibility, we undertook the full sequencing of the NBN gene in our cohort of 97 high-risk non-BRCA1 and -BRCA2 breast cancer families, along with 74 healthy unrelated controls, also from the French Canadian population. In silico programs (ESEfinder, NNSplice, Splice Site Finder and MatInspector) were used to assess the putative impact of the variants identified. The effect of the promoter variant was further studied by luciferase gene reporter assay in MCF-7, HEK293, HeLa and LNCaP cell lines., Results: Twenty-four variants were identified in our case series and their frequency was further evaluated in healthy controls. The potentially deleterious p.Ile171Val variant was observed in one case only. The p.Arg215Trp variant, suggested to impair NBN binding to histone gamma-H2AX, was observed in one breast cancer case and one healthy control. A promoter variant c.-242-110delAGTA displayed a significant variation in frequency between both sample sets. Luciferase reporter gene assay of the promoter construct bearing this variant did not suggest a variation of expression in the MCF-7 breast cancer cell line, but indicated a reduction of luciferase expression in both the HEK293 and LNCaP cell lines., Conclusion: Our analysis of NBN sequence variations indicated that potential NBN alterations are present, albeit at a low frequency, in our cohort of high-risk breast cancer cases. Further analyses will be needed to fully ascertain the exact impact of those variants on breast cancer susceptibility, in particular for variants located in NBN promoter region.

Breast cancer is a heterogeneous disease displaying some degree of familial clustering. Highly penetrant breast cancer susceptibility genes represent approximately 20-25% of the familial aggregation of breast cancer. A significant proportion of this familial aggregation of breast cancer is thus yet to be explained by other breast cancer susceptibility genes. Given the high susceptibility conferred by the two major breast cancer predisposition genes, BRCA1 and BRCA2 and the implication of these genes in many key cellular processes, assessment of genes encoding BRCA1-interacting proteins as plausible breast cancer candidate genes is thus attractive. In this study, four genes encoding BRCA1-interacting proteins were analyzed in a cohort of 96 breast cancer individuals from high-risk non-BRCA1/BRCA2 French Canadian families. Although no deleterious truncating germline mutations or aberrant spliced mRNA species were identified, a total of 10, 4, 11 and 6 variants were found in the AURKA, BAP1, BARD1 and DHX9 genes, respectively. The allele frequency of each variant was further ascertained in a cohort of 98 healthy French Canadian unrelated women and a difference in allele frequency was observed for one BARD1 variant based on single-marker analysis. Haplotype estimation, haplotype blocks and tagging SNPs identification were then performed for each gene, providing a valuable tool for further searches of common disease-associated variants in these genes and therefore further analyses on these genes in larger cohorts is warranted in the search of low-to-moderate penetrance breast cancer susceptibility alleles.

This intervention study tested whether invented spelling plays a causal role in learning to read. Three groups of kindergarten children (mean age = 5 years 7 months) participated in a 4-week intervention. Children in the invented-spelling group spelled words as best they could and received developmentally appropriate feedback. Children in the 2 comparison groups were trained in phonological awareness or drew pictures. Invented-spelling training benefited phonological and orthographic awareness and reading of words used in the intervention. Importantly, the invented-spelling group learned to read more words in a learn-to-read task than the other groups. The finding are in accord with the view that invented spelling coupled with feedback encourages an analytical approach and facilitates the integration of phonological and orthographic knowledge, hence facilitating the acquisition of reading.

