Genetic changes occurring in different stages of pre-cancer lesions reflect causal events initiating and promoting the progression to cancer. Co-existing pre-cancerous lesions including low-and high-grade squamous intraepithelial lesion... more
Genetic changes occurring in different stages of pre-cancer lesions reflect causal events initiating and promoting the progression to cancer. Co-existing pre-cancerous lesions including low-and high-grade squamous intraepithelial lesion (LGSIL and HGSIL), and adjacent "normal" cervical epithelium from six formalin-fixed paraffin-embedded samples were selected. Tissues from these 18 samples were isolated using laser-capture microdissection, RNA was extracted and sequenced. RNA-sequencing generated 2.4 billion raw reads in 18 samples, of which~50.1% mapped to known and annotated genes in the human genome. There were 40 genes up-regulated and 3 down-regulated (normal to LGSIL) in at least one-third of the sample pairs (same direction and FDR p < 0.05) including S100A7 and KLK6. Previous studies have shown that S110A7 and KLK7 are up-regulated in several other cancers, whereas CCL18, CFTR, and SLC6A14, also differentially expressed in two samples, are up-regulated specifically in cervical cancer.These differentially expressed genes in normal to LGSIL progression were enriched in pathways related to epithelial cell differentiation, keratinocyte differentiation, peptidase, and extracellular activities. In progression from LGSIL to HGSIL, two genes were up-regulated and five down-regulated in at least two samples. Further investigations using co-existing samples, which account for all internal confounders, will provide insights to better understand progression of cervical pre-cancer.
- by David Crossman and +1
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- Gene expression, Human Genome, RNA Sequencing
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit... more
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis. Female ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2. The expression of OGG1 was suppressed in mammary tissues and in mammary tum...
Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature, evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced... more
Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature, evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17β-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-tr...
Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis.... more
Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective estrogen receptor modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ERs. In the present study, we examined the effect of long-term tamoxifen treatment in the prevention of estrogen-induced breast cancer. Female ACI rats were treated with 17β-estradiol (E2), tamoxifen or with a combination of E2 and tamoxifen for eight months. Tissue levels of oxidative stress markers 8-iso-Prostane F(2α) (8-isoPGF(2α)), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) were quantified in the mammary tissues of all the treatment groups and compared with age-matched controls. Levels...
Epidemiological evidence indicates that prolonged lifetime exposure to estrogen is associated with elevated breast cancer risk in women. Oxidative stress and estrogen receptor-associated proliferative changes are suggested to play... more
Epidemiological evidence indicates that prolonged lifetime exposure to estrogen is associated with elevated breast cancer risk in women. Oxidative stress and estrogen receptor-associated proliferative changes are suggested to play important roles in estrogen-induced breast carcinogenesis. In the present study, we investigated changes in breast morphology and oxidative stress following estrogen exposure. Female ACI rats were treated with 17β-estradiol (E 2 , 3 mg, s.c.) for either 7, 15, 120 or 240 days. Animals were sacrificed, tissues were excised, and portions of the tissues were either fixed in 10% buffered formalin or snap-frozen in liquid nitrogen. Paraffin-embedded tissues were examined for histopathologic changes. Proliferative changes appeared in the breast after 7 days of E 2 exposure. Atypical ductal proliferation and significant reduction in stromal fat were observed following 120 days of E 2 exposure. Both in situ and invasive carcinomas were observed in the majority of the mammary glands from rats treated with E 2 for 240 days. Palpable breast tumors were observed in 82% of E 2 -treated rats after 228 days, with the first palpable tumor appearing after 128 days. No morphological changes were observed in the livers, kidneys, lungs or brains of rats treated with E 2 for 240 days compared to controls. Furthermore, 8-isoprostane (8-isoPGF 2α ) levels as well as the activities of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase and catalase, were quantified in the breast tissues of rats treated with E 2 for 7, 15, 120 and 240 days and compared to activity levels in age-matched controls. 8-isoPGF 2α levels displayed time-dependent increases upon E 2 treatment and were significantly higher than control levels at the 15, 120 and 240 day time-points. 8-isoPGF 2α levels observed in E 2 -induced mammary tumors were significantly higher than levels found in control mammary tissue from age-matched animals. Similarly, alterations in glutathione peroxidase and superoxide dismutase activities were detected in both mammary and tumor tissue from E 2 -treated rats. Taken together, our data reveal that proliferative changes in the breast tissue of ACI rats are associated with increases in 8-isoPGF 2α formation as well as changes in the activities of antioxidant enzymes. These oxidative changes appear to be a function of E 2 exposure and occur prior to tumor development.
