A Homozygous Deep Intronic Mutation Alters the Splicing of Nebulin Gene in a Patient With Nemaline Myopathy.

Nemaline myopathy is a rare disorder affecting the muscle sarcomere. Mutations in nebulin gene (NEB) are known to be responsible for about 50% of nemaline myopathy cases. Nebulin is a giant protein which is formed integrally with the sarcomeric thin filament. This complex gene is under extensive alternative splicing giving rise to multiple isoforms. In this study, we report a 6-year-old boy presenting with general muscular weaknesses. Identification of rod-shaped structures in the patient' biopsy raised doubt about the presence of a nemaline myopathy. Next-generation sequencing was used to identify a causative mutation for the patient syndrome. A homozygous deep intronic substitution was found in the intron 144 of the NEB. The variant was predicted by in silico tools to create a new donor splice site. Molecular analysis has shown that the mutation could alter splicing events of the nebulin gene leading to a significant decrease of isoforms level. This change in the expression level of nebulin could give rise to functional consequences in the sarcomere. These results are consistent with the phenotypes observed in the patient. Such a discovery of variants in this gene will allow a better understanding of the involvement of nebulin in neuromuscular diseases and help find new treatments for the nemaline myopathy.

A founder mutation in the PLPBP gene in families from Saguenay-Lac-St-Jean region affected by a pyridoxine-dependent epilepsy.

Pyridoxine-dependent epilepsy (PDE) is a relatively rare subgroup of epileptic disorders. They generally present in infancy as an early onset epileptic encephalopathy or seizures, refractory to standard treatments, with rapid and variable responses to vitamin B6 treatment. Whole exome sequencing of three unrelated families identified homozygous pathogenic mutation c.370_373del, p.Asp124fs in PLPBP gene in five persons. Haplotype analysis showed a single shared profile for the affected persons and their parents, leading to a hypothesis about founder effect of the mutation in Saguenay-Lac-St-Jean region of French Canadians. All affected probands also shared one single mitochondrial haplotype T2b3 and two rare variations in the mitochondrial genome m.801A>G and m.5166A>G suggesting that a single individual female introduced PLPBP mutation c.370_373del, p.Asp124fs in Quebec. The mutation p.Asp124fs causes a severe disease phenotype with delayed myelination and cortical/subcortical brain atrophy. The most noteworthy radiological finding in this Quebec founder mutation is the presence of the temporal cysts that can be used as a marker of the disease. Also, both patients, who are alive, had a history of prenatal supplements taken by their mothers as antiemetic medication with high doses of pyridoxine. In the context of suspected PDE in patients with neonatal refractory seizures, treatment with pyridoxine and/or Pyridoxal-5-phophate has to be started immediately and continued until the results of genetic analysis received. Even with early appropriate treatment, neurological outcome of our patient is still poor.

Whole-exome sequencing identifies homozygous mutation in TTI2 in a child with primary microcephaly: a case report.

BACKGROUND: Primary microcephaly is defined as reduced occipital-frontal circumference noticeable before 36 weeks of gestation. Large amount of insults might lead to microcephaly including infections, hypoxia and genetic mutations. More than 16 genes are described in autosomal recessive primary microcephaly. However, the cause of microcephaly remains unclear in many cases after extensive investigations and genetic screening. CASE PRESENTATION: Here, we described the case of a boy with primary microcephaly who presented to a neurology clinic with short stature, global development delay, dyskinetic movement, strabismus and dysmorphic features. We performed microcephaly investigations and genetic panels. Then, we performed whole-exome sequencing to identify any genetic cause. Microcephaly investigations and genetic panels were negative, but we found a new D317V homozygous mutation in TELOE-2 interacting protein 2 (TTI2) gene by whole-exome sequencing. TTI2 is implicated in DNA damage response and mutation in that gene was previously described in mental retardation, autosomal recessive 39. CONCLUSIONS: We described the first French Canadian case with primary microcephaly and global developmental delay secondary to a new D317V homozygous mutation in TTI2 gene. Our report also highlights the importance of TTI2 protein in brain development.

Identification of a gene signature for different stages of breast cancer development that could be used for early diagnosis and specific therapy.

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.

Transcriptional signature of lymphoblastoid cell lines of BRCA1, BRCA2 and non-BRCA1/2 high risk breast cancer families.

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.

Genome-wide methylation analysis of DNMT3B gene isoforms revealed specific methylation profiles in breast cell lines.

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.

Analysis of a FANCE Splice Isoform in Regard to DNA Repair.

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.

The Role of Methylation in Breast Cancer Susceptibility and Treatment.

