Alterations in candidate genes PHF2, FANCC, PTCH1 and XPA at chromosomal 9q22.3 region: Pathological significance in early- and late-onset breast carcinoma

Introduction: Younger women with breast carcinoma (BC) exhibits more aggressive pathologic features compared to older women; young age could be an independent predictor of adverse prognosis. To find any existing differences in the molecular pathogenesis of BC in both younger and older women, alterations at chromosomal (chr.) 9q22.32-22.33 region were studied owing to its association in wide variety of tumors. Present work focuses on comparative analysis of alterations of four candidate genes; PHF2, FANCC, PTCH1 and XPA located within 4.4 Mb region of the afore-said locus in two age groups of BC, as well as the interrelation and prognostic significance of alterations of these genes. Methods: Deletion analysis of PHF2, FANCC, PTCH1 and XPA were examined in a subset of 47 early-onset (group-A: ≤ 40 years) and 59 late-onset (group-B: > 40 years) breast carcinomas using both microsatellite and exonic markers. Methylation Sensitive Restriction analysis (MSRA) was done to check for promoter methylation. Quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemisty (IHC) was done in some genes to see their relative mRNA and protein expressions respectively. Clinico-pathological correlation of different parameters as well as patient survival was calculated using different statistical softwares like EpiInfo 6.04b, SPSS 10.0 etc. Results: Either age group exhibited high frequency of overall alterations in PHF2, FANCC and PTCH1 compared to XPA. Samples with alteration (deletion/methylation) in these genes showed reduced level of mRNA expression as seen by Q-PCR. Immunohistochemical analysis of FANCC and PTCH1 also supported this observation. Poor patient survival was noted in both age groups having alterations in FANCC. Similar result was also seen with PTCH1 and XPA alterations in group-A and PHF2 alterations in group-B. This reflected their roles as prognostic tools in the respective groups in which they were altered. Conclusion: Overall alterations of PHF2, FANCC and PTCH1 were comparatively higher than XPA. Differential association of alterations in FANCC and PTCH1 with that of PHF2, XPA and two breast cancer susceptibility genes (BRCA1/BRCA2) in the two age groups suggests differences in their molecular pathogenesis and dysregulation of multiple DNA repair pathways as well as hedgehog dependent stem cell renewal pathway

DNA linkage based diagnosis of Wilson disease in asymptomatic siblings

Wilson disease (WD) is an autosomal recessive disorder caused by defects in ATP7B gene located in chromosome 13q14, and manifested as hepatolenticular degeneration as a result of accumulation of copper. No information on the mutation in the ATP7B gene and haplotypes using linked markers is available for WD patients in India. Hence, the present study was undetaken to identify, by a PCR-based molecular diagnostic test, presymptomatic siblings of WD affected individuals in families with multiple offspring. Methods: Genomic DNA was prepared from the peripheral blood of the patients, siblings and his/her first degree relatives. The repeat-markers flanking WD locus were amplified by PCR using fluorescent labeled primers. Amplified DNA fragments were analyzed by polyacrylamide gel electrophoresis in ABI 377 DNA sequencing system. Genotypes of the samples were determined using Genescan software. Haplotypes were determined based on segregation of the alleles in the families under study. Results: Among 15 WD affected families with multiple children, 4 cases were identified where younger siblings shared same genotype as the patient at all three markers analyzed. Further, eight different haplotypes were detected in the four patients. Interpretation & conclusion: The siblings of the WD patients carrying the same genotype at the markers linked to WD locus were presymptomatically diagnosed individuals. Presence of eight different haplotypes in the four patients suggested mutational heterogeneity at the WD locus. The test helps clinicians for therapeutic intervention in suspect WD cases by copper chelating agents prior to manifestation of overt clinical symptoms. Key words ATP7B - genotype - haplotype - microsatellite - Wilson disease (WD) is a genetic disorder, which manifests as hepatolenticular degeneration as a result of accumulation of copper in the brain, liver, kidney and cornea due to its deranged biliary excretion1. In 1912, a WD was described as a familial syndrome of progressive lenticular degeneration associated with cirrhosis of the liver2. The etiological role of copper in the pathogenesis of WD was recognized much late

Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death

Background: Nucleoporins mediate nucleocytoplasmic exchange of macromolecules and several have been assigned active mitotic functions. Nucleoporins can participate in various mitotic functions like spindle assembly, kinetochore organisation and chromosome segregation- important for genome integrity. Pathways to genome integrity are frequently deregulated in cancer and many are regulated in part by microRNAs. Indeed, altered levels of numerous microRNAs have frequently been associated with tumorigenesis. Here, we unveil a microRNA-mediated regulation of the nucleoporin Nup214 and its downstream effect on genome integrity. Methods: Databases/bioinformatic tools such as miRBase, Oncomine and RNAhybrid predicted Nup214 as a miR-133b target. To validate this, we used luciferase reporter assays, Real-Time PCR and immuno-blotting. Flow cytometry and immuno-blots of mitotic markers were used to analyse cell cycle pattern upon thymidine synchronization and miR-133b treatment. Mitotic indices and chromosomal abnormalities were assessed by immuno-fluorescence for FITC-tagged phospho-H3 as well as video-microscopy for GFP-tagged histone H4. Annexin V/propidium iodide staining, caspase3/ PARP cleavage and colony formation assays were done to investigate cell death upon either miR-133b transfection or NUP214 knockdown by siRNA. UPCI:SCC084, HCT116, HeLa-H4-pEGFP and HEK293 (human oral squamous cell carcinoma, colorectal, cervical carcinomas and embryonic kidney cell lines, respectively) were used. miR-133b and NUP214 expressions were validated in cancer cell lines and tissues by Real-Time PCR. Results: Examination of head and neck tumour tissues and cancer cell lines revealed that Nup214 and miR-133b expressions are negatively correlated. In vitro, Nup214 was significantly downregulated by ectopic miR-133b. This downregulation elevated mitotic indices and delayed degradation of mitotic marker proteins cyclinB1 and cyclinA and dephosphorylation of H3. Moreover, this mitotic delay enhanced chromosomal abnormalities and apoptosis. Conclusions: We have identified NUP214, a member of the massive nuclear pore complex, as a novel miR-133b target. Thus, we have shown a hitherto unknown microRNA regulation of mitosis mediated by a member of the nucleoporin family. Based on observations, we also raise some hypotheses regarding transport-dependent/independent functions of Nup214 in this study. Our results hence attempt to explain why miR-133b is generally downregulated in tumours and lay out the potential for Nup214 as a therapeutic target in the treatment of cancer

Skin mediated human papillomavirus infection in breast: A report of four cases

To address the ambiguity of different modes of human papillomavirus (HPV) transmission in breast, the immunohistochemical expression of two oncoproteins E6/E7 of HPV16 was analyzed in primary breast cancer (BC) and adjacent normal skin of 4 samples. The patients were of 35–55 years old having no previous history of cancer. The E6/E7 expressions were evident in both skin and BC. In skin, high/moderate cytoplasmic expressions of E6/E7 proteins were seen in all samples, whereas in BC, high/moderate cytoplasmic expressions of the proteins were observed in 2–3 samples. Thus, it seems that HPV infection in the breast may occur through the skin

Fundamental genomic unity of ethnic India is revealed by analysis of mitochondrial DNA

Mitochondrial DNA (mtDNA) profiles of 23 ethnic populations of India drawn from diverse cultural, linguistic and geographical backgrounds are presented. There is extensive sharing of a small number of mtDNA haplotypes, reconstructed on the basis of restriction fragment length polymorphisms, among the populations. This indicates that Indian populations were founded by a small number of females, possibly arriving on one of the early waves of out-of-Africa migration of modern humans; ethnic differentiation occurred subsequently through demographic expansions and geographic dispersal. The Asian-specific haplogroup M is in high frequency in most populations, especially tribal populations and Dravidian populations of southern India. Populations in which the frequencies of haplogroup M are relatively lower show higher frequencies of haplogroup U; such populations are primarily caste populations of northern India. This finding is indicative of a higher Caucasoid admixture in northern Indian populations. By examining the sharing of haplotypes between Indian and south-east Asian populations, we have provided evidence that south-east Asia was peopled by two waves of migration, one originating in India and the other originating in southern China. These findings have been examined and interpreted in the light of inferences derived from previous genomic and historical studies