The genome project increased appreciation of genetic complexity underlying disease phenotypes: many genes contribute each phenotype and each gene contributes multiple phenotypes. The aspiration of predicting common disease in individuals... more
The genome project increased appreciation of genetic complexity underlying disease phenotypes: many genes contribute each phenotype and each gene contributes multiple phenotypes. The aspiration of predicting common disease in individuals has evolved from seeking primary loci to marginal risk assignments based on many genes. Genetic interaction, defined as contributions to a phenotype that are dependent upon particular digenic allele combinations, could improve prediction of phenotype from complex genotype, but it is difficult to study in human populations. High throughput, systematic analysis of S. cerevisiae gene knockouts or knockdowns in the context of disease-relevant phenotypic perturbations provides a tractable experimental approach to derive gene interaction networks, in order to deduce by cross-species gene homology how phenotype is buffered against disease-risk genotypes. Yeast gene interaction network analysis to date has revealed biology more complex than previously imagi...
- by John Hartman and +2
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- Genes
Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which -owing to extensive interparalog sequence... more
Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which -owing to extensive interparalog sequence exchange -closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2-and PMS2CL-specific sequences. We selected eight such reference samples -all publicly available -and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5 0 breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations.
- by Ludwine Messiaen and +2
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- Adolescent, Biological Sciences, Child, Neurofibromatosis
The expression of a gene requires not only a normal coding sequence but also intact regulatory regions, which can be located at large distances from the target genes, as demonstrated for an increasing number of developmental genes. In... more
The expression of a gene requires not only a normal coding sequence but also intact regulatory regions, which can be located at large distances from the target genes, as demonstrated for an increasing number of developmental genes. In previous mutation studies of the role of FOXL2 in blepharophimosis syndrome (BPES), we identified intragenic mutations in 70% of our patients. Three translocation breakpoints upstream of FOXL2 in patients with BPES suggested a position effect. Here, we identified novel microdeletions outside of FOXL2 in cases of sporadic and familial BPES. Specifically, four rearrangements, with an overlap of 126 kb, are located 230 kb upstream of FOXL2, telomeric to the reported translocation breakpoints. Moreover, the shortest region of deletion overlap (SRO) contains several conserved nongenic sequences (CNGs) harboring putative transcription-factor binding sites and representing potential long-range cis-regulatory elements. Interestingly, the human region orthologous to the 12-kb sequence deleted in the polled intersex syndrome in goat, which is an animal model for BPES, is contained in this SRO, providing evidence of human-goat conservation of FOXL2 expression and of the mutational mechanism. Surprisingly, in a fifth family with BPES, one rearrangement was found downstream of FOXL2. In addition, we report nine novel rearrangements encompassing FOXL2 that range from partial gene deletions to submicroscopic deletions. Overall, genomic rearrangements encompassing or outside of FOXL2 account for 16% of all molecular defects found in our families with BPES. In summary, this is the first report of extragenic deletions in BPES, providing further evidence of potential long-range cis-regulatory elements regulating FOXL2 expression. It contributes to the enlarging group of developmental diseases caused by defective distant regulation of gene expression. Finally, we demonstrate that CNGs are candidate regions for genomic rearrangements in developmental genes.
Repeated occurrence of a hitherto unrecognized form of spondyloepiphyseal dysplasia tarda (SED tarda) has been studied in two independent families. Because parental consanguinity was also present in one family, autosomal recessive... more
Repeated occurrence of a hitherto unrecognized form of spondyloepiphyseal dysplasia tarda (SED tarda) has been studied in two independent families. Because parental consanguinity was also present in one family, autosomal recessive inheritance is proposed. The onset was in late childhood. The slowly evolving disorder shared several features of the already known types of SED tarda. The radiographic abnormalities were limited to the spine and proximal femora. The patients' hands were normal. The entity described is set apart not only from the X-linked and autosomal-dominant forms of SED tarda but also from the already delineated autosomal recessive types by significant clinical and radiographic differences. Final genotypic characterization must await the results of genetic linkage studies and of appropriate molecular genetics investigations. ß
- by Ludwine Messiaen and +1
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- Genetics, Spine, Adolescent, Child
Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic... more
Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.
