Yvan Labrie

Mutation analysis and characterization of ATR sequence variants in breast cancer cases from high-risk French Canadian breast/ovarian cancer families

BackgroundAtaxia 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.MethodsATR 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.ResultsThe 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.ConclusionAlthough 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 Δ33 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.

Androgens induce expression of SPAK, a STE20/SPS1-related kinase, in LNCaP human prostate cancer cells.

Genes that are regulated by androgens in the human prostate are believed to play an essential role in prostate physiology and they may also be involved in the proliferative response of prostate cancer cells to androgens. We used a cDNA subtraction approach to identify novel androgen-regulated transcripts in LNCaP cells that were exposed to 0.1 nM R1881 for 24 h. We report here that SPAK, a recently identified STE20/SPS1-related kinase that modulates p38 MAP kinase activity, exhibited increased expression in androgen-treated LNCaP cells. Androgen regulation of SPAK was both dose- and time-dependent. R1881-induced SPAK expression was completely abrogated by the antiandrogen casodex and by actinomycin D indicating that androgen induction of SPAK requires the androgen receptor and transcription. Cycloheximide caused a partial inhibition of R1881-induced SPAK expression which suggests that androgen induction of SPAK expression may require synthesis of additional proteins. Northern blot and ribonuclease protection assays demonstrated that SPAK is expressed at high levels in normal human testes and prostate, as well as in a number of breast and prostate cancer cell lines. These results identify SPAK, a member of a key cell signalling pathway, as an androgen-responsive gene in LNCaP cells. We hypothesize that SPAK may mediate androgen action in the normal and cancerous prostate gland.

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

AbstractThe 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.

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.

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

BackgroundThe 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.MethodsIn 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.ResultsTwenty-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 γ-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.ConclusionOur 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.

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 ∼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.

Mapping of the HSD17B2 gene encoding type II 17β-hydroxysteroid dehydrogenase close to D16S422 on chromosome 16q24.1-q24.2

The enzymes of the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17 beta-hydroxysteroid counterparts. The structure of human type II 17 beta-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of delta 4-androstenedione and testosterone, androstanedione and dihydrotestosterone, and estrone and 17 beta-estradiol, whereas type I 17 beta-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 bp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Généthon. The maximal pairwise lod score (Zmax = 33.3) with a maximal recombination fraction (theta max) of 0.008 was obtained with the marker D16S422 located on 16q24.1-q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Généthon markers. The order of the HSD17B2 gene and markers is qter-D16S516-D16S504-D16S507-D16S505-D16S511+ ++-[HSD17B2-D16S422]-D16S520- D16S413-tel.

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.

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.

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.