The BRIP1 gene encodes a helicase interacting with BRCA1, which contributes to BRCA1-associated DNA repair function. Germ-line BRIP1 mutations affecting the helicase domain activity have been identified in early onset breast cancer patients. In addition, BRIP1 was recently identified as deficient in Fanconi anemia (FA) complementation group J. Given the growing evidence now linking BRCA1, BRCA2, and the FA pathway, as well as the involvement of FA proteins (BRCA2/FANCD1 and PALB2/FANCN) in breast cancer susceptibility, we sought to evaluate the contribution of FANCJ gene alterations regarding breast cancer susceptibility among our cohort of 96 breast cancer individuals from high-risk non-BRCA1/2 French Canadian families. No deleterious mutation, exon deletion, or retention of intronic portions could be identified. However, extensive analysis of the promoter and whole exonic and flanking intronic regions of FANCJ led to the identification of 42 variants, including 22 novel variants not previously reported, four of which were located in the promoter region. Transcription factors analysis revealed a potential involvement of FANCJ promoter variants in regulation of FANCJ expression, and reporter gene assays were performed. The allelic frequency was assessed in a cohort of 73 unaffected French Canadian individuals, and haplotype analysis and tagging single nucleotide polymorphism (SNP) identification were also performed. Although our study unlikely involves FANCJ as a high-risk predisposition gene in non-BRCA1/2 high-risk French Canadian families, the possible association of FANCJ missense variants with phenotypes associated with FA, such as childhood cancer, cannot be excluded.

Our current understanding of breast cancer susceptibility involves mutations in the 2 major genes BRCA1 and BRCA2, found in about 25% of high-risk families, as well as few other low penetrance genes such as ATM and CHEK2. Approximately two-thirds of the multiple cases families remain to be explained by mutations in still unknown genes. In a candidate gene approach to identify new genes potentially involved in breast cancer susceptibility, we analyzed genomic variants in the ZBRK1 gene, a co-repressor implicated in BRCA1-mediated repression of GADD45. Direct sequencing of ZBRK1 entire coding region in affected breast cancer individuals from 97 high-risk French Canadian breast/ovarian cancer families and 94 healthy controls led to the identification of 18 genomic variants. Haplotype analyses, using PHASE, COCAPHASE and HaploStats programs, put in evidence 3 specific haplotypes which could potentially modulate breast cancer risk, and among which 2 that are associated with a potential protective effect (p = 0.01135 and p = 0.00268), while another haplotype is over-represented in the case group (p = 0.00143). Further analyses of these haplotypes indicated that a strong component of the observed difference between both groups emerge from the first 5 variants (out of 12 used for haplotype determination). The present study also permitted to determine a set of tagging SNPs that could be useful for subsequent analyses in large scale association studies. Additional studies in large cohorts and other populations will however be needed to further evaluate if common and/or rare ZBRK1 sequence variants and haplotypes could be associated with a modest/intermediate breast cancer risk., (Copyright 2007 Wiley-Liss, Inc.)

GADD45A is an evolutionary conserved gene whose expression is regulated by two major tumor suppressor proteins involved in breast cancer etiology, namely, p53 and BRCA1, and which acts primarily in the control of the G2/M cell-cycle transition, apoptosis, and DNA repair. Following genotoxic stress, the p53 protein activates GADD45A transcription, whereas in absence of DNA damage, BRCA1 represses GADD45A expression through interaction with the zinc finger protein ZNF350. Moreover, BRCA1 can activate GADD45A gene expression through interactions with transcription factors binding to the gene promoter. On the basis of the intricate network of interactions between GADD45A, p53, and BRCA1, and the fact that both BRCA1 or TP53 mutations are involved in breast cancer tumorigenesis, we undertook the characterization of the entire coding sequence, intron/exon boundaries, and p53- and ZNF350-binding sequences of this potential breast cancer susceptibility candidate gene in a sample set of 96 women affected with breast cancer from non-BRCA1 and BRCA2 French Canadian families with a high risk of breast cancer and 95 healthy controls from the same population. Although none of the 12 identified sequence variations show a significant difference in frequency between both sample sets, haplotype phasing and frequency estimations identified a common haplotype displaying a higher frequency among the control group. As the variants present on this particular haplotype are noncoding variants in either intron 2 or 3, this finding will have to be further investigated in larger cohorts and other populations. In this regard, our study also identified tagging single nucleotide polymorphisms (tSNPs), providing useful data for other large-scale association studies.