Genetic Variants Associated with Arsenic Susceptibility: Study of Purine Nucleoside Phosphorylase, Arsenic (+3) Methyltransferase, and Glutathione S-Transferase Omega Genes

BACKGROUND: Individual variability in arsenic metabolism may underlie individual susceptibility toward arsenic-induced skin lesions and skin cancer. Metabolism of arsenic proceeds through sequential reduction and oxidative methylation being mediated by the following genes: purine nucleoside phosphorylase (PNP), arsenic (+3) methyltransferase (As3MT), glutathione S-transferase omega 1 (GSTO1), and omega 2 (GSTO2). PNP functions as arsenate reductase; As3MT methylates inorganic arsenic and its metabolites; and both GSTO1 and GSTO2 reduce the metabolites. Alteration in functions of these gene products may lead to arsenic-specific disease manifestations. OBJECTIVES: To find any probable association between arsenicism and the exonic single nucleotide polymorphisms (SNPs) of the above-mentioned arsenic-metabolizing genes, we screened all the exons in those genes in an arsenic-exposed population. METHODS: Using polymerase chain reaction restriction fragment length polymorphism analysis, we screened the exons in 25 cases (individuals with arsenic-induced skin lesions) and 25 controls (individuals without arsenic-induced skin lesions), both groups drinking similar arsenic-contaminated water. The exonic SNPs identified were further genotyped in a total of 428 genetically unrelated individuals (229 cases and 199 controls) for association study. RESULTS: Among four candidate genes, PNP, As3MT, GSTO1, and GSTO2, we found that distribution of three exonic polymorphisms, His20His, Gly51Ser, and Pro57Pro of PNP, was associated with arsenicism. Genotypes having the minor alleles were significantly overrepresented in the case group: odds ratio (OR) = 1.69 [95% confidence interval (CI), 1.08–2.66] for His20His; OR = 1.66 [95% CI, 1.04–2.64] for Gly51Ser; and OR = 1.67 [95% CI, 1.05–2.66] for Pro57Pro. CONCLUSIONS: The results indicate that the three PNP variants render individuals susceptible toward developing arsenic-induced skin lesions. KEY WORDS: arsenic, As3MT, GSTO1, GSTO2, PNP, skin lesion, susceptibility. Environ Health Perspect 116:501–505 (2008). doi:10.1289/ehp.10581 available via http://dx.doi.org/ [Online 14 January 2008

DNA damage induced p53 downregulates Cdc20 by direct binding to its promoter causing chromatin remodeling

CDC20 is a critical molecule in the Spindle Assembly Checkpoint (SAC). It activates the Anaphase promoting complex and helps a dividing cell to proceed towards Anaphase. CDC20 is overexpressed in many tumor cells which cause chromosomal instability. There have been limited reports on the mechanism of SAC's response to genotoxic stress. We show that ectopically expressed p53 or DNA damage induced endogenous p53 can downregulate Cdc20 transcriptionally. We have identified a consensus p53-binding site on the Cdc20 promoter and have shown that it is being used by p53 to bind the promoter and bring about chromatin remodeling thereby repressing Cdc20. Additionally, p53 also downregulates Cdc20 promoter through CDE/CHR element, but in a p21 independent manner. This CDE/CHR element-mediated downregulation occurs only under p53 overexpressed condition but not in the context of DNA damage. The present results suggest that the two CCAAT elements in the Cdc20 promoter are not used by p53 to downregulate its activity, as reported earlier

LIMD1 is more frequently altered than RB1 in head and neck squamous cell carcinoma: clinical and prognostic implications

