Predicting the molecular role of MEIS1 in esophageal squamous cell carcinoma.

The three amino acid loop extension (TALE) class myeloid ecotropic viral integration site 1 (MEIS1) homeobox gene is known to play a crucial role in normal and tumor development. In contrast with its well-described cancer stemness properties in hematopoietic cancers, little is known about its role in solid tumors like esophageal squamous cell carcinoma (ESCC). Here, we analyzed MEIS1 expression and its clinical relevance in ESCC patients and also investigated its correlation with the SOX2 self-renewal master transcription factor in the ESCC samples and in the KYSE-30 ESCC cell line. MEIS1 mRNA and protein expression were significantly decreased in ESCC disease (P < 0.05). The inverse correlation between MEIS1 mRNA expression and tumor cell metastasis to the lymph nodes (P = 0.004) was significant. Also, MEIS1 protein levels inversely correlated to lymph node involvement (P = 0.048) and high tumor stage (stages III/IV, P = 0.030). The low levels of DNA methylation in the MEIS1 promoter showed that this suppression does not depend on methylation. We showed that downregulation of EZH2 restored MEIS1 expression significantly. Also, we investigated that MEIS1 downregulation is concomitant with increased SOX2 expression. To the best of our knowledge, this is the first report on the MEIS1 gene in ESCC. The inverse correlation of MEIS1 with metastasis, tumor staging, and the role of EZH2 in methylation, together with its correlation with stemness factor SOX2 expression, led us to predict cancer stemness properties for MEIS1 in ESCC.

Two novel mutations in CYP11B1 and modeling the consequent alterations of the translated protein in classic congenital adrenal hyperplasia patients.

Mutations in the 11ß-hydroxylase (CYP11B1) gene are the second leading cause of congenital adrenal hyperplasia (CAH), an autosomal recessive disorder characterized by adrenal insufficiency, virilization of female external genitalia, and hypertension with or without hypokalemic alkalosis. Molecular analysis of CYP11B1 gene in CAH patients with 11ß-hydroxylase deficiency was performed in this study. Cycle sequencing of 9 exons in CYP11B1 was performed in 5 unrelated families with 11ß-hydroxylase deficient children. Three-dimensional models for the normal and mutant proteins and their affinity to their known substrates were examined. Analysis of the CYP11B1 gene revealed two novel mutations, a small insertion in exon 7 (InsAG393) and a small deletion in exon 2 (DelG766), and three previously known missense mutations (T318M, Q356X, and R427H). According to docking results, the affinity of the protein to its substrates is highly reduced by these novel mutations. DelG766 has more negative impact on the protein in comparison to InsAG393. The novel mutations, InsAG393 and DelG766, change the folding of the protein and disrupt the enzyme's active site as it was measured in the protein modeling and substrate binding analysis. Molecular modeling and sequence conservation were predictive of clinical severity of the disease and correlated with the clinical diagnosis of the patients.

p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in esophageal squamous cell carcinoma.

BACKGROUND: Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. METHODS: Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. RESULTS: Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62%) of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P < 0.001). p16INK4a aberrant methylation was significantly associated with decreased p16 protein expression (P = 0.033), as well as the overexpression of p53 (P = 0.020). CONCLUSIONS: p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development.

Interactions between Glutathione-S-Transferase M1, T1 and P1 polymorphisms and smoking, and increased susceptibility to esophageal squamous cell carcinoma.

BACKGROUND: A complex of genetic and environmental factors is involved in carcinogenesis of the esophageal squamous cell carcinoma (ESCC). Glutathione-S-Transferases (GSTs) are phase-II enzymes playing role in detoxification of carcinogen electrophiles. Genetic polymorphisms of GSTM1, GSTT1 and GSTP1 in association with some environmental factors and their impact on esophageal cancer susceptibility were assessed in the Iranian population. METHODS: Genomic DNA of peripheral blood leukocytes from 148 confirmed esophageal cancer cases and 137 healthy individuals as control group was assayed for restriction fragment length polymorphisms in the GSTP1 loci by PCR amplification followed by digestion with Alw26I. Deletion of the GSTM1 and GSTT1 genes was detected by multiplex PCR. A data-mining method based on decision trees was applied to produce a predictive model of interactions between genotypes. RESULTS: Smoking was independently associated with ESCC (p<0.05, OR: 2.286, 95% CI=1.311-3.983). Smoking along with GSTP1 Val/Val genotype was associated to ESCC (p<0.001, OR: 3.886, 95% CI=1.830-8.251), while non-smokers with GSTP1 Val/Val were significantly more frequent in non-cancerous group. (p=0.007, OR: 0.507, 95% CI=0.309-0.830). CONCLUSIONS: Data-mining methods are useful tools to map out a scheme for predicting complex relations and combinations of different genotypes. Genotyping analysis of GSTP1 together with assessment of smoking seems to be important in determining the risk of ESCC in the Iranian population.

p16 promoter hypermethylation: a useful serum marker for early detection of gastric cancer.

AIM: To determine p16 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS: Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction (MSP) was used to evaluate methylation status of p16 promoter. p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS: Methylation was detected in 44.2% (23/52) of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients (14/52). Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases (32/52), and was significantly associated with promoter hypermethylation (P < 0.001). Methylation was significantly more frequent in higher pathological grades (P < 0.05). Methylation was not associated with other clinicopathological features and environmental factors including H pylori infection and smoking. CONCLUSION: p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.

Three novel CYP21A2 mutations and their protein modelling in patients with classical 21-hydroxylase deficiency from northeastern Iran.

OBJECTIVE: Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders frequently caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). We describe three novel CYP21A2 mutations in CAH patients. DESIGN AND METHODS: Sequence analysis of the entire CYP21A2 gene followed by molecular modelling was performed in three unrelated classical CAH patients of northeastern Iranian origin. The active (CYP21A2) and pseudogene (CYP21A1P) alleles were screened for the presence of the new variations in controls. RESULTS: Two novel missense mutations, F404S in exon 9 and T450P in exon 10, were found in homozygous forms in two female patients with a salt-wasting (SW) phenotype. These novel variants were screened by allele-specific polymerase chain reaction (PCR) and excluded in 100 unrelated normal alleles. Prediction of clinical severity, based on molecular modelling and sequence conservation, correlates well with the clinical diagnosis of the patients carrying these mutations. The third novel mutation, a small 10-bp deletion in exon 1, g.19_28del, was found in a female patient with a simple virilizing phenotype in a compound heterozygous form with the common intron 2 splice mutation (IVS2-13A/C>G). This frameshift mutation causes a premature stop codon at amino acid position 48, L48X, resulting in a nonfunctional protein. The CYP21A1P pseudogene alleles were also screened and none of these novel mutations could be detected. CONCLUSIONS: Three novel mutations were found in the CYP21A2 gene and predicted to drastically impair enzyme activity resulting in severe classic CAH. None of these mutations occurs in the CYP21A1P pseudogene.