Cowden syndrome is a disease associated with an increase in breast cancer susceptibility. Alleles in PTEN and other breast cancer susceptibility genes would be responsible for approximately 25% of the familial component of breast cancer risk, BRCA1 and BRCA2 being the two major genes responsible for this inherited risk. In order to evaluate the proportion of high-risk French Canadian non-BRCA1/BRCA2 breast/ovarian cancer families potentially harboring a PTEN germline mutation, the whole coding and flanking intronic sequences were analyzed in a series of 98 breast cancer cases. Although no germline mutation has been identified in the coding region, our study led to the identification of four intronic variants. Further investigations were performed to analyze the effect of these variants, alone and/or in combination, on splicing and PTEN protein levels. Despite suggestive evidence emerging from in silico analyses, the presence of these intronic variants do not seem to alter RNA splicing or PTEN protein levels. In addition, as loss of PTEN or part of it has been reported, Western blot analysis has also been performed. No major deletion could be identified in our cohort. Therefore, assuming a Poisson distribution for the frequency of deleterious mutation in our cohort, if the frequency of such deleterious mutation was 2%, we would have had a 90% or greater chance of observing at least one such mutation. These results suggest that PTEN germline mutations are rare and are unlikely to account for a significant proportion of familial breast cancer cases in the French Canadian population.

The poly(ADP-ribose) polymerase (PARP/ADPRT) protein family catalyzes the synthesis of cellular poly(ADP-ribose) following DNA damage and is involved in genomic integrity by regulating cellular responses to DNA damage and apoptosis. Moreover, ADPRT inhibition contributes to a protective effect against cancer development. These findings render ADPRT an attractive candidate susceptibility gene for breast cancer, and thus the goal of this study was to evaluate the possible involvement of ADPRT sequence variations in breast cancer susceptibility. The complete sequence of the 23 exons and flanking intronic sequences of the ADPRT gene was analyzed in 54 affected individuals from distinct high-risk non-BRCA1/2 French Canadian families. No deleterious truncating mutation was identified in the coding region. However, 34 sequence variations were identified, among which seven are coding variants and seven are novel changes. All coding variants and intronic changes located in the vicinity of the coding variants identified in the case series were also analyzed in a cohort of 73 unrelated healthy French Canadian individuals. Interestingly, one missense variant (Pro377Ser) was observed in three different breast cancer cases but was not present among unaffected individuals. We have conducted here an exhaustive detailed mutation and haplotype tagging analysis of the ADPRT gene with regard to breast cancer, providing useful data for other large-scale association studies. Additional studies in other cohorts and other populations are however needed to further evaluate the implication of the Pro377Ser missense variant with regard to breast cancer susceptibility.

Background: Ataxia telangiectasia-mutated and Rad3-related (ATR) is a member of the PIK-related family which plays, along with ATM, a central role in cell-cycle regulation. ATR has been shown to phosphorylate several tumor suppressors like BRCA1, CHEK1 and TP53. ATR appears as a good candidate breast cancer susceptibility gene and the current study was designed to screen for ATR germline mutations potentially involved in breast cancer predisposition., Methods: ATR direct sequencing was performed using a fluorescent method while widely available programs were used for linkage disequilibrium (LD), haplotype analyses, and tagging SNP (tSNP) identification. Expression analyses were carried out using real-time PCR., Results: The complete sequence of all exons and flanking intronic sequences were analyzed in DNA samples from 54 individuals affected with breast cancer from non-BRCA1/2 high-risk French Canadian breast/ovarian families. Although no germline mutation has been identified in the coding region, we identified 41 sequence variants, including 16 coding variants, 3 of which are not reported in public databases. SNP haplotypes were established and tSNPs were identified in 73 healthy unrelated French Canadians, providing a valuable tool for further association studies involving the ATR gene, using large cohorts. Our analyses led to the identification of two novel alternative splice transcripts. In contrast to the transcript generated by an alternative splicing site in the intron 41, the one resulting from a deletion of 121 nucleotides in exon 33 is widely expressed, at significant but relatively low levels, in both normal and tumoral cells including normal breast and ovarian tissue., Conclusion: Although no deleterious mutations were identified in the ATR gene, the current study provides an haplotype analysis of the ATR gene polymorphisms, which allowed the identification of a set of SNPs that could be used as tSNPs for large-scale association studies. In addition, our study led to the characterization of a novel Delta33 splice form, which could generate a putative truncated protein lacking several functional domains. Additional studies in large cohorts and other populations will be needed to further evaluate if common and/or rare ATR sequence variants can be associated with a modest or intermediate breast cancer risk.