Members of the steroid-thyroid-retinoid receptor superfamily regulate a spectrum of cellular functions, including metabolism and growth and differentiation. We sought to isolate novel members of this family by using degenerate... more
Members of the steroid-thyroid-retinoid receptor superfamily regulate a spectrum of cellular functions, including metabolism and growth and differentiation. We sought to isolate novel members of this family by using degenerate oligonucleotide primers directed to sequences encoding the AF-2 domain of these molecules in a PCR-based approach. The AF-2 domain serves a critical function in recruiting coregulatory molecules and in transcriptional activation. We report the cloning and initial characterization of a novel gene, WDR10, which encodes a 140-kD protein that is highly expressed in pituitary and testis. This protein, WDR10p, contains an AF-2 domain as well as seven N-terminal WD repeats and is highly conserved through evolution. Chromosomal localization studies placed WDR10 at 3q21, near a locus for the Moebius syndrome, Hailey-Hailey disease, and rhodopsin, which is involved in several forms of retinitis pigmentosa. The expression pattern of WDR10 and its chromosomal location makes this novel gene a candidate gene for the hypogonadism associated with some forms of retinitis pigmentosa and the Moebius syndrome. 41
Objective: To evaluate the use of multiple displacement amplification (MDA) for whole-genome amplification in the preimplantation genetic diagnosis (PGD) of Marfan syndrome. Design: Multiple displacement amplification was used to amplify... more
Objective: To evaluate the use of multiple displacement amplification (MDA) for whole-genome amplification in the preimplantation genetic diagnosis (PGD) of Marfan syndrome. Design: Multiple displacement amplification was used to amplify the whole-genome directly from a single cell. The MDA product was used for polymerase chain reaction (PCR) analysis of five different loci. At this point MDA was used to develop a PGD-Marfan syndrome program. Setting: Fertility and gynecology private center in Alicante, Spain. Patient(s): A couple in which the husband is affected by Marfan syndrome and carries a novel mutation in the FBN-1 gene. Intervention(s): The MDA of single cells and PCR tests for PGD. Main Outcome Measure(s): Allele drop-out (ADO), amplification efficiency rates, and the ability to detect Marfan syndrome using MDA.
About 4% of all BRCA1 and BRCA2 alterations reported to the Breast Information Core database are splice site variants. Only a limited number of them have been studied at the RNA level. By BRCA1 and BRCA2 mutation analysis of... more
About 4% of all BRCA1 and BRCA2 alterations reported to the Breast Information Core database are splice site variants. Only a limited number of them have been studied at the RNA level. By BRCA1 and BRCA2 mutation analysis of breast/ovarian cancer families, we identified two novel and eight previously reported potential splice site mutations, never characterized at the cDNA level before. RT-PCR was performed to determine whether these variants disrupted correct splicing. To ensure efficient detection of transcripts containing premature termination codons, a nonsense-mediated mRNA decay inhibitor was added to the lymphoblastoid cell lines of the patients before RNA extraction. We found that BRCA1 IVS3ϩ3AϾC, 4304GϾA (in the last codon of exon 12), and IVS19ϩ2delT and BRCA2 IVS6ϩ1GϾA, IVS23Ϫ2AϾG, and IVS24ϩ1GϾA lead to aberrant transcripts in lymphocytes. Therefore, they were considered to be true pathogenic mutations, predisposing carriers to cancers of the hereditary breast/ovarian cancer syndrome. BRCA2 IVS24Ϫ16TϾC is a frequent polymorphism in linkage disequilibrium, with a polymorphic stop codon in exon 27, K3326X. BRCA1 IVS2Ϫ14CϾT and BRCA2 IVS9Ϫ5insT and IVS25ϩ9AϾC represent rare variants, not disrupting normal splicing in blood lymphocytes. However, some of the alterations may act differently, qualitatively and/or quantitatively, in breast or ovarian tissues. The data provided in this paper allowed more accurate risk estimation of patients and relatives carrying the mutations described herein and have facilitated genetic counseling. Furthermore, our study is important for a better understanding of splicing mechanisms and revealed new patterns of alternative splicing in BRCA1 and BRCA2.