Introduction: To understand the role of two interacting proteins LIMD1 and pRB in development of head and neck squamous cell carcinoma (HNSCC), alterations of these genes were analyzed in 25 dysplastic head and neck lesions, 58 primary HNSCC samples and two HNSCC cell lines. Methods: Deletions of LIMD1 and RB1 were analyzed along with mutation and promoter methylation analysis of LIMD1. The genotyping of LIMD1 linked microsatellite marker, hmlimD1, was done to find out any risk allele. The mRNA expression of LIMD1 and RB1 were analyzed by Q-PCR. Immunohistochemical analysis of RB1 was performed. Alterations of these genes were correlated with different clinicopathological parameters. Results: High frequency [94% (78/83)] of LIMD1 alterations was observed in the samples studied. Compare to frequent deletion and methylation, mutation of LIMD1 was increased during tumor progression (P = 0.007). Six novel mutations in exon1 and one novel intron4/exon5 splice-junction mutation were detected in LIMD1 along with a susceptible hmlimD1 (CA)20 allele. Some of these mutations [42% (14/33)] produced non-functional proteins. RB1 deletion was infrequent (27%). Highly reduced mRNA expression of LIMD1 (25.1 ± 19.04) was seen than RB1 (3.8 ± 8.09), concordant to their molecular alterations. The pRB expression supported this data. Tumors with LIMD1 alterations in tobacco addicted patients without HPV infection showed poor prognosis. Co-alterations of these genes led the worse patients’ outcome. Conclusions: Our study suggests LIMD1 inactivation as primary event than inactivation of RB1 in HNSCC development

IL1B Induced Smad 7 Negatively Regulates Gastrin Expression

BACKGROUND: Helicobacter pylori elicited IL1B is one of the various modulators responsible for perturbation of acid secretion in gut. We have earlier reported that IL1B activated NFkB downregulates gastrin, a major modulator of acid secretion. However, we hypothesized that regulation of gastrin by IL1B would depend on the cell's ability to integrate inputs from multiple signaling pathways to generate appropriate biological response. PRINCIPAL FINDING: In this study, we report that IL1B induces Smad 7 expression by about 4.5 fold in gastric carcinoma cell line, AGS. Smad 7 resulted in transcriptional repression of gastrin promoter by about 6.5 fold when co-transfected with Smad 7 expression vector and gastrin-promoter luciferase in AGS cells. IL1B inhibited phosphorylation of Smad 3 and subsequently interfered with nuclear translocation of the positive Smad complex, thus occluding it off the gastrin promoter. IL1B promoter polymorphisms (-511T/-31C IL1B) are known to be associated with H. pylori associated gastro-duodenal ulcer. We observed that IL1B expressed from -31T promoter driven IL1B cDNA elicited 3.5 fold more Smad 7 than that expressed from the IL1B-31C variant in AGS cells. This differential activation of Smad 7 by IL1B promoter variants translated into differential downregulation of gastrin expression. We further analyzed Smad 7, NFkB, IL1B and gastrin expression in antral gut biopsy samples of patients with H. pylori associated duodenal ulcer and normal individuals. We observed that individuals with duodenal ulcer had significantly lower levels of IL1B, Smad 7, NFkB and corresponding higher level of gastrin expression. CONCLUSION: Pro-inflammatory cytokine IL1B repress gastrin expression by activating Smad 7 and subsequent inhibition of nuclear localization of Smad 3/4 complex. Polymorphic promoter variants of IL1B gene can modulate the IL1B expression which resulted in differential activation Smad 7 and consequent repression of gastrin expression, respectively. Analysis of H. pylori infected duodenal ulcer patient's gut biopsy samples also supported this observation

NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

Helicobacter pylori is a major pathogen associated with the development of gastroduodenal diseases. It has been reported that H. pylori induced pro-inflammatory cytokine IL1B is one of the various modulators of acid secretion in the gut. Earlier we reported that IL1B-activated NFkB down-regulates gastrin, the major hormonal regulator of acid secretion. In this study, the probable pathway by which IL1B induces NFkB and affects gastrin expression has been elucidated. IL1B-treated AGS cells showed nine-fold activation of MyD88 followed by phosphorylation of TAK1 within 15 min of IL1B treatment. Furthermore, it was observed that activated TAK1 significantly up-regulates the NFkB subunits p50 and p65. Ectopic expression of NFkB p65 in AGS cells resulted in about nine-fold transcriptional repression of gastrin both in the presence and absence of IL1B. The S536A mutant of NFkB p65 is significantly less effective in repressing gastrin. These observations show that a functional NFkB p65 is important for IL1B-mediated repression of gastrin. ChIP assays revealed the presence of HDAC1 and NFkB p65 along with NCoR on the gastrin promoter. Thus, the study provides mechanistic insight into the IL1B-mediated gastrin repression via NFk