The interests of First Nations communities in Canada have traditionally had little voice at the various points of authority that maintain the equilibrium or balance necessary to get environmental protection laws ratified, regulations distributed, and enforcement actions initiated on First Nations lands. (First Nations is the term commonly used in Canada to describe the various societies of indigenous peoples who are accorded status as "Indians" by the Indian Act of 1985 and who are not of Inuit or Métis descent.) Along with a lack of adequate funding to address human and environmental issues-as well as past industrial exploitation of First Nations lands-the safety and acceptability of many solid waste management practices in Canadian First Nations communities have become a serious concern for many members from both human and environmental health perspectives. A history of poor management, monitoring, and remediation of solid waste facilities across Canada's First Nations Communities and the lack of current resolve over this issue has left First Nations people feeling the consequences of pollution to their environment: rivers, land, and air. First Nations people are traditionally connected to the land, and consequently the degradation of the environment also leads to a decline in a way of life for the people and thus a decline in the cultural health of communities. This article examines the issues surrounding waste management on First Nations communities, looks at how First Nations are trying to handle their solid waste, and considers the larger issues of environmental degradation that First Nations communities face throughout Canada.

The relations among articulation accuracy, speech perception, and phoneme awareness were examined in a sample of 97 typically developing children ages 48 to 66 months. Of these 97 children, 46 were assessed twice at ages 4 and 5 years. Children completed two tasks for each of the three skills, assessing these abilities for the target phoneme /r/ and the control phoneme /m/ in the word-initial position. Concurrent analyses revealed that phoneme-specific relations existed among articulation, awareness, and perception. Articulation accuracy of /r/ predicted speech perception and phoneme awareness for /r/ after controlling for age, vocabulary, letter-word knowledge, and speech perception or phoneme awareness for the control phoneme /m/. The longitudinal analyses confirmed the pattern of relations. The findings are consistent with a model whereby children's articulation accuracy affects preexisting differences in phonological representations and, consequently, affects how children perceive, discriminate, and manipulate speech sounds.

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.

We performed an analysis of inbreeding and kinship among the ascending genealogies of 205 autopsy-confirmed Alzheimer disease (AD) subjects recruited in the Saguenay area of Québec. We hypothesized that if some traits pertaining to the disease were determined by inherited factors, and if the corresponding genes were not too frequent in the population, it might be possible to detect some clusters of patients related to common ancestors and presenting a level of kinship and/or inbreeding higher than is observed in the unaffected population of the same age. In view of the heterogeneity of the disease, we also verified if some of the factors investigated could be associated more specifically with subsets of cases based on age of onset and on apolipoprotein E (APOE) genotype. Results were compared with those obtained on 205 controls matched for gender, place and year of birth. We found that late-onset AD cases with an APOE-epsilon 4 were significantly more inbred than controls and that this increase was explained by the high level of inbreeding of a few cases whose parents were related at the first-cousin level. This could possibly indicate the implication of a recessive element in a small subset of AD cases in the Saguenay population. We also found that late-onset epsilon 4+ cases were significantly more closely related among themselves than with controls. This increase in kinship may be attributable to the presence of the epsilon 4 allele or to some other unidentified genetic factor possibly acting in conjunction with APOE-epsilon 4.

In a series of 33 intertrochanteric femoral fractures in patients suffering from classic rheumatoid arthritis (RA), the typical patient was about 70 years of age, osteopenic, and severely affected by longstanding polyarticular RA of joints other than the hip. Prefracture involvement of the hip joint (other than osteopenia) was apparent radiographically in only 9.1% of patients. There was a higher rate of avascular necrosis (9.7%) and nonunion (6.5%) than expected in the nonrheumatoid general population. There was also a high rate of secondary displacement. Of three patients with avascular necrosis, two had had corticosteroid treatment.