Neurofibromatosis type I (NF1) is an autosomal dominant familial tumor syndrome characterized by the presence of multiple benign neurofibromas. In 95% of NF1 individuals, a mutation is found in the NF1 gene, and in 5% of the patients, the... more
Neurofibromatosis type I (NF1) is an autosomal dominant familial tumor syndrome characterized by the presence of multiple benign neurofibromas. In 95% of NF1 individuals, a mutation is found in the NF1 gene, and in 5% of the patients, the germline mutation consists of a microdeletion that includes the NF1 gene and several flanking genes. We studied the frequency of loss of heterozygosity (LOH) in the NF1 region as a mechanism of somatic NF1 inactivation in neurofibromas from NF1 patients with and without a microdeletion. There was a statistically significant difference between these two patient groups in the proportion of neurofibromas with LOH. None of the 40 neurofibromas from six different NF1 microdeletion patients showed LOH, whereas LOH was observed in 6/28 neurofibromas from five patients with an intragenic NF1 mutation (P ¼ 0.0034, Fisher's exact). LOH of the NF1 microdeletion region in NF1 microdeletion patients would de facto lead to a nullizygous state of the genes located in the deletion region and might be lethal. The mechanisms leading to LOH were further analyzed in six neurofibromas. In two out of six neurofibromas, a chromosomal microdeletion was found; in three, a mitotic recombination was responsible for the observed LOH; and in one, a chromosome loss with reduplication was present. These data show an important difference in the mechanisms of second hit formation in the 2 NF1 patient groups. We conclude that NF1 is a familial tumor syndrome in which the type of germline mutation influences the type of second hit in the tumors. V V C
- by Ludwine Messiaen and +1
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- Phenotype, Genes
Neurofibromatosis type 1 (NF1), the most common tumor-predisposing disorder in humans, is caused by defects in the NF1 tumor-suppressor gene. Comprehensive mutation analysis applying RNA-based techniques complemented with FISH analysis... more
Neurofibromatosis type 1 (NF1), the most common tumor-predisposing disorder in humans, is caused by defects in the NF1 tumor-suppressor gene. Comprehensive mutation analysis applying RNA-based techniques complemented with FISH analysis achieves mutation detection rates of $95% in NF1 patients. The majority of mutations are minor lesions, and $5% are total gene deletions. We found 13 single-and/or multiexon deletions/duplications out of 1,050 detected mutations using our RNA-based approach in a cohort of 1,100 NF1 patients and confirmed these changes using multiplex ligation-dependent probe amplification (MLPA). With MLPA, we found another 12 novel multiexon deletion/duplications in 55 NF1 patients for whom analysis with multiple assays had not revealed a NF1 mutation, including 50 previously analyzed comprehensively. The extent of the 22 deletions and 3 duplications varied greatly, and there was no clustering of breakpoints. We also evaluated the sensitivity of MLPA in identifying deletions in a mosaic state. Furthermore, we tested whether the MLPA P122 NF1 area assay could distinguish between type I deletions, with breakpoints in low-copy repeats (NF1-LCRs), and type II deletions, caused by aberrant recombination between the JJAZ gene and its pseudogene. Our study showed that intragenic deletions and/or duplications represent only $2% of all NF1 mutations. Although MLPA did not substantially increase the mutation detection rate in NF1 patients, it was a useful first step in a comprehensive mutation analysis scheme to quickly pinpoint patients with single-or multiexon deletions/duplications as well as patients with a total gene deletion who will not need full sequencing of the complete coding region.
Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by congenital nystagmus and photodysphoria, moderate to severe reduction of visual acuity, hypopigmentation of the retina, and the presence of macromelanosomes in... more
Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by congenital nystagmus and photodysphoria, moderate to severe reduction of visual acuity, hypopigmentation of the retina, and the presence of macromelanosomes in the skin and eyes. We have previously isolated the gene for OA1 and characterized its protein product as melanosomal membrane glycoprotein displaying structural and functional features of G proteincoupled receptors. We and others have identified mutations of various types within the OA1 gene in patients with this disorder, including deletions and splice site, frameshift, nonsense, and missense mutations. However, different prevalences of large intragenic deletions have been reported, ranging from 10% to 50% in independent studies. To determine whether these differences might be related to the geographic origin of the OA1 families tested, we performed a further extensive mutation analysis study leading to the identification of pathogenic mutations in 30 unrelated OA1 patients mainly from Europe and North America. These results, together with our earlier mutation reports on OA1, allow us to resolve the apparent discrepancies between previous studies and point to a substantial difference in the frequency of large intragenic deletions in European (<10%) compared with North American (>50%) OA1 families. These observations and our overall refinement of point mutation distribution within the OA1 gene have important implications for the molecular diagnosis