Optimization of degenerate oligonucleotide primed PCR for amplification of microdissected chromosome segments

The degenerate oligonucleotide primer of sequence, 5\u27 CCGACTCGAGNNNNNNATGTGG 3\u27, contains a specific 3\u27 hexanucleotide , a central random hexanucleotide and an Xhol site towards it\u27s 5\u27 end. This degenerate primer (Telenius et al, 1992) has been developed for the amplification and subsequent cloning of any source of target DNA, particularly the human chromosome, in a process termed degenerate oligonucleotide primed PGR (DOP-PCR). In the present study, bacteriophage lambda DNA was employed as a model target DNA to test a variety of DOPPCR procedures. To achieve the highest efficiency, different concentrations of primer and Taq DNA polymerase and also a variety of PGR program cycles were tested. After achieving the optimal concentration of the DOP primer and Taq DNA polymerase, two different protocols were investigated. The first reUes on low-temperature pre-amplification of the target DNA by modified T7 DNA polymerase (Sequenase) and uses the amphfied segments for a subsequent amplification catalysed by Taq DNA polymerase. With this protocol, amplified DNA was obtained with down to 0.2 pg DNA of template. The other procedure employs Taq DNA polymerase for all stages of amphfication. We have developed a modified form of this strategy by boosting Taq DNA polymerase in the middle of the amphfication program. This modification increases the efficiency and sensitivity of the procedure and enables us to ampUfy as little as 0.02 pg DNA. The latter method should be of considerable value for amphfication of DNA from a wide variety of sources, particularly human chromosomes, since it should reduce to a minimum the number of dissected chromosome fragments required. In fact, following the completion of this work, the method has been successfully applied to the analysis of a human chromosome translocation in collaboratio

Evaluation of biochemical constituents and inhibitory effect of tea clone 100 on colorectal cancer cell line HCT- 116

Purpose: To evaluate the total content of polyphenols and the free radical scavenging activity of three different extracts of three types of tea clone 100 (black, green and white), and their anti-proliferative effects on colorectal cancer.Methods: Five major polyphenols, viz, (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate, were identified using thin layer chromatography (TLC). Catechins were quantified by high performance liquid chromatography (HPLC). Antioxidant activity was measured by DPPH radical scavenging method, while 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay was employed for the determination of cell viability of colon cell line HCT-116 after 24 and 48 h.Results: The aqueous methanol (70 %) extract of white tea yielded the highest amount of polyphenols (36.67 ± 0.54 mg GAE/g dry weight). The DPPH radical scavenging activity of white tea was 71.74 ± 0.42 %, and it produced high anti-proliferation activity against colorectal cancer cell line HCT-116 (86.06 ± 0.54 %).Conclusion: White tea extract possesses high DPPH radical scavenging activity, and exerts good antiproliferative effects against colorectal cancer cell line HCT-116, most likely due to its catechin content.Keywords: Camellia sinensis L., Catechin, DPPH radical scavenging, Anti-cance

Prenatal diagnosis of Sex determining region Y -box transcription factor 2 anophthalmia syndrome caused by germline mosaicism using next-generation sequencing: A case report

Background: Sex determining region Y box transcription factor 2 (SOX2) mutations lead to bilateral anophthalmia with autosomal dominant human inheritance. SOX2 mutations could result in severe ocular phenotypes usually associated with variable systemic defects. Most patients described with SOX2 anophthalmia syndrome possessed de novo mutations in this gene. Case Presentation: In this case report, we describe 2 brothers with mental retardation and bilateral anophthalmia caused due to SOX2 germline mosaicism in unaffected parents. Next-generation DNA sequencing was carried out to determine the family’s possible cause of genetic mutation. Sanger sequencing was performed on the patients and their parents. Prenatal diagnosis was done in both pregnancies of the older brother’s wife via chorionic villus sampling. A novel heterozygous pathogenic frameshift deletion variant (exon1:c.58_80del:p.G20fs) was identified in the SOX2 gene, which was confirmed by Sanger sequencing in both affected brothers and did not exist in healthy parents, indicating germline mosaicism. Conclusion: Most SOX2 mutations known look to arise de novo in probands and are diagnosed through anophthalmia or microphthalmia. Prenatal diagnosis should be offered to healthy parents with a child with SOX2 mutation every pregnancy. Key words: Anophthalmos, SOX2 anophthalmia syndrome, Mosaicism

MRP1 but Not MDR1 Is Associated with Response to Neoadjuvant Chemotherapy in Breast Cancer Patients

A major problem in the treatment of breast cancer is the development of resistance to chemotherapeutic agents. Although the role of multidrug resistance 1 (MDR1) and multidrug resistance associated protein 1 (MRP1) in inducing drug resistance in many cancers has been widely investigated the clinical significance of expression of these genes in breast cancer remains unclear and the data is still controversial. We investigated the expression of MDR1 and MRP1 in breast cancer patients as well as the possible correlation between MDR1 and MRP1 and clinical response to chemotherapy. In the present study, MDR1 and MRP1 gene expression were investigated by real time reverse transcription polymerase chain reaction (RT-PCR) assay in 54 breast cancer tumors and in corresponding adjacent normal tissues before neoadjuvant chemotherapy. The expression level of MDR1 and MRP1 were significantly higher in breast tumors than normal breast tissues. Although a significant relationship was found between the MRP1 expression and response to treatment no association was observed between MDR1 expression and response to treatment. MDR1 and MRP1 expression levels have been shown to be independent of tumor size, histological grade and the status of progesterone or estrogen receptor