We present a method of fabricating nonimaging light concentrators from total internal reflection film. A prototype has been made and tested and found to operate in agreement with predictions of ray-tracing codes. The performance of the prototype is comparable with that of concentrators made from specular reflecting materials.

We have previously shown that descendants of CHO-derived hprt or aprt mutants induced by ethyl methanesulphonate usually undergo a rapid loss of the mutant phenotype during the 10 generations or so of culture in non-selective medium immediately following mutagenesis (Bradley, 1980; Bradley and Laviolette, 1989). We now present an analysis of several mutants and their descendants which have lost the mutant phenotype, or 'reversed'. The drug-resistance properties of reversed cells were generally intermediate between W.T. and mutant, and message level and enzyme-specific activity were also intermediate, correlating with the phenotype. Although this was consistent with a model of inactivation-reactivation of the target gene to explain the reversal phenomenon, the model was ruled out by Northern blot analysis of several induced mutants, which showed no correlation between level of message and tendency of the mutant to lose its phenotype. Karyotype analysis showed that three out of four reversed lines were near-tetraploid and the fourth had a substantial proportion of near-tetraploid cells. This suggests cell fusion between a mutant and a W.T. cell may explain the phenomenon. A prediction of this model, namely that mutagen treatment increases cell hybrid formation, was tested and found to be true.

We reviewed the records and radiographs from 10 hospitals to identify 50 patients with rheumatoid arthritis (RA) who had sustained 52 femoral neck fractures. Most patients were female (88%), elderly (mean age 66 years) and had had severe polyarticular disease for a mean duration of 16 years. Over half had taken systemic corticosteroids, nearly all were severely osteopenic but few had rheumatoid changes in the hip. Of the 20 fractures treated by internal fixation 12 had complications including nonunion (5), osteonecrosis (5), infection (1), and intertrochanteric fracture (1). Only one of the nine undisplaced fractures required reoperation, but seven of the 11 displaced fractures had revision surgery. Twenty fractures were treated by primary total hip arthroplasty with only one early complication. The other 12 fractures had been treated by hemiarthroplasty (9), hip excision (1) or non-operatively (2). Our results suggest that, in elderly rheumatoid patients, severely displaced femoral neck fractures should be treated by total hip replacement.

We established for human fetal liver cells (cultured for 2 wk) in a hormonally defined medium, optimal conditions for attachment, multiplication, and preservation of epithelial morphology as well as production and secretion of serum proteins characteristic of fetal (alpha l-fetoprotein, AFP) and adult (albumin and hemopexin) life. Conditions were considered optimal when cell number, albumin, and hemopexin levels were maintained throughout the 2-wk culture period. However, the decrease in AFP concentration, which occurred after a few days of culture, could not be reversed. The culture system developed is a suitable model for studying regulatory mechanisms governing structure and function during differentiation and may prove useful for testing the effect of toxic agents during fetal development of the human liver.

Patterns of methylation of CpG dinucleotides in the promoter region of the thymidine kinase (TK) gene in wild-type and TK-deficient Chinese hamster cell lines were studied. Whereas wild-type cells were unmethylated, three conventionally derived TK-deficient cell lines were all almost completely methylated in the promoter region. Demethylation at a number of different CpG sites was observed upon selection for reexpression of the TK gene. Of thirteen HhaI (GCGC) or HpaII (CCGG) sites studied, the highest correlation between absence of methylation and at least partial TK activity was obtained at one HhaI site within 20 bp of the putative cap site. Silencing in the three conventionally derived mutants is therefore accompanied by hypermethylation of the promoter-associated CpG-rich island. We contrast this situation with another type of silencing event, in which TK was coordinately inactivated at a high frequency with at least one other linked allele. Methylation of the promoter region of TK was not associated with this event, but two lines of evidence suggested a role for methylation at sites other than in the promoter region of TK.