DNA methylation is a critical mechanism of epigenetic modification involved in gene expression programming, that can promote the development of several cancers, including breast cancer. The methylation of CpG islands by DNA methyltransferases is reversible and has been shown to modify the transcriptional activity of key proliferation genes or transcription factors involved in suppression or promotion of cell growth. Indeed, aberrant methylation found in gene promoters is a hallmark of cancer that could be used as non-intrusive biomarker in body fluids such as blood and plasma for early detection of breast cancer. Many biomarker genes have been evaluated for breast cancer detection. However, in the absence of a unique biomarker having the sufficient specificity and sensitivity, a panel of multiple genes should be used. Treatments targeting aberrant methylation by DNA methyltransferase inhibitors, which trigger re-expression of silenced genes, are now available and allow for better treatment efficiency.

Analysis of ZNF350/ZBRK1 promoter variants and breast cancer susceptibility in non-BRCA1/2 French Canadian breast cancer families.

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.

Polymorphic variations in the FANCA gene in high-risk non-BRCA1/2 breast cancer individuals from the French Canadian population.

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.

Evaluation of the contribution of the three breast cancer susceptibility genes CHEK2, STK11, and PALB2 in non-BRCA1/2 French Canadian families with high risk of breast cancer.

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.

Variations in the NBN/NBS1 gene and the risk of breast cancer in non-BRCA1/2 French Canadian families with high risk of breast cancer.

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.

Genetic sequence variations of BRCA1-interacting genes AURKA, BAP1, BARD1 and DHX9 in French Canadian families with high risk of breast cancer.

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.

Fine temporal analysis of DHT transcriptional modulation of the ATM/Gadd45g signaling pathways in the mouse uterus.

In rodents, the uterus of a mature female undergoes changes during the uterine cycle, under the control of steroid hormones. 5alpha-Dihydrotestosterone (DHT) is recognized to play an important role in the regulation of androgen action in normal endometrium. Using microarray technology, a screening analysis of genes responding to DHT in the uterus of ovariectomized mice, has allowed us to highlight multiple genes of the ATM/Gadd45g pathway that are modulated following exposure to DHT. Two phases of regulation were identified. In the early phase, the expression of genes involved in the G2/M arrest is rapidly increased, followed by the repression of genes of the G1/S checkpoint, and by the induction of transcriptional regulators. Later, i.e. from 12 to 24 hr, genes involved in G2/M transition, cytoarchitectural and lipid-related genes are stimulated by DHT while immunity-related genes appear to be differentially regulated by the hormone. These results show that a physiological dose of DHT induces the transcription of genes promoting the cell cycle progression in mice. Profile determination of temporal uterine gene expression at the transcriptional level enables us to suggest that the DHT modulation of genes involved in ATM/Gadd45g signaling in an ATM- or p53-independent manner, could play an important role in the cyclical changes of uterine cells in the mouse uterus.

Mutational analysis of the breast cancer susceptibility gene BRIP1 /BACH1/FANCJ in high-risk non-BRCA1/BRCA2 breast cancer families.

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.

Analysis of GADD45A sequence variations in French Canadian families with high risk of breast cancer.

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.

Genetic variants and haplotype analyses of the ZBRK1/ZNF350 gene in high-risk non BRCA1/2 French Canadian breast and ovarian cancer families.

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.

Genetic sequence variations and ADPRT haplotype analysis in French Canadian families with high risk of breast cancer.

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.

Germline mutations in the breast cancer susceptibility gene PTEN are rare in high-risk non-BRCA1/2 French Canadian breast cancer families.

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.

Temporal analysis of E2 transcriptional induction of PTP and MKP and downregulation of IGF-I pathway key components in the mouse uterus.

17beta-Estradiol (E2) is well known to be associated with uterine cancer, endometriosis, and leiomyomas. Although insulin-like growth factor I (IGF-I) has been identified as a mediator of the uterotrophic effect of E2 in several studies, this mechanism is still not well understood. In the present study, identification of the genes modulated by a physiological dose of E2, in the uterus, has been done in ovariectomized mice using Affymetrix microarrays. The E2-induced genomic profile shows that multiple genes belonging to the IGF-I pathway are affected after exposure to E2. Two phases of regulation could be identified. First, from 0 to 6 h, the expression of genes involved in the cell cycle, growth factors, protein tyrosine phosphatases, and MAPK phosphatases is quickly upregulated by E2, while IGF-I receptor and several genes of the MAPK and phosphatidylinositol 3-kinase pathways are downregulated. Later, i.e., from 6 to 24 h, transporters and peptidases/proteases are stimulated, whereas defense-related genes are differentially regulated by E2. Finally, cytoarchitectural genes are modulated later. The present data show that a physiological dose of E2 induces, within 24 h, a series of transcriptional events that promote the uterotrophic effect. Among these, the E2-mediated activation of the IGF-I pathway seems to play a pivotal role in the uterotrophic effect. Furthermore, the protein tyrosine phosphatases and MAPK phosphatases are likely to modulate the estrogenic uterotrophic action by targeting, at different steps, the IGF-I pathway.