Identification of two novel homozygous nonsense mutations in TRAPPC9 in two unrelated consanguineous families with intellectual Disability from Iran

Background: Pathogenic mutations in TRAPPC9 are associated with autosomal recessive Intellectual Disability (ID), a major public health issue that affects about 1–3% of children worldwide. Method: Clinical evaluation, magnetic resonance imaging, peripheral blood karyotype, Multiplex ligation-dependent probe amplification (MLPA), array CGH, and whole-exome sequencing were used to characterize etiology in three patients from two unrelated consanguineous families of Iranian descent with intellectual disability. Results: Whole-exome sequencing showed two novel homozygous nonsense mutations (c.937C>T) in exon 3 and (c.3103C>T) in exon 19 of TRAPPC9 (NM_031466.7) in two unrelated consanguineous families. Conclusion: The two novel variants found in TRAPPC9 caused truncated protein and clinical manifestations such as ID, developmental delay, microcephaly, and brain abnormalities in three patient

Aberrant DNA Methylation of Two Tumor Suppressor Genes, p14ARF and p15INK4b, after Chronic Occupational Exposure to Low Level of Benzene

Background: Exposure to benzene would be associated with many diseases including leukemia. Epigenetic alterations seem to be among the main mechanisms involved. Objective: To determine if chronic occupational exposure to low level of benzene would be associated with DNA methylation. Methods: Global DNA methylation and promoter-specific methylation of the two tumor suppressor genes, p14ARF and p15INK4b, were assessed employing methylation-specific PCR using the DNA extracted from 40 petrochemical workers exposed to ambient benzene levels of <1 ppm, and 31 office workers not exposed to benzene or its derivatives. Results: While an increase in global DNA methylation of 5% in p14ARF (p=0.501) and 28% in p15INK4b (p=0.02) genes was observed in the exposed group, no hypermethylation in either of the studied genes was observed in the unexposed group. No significant association was found between the frequency of aberrant methylation and either of age, work experience, and smoking habit in the exposed group. Conclusion: Chronic occupational exposure to lower than the permissible exposure limit of benzene may still result in DNA methylation of tumor suppressor genes that may ultimately lead to development of cancer

A Panel of Cancer Testis Antigens and Clinical Risk Factors to Predict Metastasis in Colorectal Cancer

Colorectal cancer (CRC) is the third common carcinoma with a high rate of mortality worldwide and several studies have investigated some molecular and clinicopathological markers for diagnosis and prognosis of its malignant phenotypes. The aim of this study is to evaluate expression frequency of PAGE4, SCP-1, and SPANXA/D cancer testis antigen (CTA) genes as well as some clinical risk markers to predict liver metastasis of colorectal cancer patients. The expression frequency of PAGE4, SCP-1, and SPANXA/D cancer/testis antigen (CTA) genes was obtained using reverse transcription polymerase chain reaction (RT-PCR) assay in 90 colorectal tumor samples including both negative and positive liver metastasis tumors. Statistical analysis was performed to assess the association of three studied genes and clinical risk factors with CRC liver metastasis. The frequency of PAGE4 and SCP-1 genes expression was significantly higher in the primary tumours with liver metastasis when statistically compared with primary tumors with no liver metastasis (P<0.05). Among all clinical risk factors studied, the lymph node metastasis and the depth of invasion were statistically correlated with liver metastasis of CRC patients. In addition, using multiple logistic regression, we constructed a model based on PAGE4 and lymph node metastasis to predict liver metastasis of CRC

Expression of MRP1 gene in acute leukemia

CONTEXT AND OBJECTIVE: Overexpression of the multidrug resistance-associated protein 1 (MRP1) gene has been linked with resistance to chemotherapy in vitro, but little is known about its clinical impact on acute leukemia patients. Our aim was to investigate the possible association between MRP1 gene expression level and clinical outcomes among Iranian leukemia patients. DESIGN AND SETTING: This was an analytical cross-sectional study on patients referred to the Hematology, Oncology and Stem Cell Research Center, Sharyatee Public Hospital, whose diagnosis was acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL). All molecular work was performed at NIGEB (public institution). METHODS: To correlate with prognostic markers and the clinical outcome of acute leukemia, MRP1 gene expression was assessed in 35 AML cases and 17 ALL cases, using the quantitative real-time polymerase chain reaction and comparing this to the chemotherapy response type. RESULTS: Mean expression in AML patients in complete remission (0.032 ± 0.031) was significantly lower than in relapsed cases (0.422 ± 0.297). In contrast, no significant difference in MRP1 mRNA level was observed between complete remission and relapsed ALL patients. There was a difference in MRP1 expression between patients with unfavorable and favorable cytogenetic prognosis (0.670 ± 0.074 and 0.028 ± 0.013, respectively). MRP1 expression in M5 was significantly higher (p-value = 0.001) than in other subtypes. CONCLUSIONS: The findings suggest that high MRP1 expression was associated with poor clinical outcome and was correlated with the M5 subtype and poor cytogenetic subgroups among AML patients but not among ALL patients