CRISPR/Cas9 RET Gene Knockout in Medullary Thyroid Carcinoma Cell-lines: Optimization and Validation.

Background: Medullary Thyroid Cancer (MTC) is a very aggressive type of thyroid carcinoma. Mutation in RET proto-oncogene is demonstrated in MTC development. We aimed to knock-out of RET-oncogene using CRISPR/Cas9 genome editing method in MTC cell-lines. Methods: This research was conducted in Shahid Beheshti University of Medical Sciences, Tehran, Iran during 2019-2020. Four different sgRNAs were designed to target exons one, two, and four of RET-oncogene in TT and MZ-CRC-1 cell-lines using bioinformatics tools, then the CRISPR/Cas9 constructs was made. About 72-hours after cell transfection, T7EI method and DNA sequencing were used to confirm the knock-out of RET-oncogene. Expression of RET, Calcitonin genes and RET protein were evaluated by Real-time PCR and ELISA, respectively. Results: The results of T7E1, and DNA sequencing of transfected cells confirmed RET gene knock-out by CRISPR/Cas9. There was a significant decrease in RET gene expression and RET protein in transfected TT and MZ cells compared to controls. The rate of cell apoptosis in transfected cells was significantly increased. Calcitonin gene expression was also significantly reduced in transfected cells. p-RET, p-PI3K, p-AKT, p-MEK, p-ERK protein levels were significantly reduced in TT and MZ transfected cells. Conclusion: For the first time, knock-out of RET gene was performed and confirmed using CRISPR/Cas9. Inhibition of this gene leads to inhibition of the tyrosine kinase RET signal transduction pathway. Therefore, it can be one of the most effective and specific therapeutic goals in the field of Personalized Medicine in the treatment of diseases caused by over activity of RET molecular pathway.

CpG island status as an epigenetic alteration for NIS promoter in thyroid neoplasms; a cross-sectional study with a systematic review.

BACKGROUND: Gene silence via methylation of the CpG islands is cancer's most common epigenetic modification. Given the highly significant role of NIS in thyroid cancer (TC) differentiation, this cross-sectional study aimed to investigate the DNA methylation pattern in seven CpG islands (CpG1-7 including +846, +918, +929, +947, +953, +955, and +963, respectively) of the NIS promoter in patients diagnosed with papillary (PTC), follicular (FTC), and multinodular goiter (MNG). Additionally, a systematic review of the literature was conducted to compare our results with studies concerning methylation of the NIS gene promoter. METHODS: Thyroid specimens from 64 patients met the eligibility criteria, consisting of 28 PTC, 9 FTC, and 27 benign MNG cases. The mRNA of NIS was tested by qRT-PCR. The bisulfite sequencing PCR (BSP) technique was performed to evaluate the promoter methylation pattern of the NIS gene. Sequencing results were received in chromatograph, FASTA, SEQ, and pdf formats and were analyzed using Chromas. The methylation percentage at each position and for each sample was calculated by mC/(mC+C) formula for all examined CpGs; following that, the methylation percentage was also calculated at each CpG site. Besides, a literature search was conducted without restricting publication dates. Nine studies met the eligibility criteria after removing duplicates, unrelated articles, and reviews. RESULTS: NIS mRNA levels decreased in tumoral tissues of PTC (P = 0.04) and FTC (P = 0.03) patients compared to their matched non-tumoral ones. The methylation of NIS promoter was not common in PTC samples, but it was frequent in FTC (P < 0.05). Significant differences were observed in the methylation levels in the 4th(+ 947), 6th(+ 955), and 7th(+ 963) CpGs sites in the forward strand of NIS promoter between FTC and MNG tissues (76.34 ± 3.12 vs 40.43 ± 8.42, P = 0.004, 69.63 ± 3.03 vs 23.29 ± 6.84, P = 0.001 and 50.33 ± 5.65 vs 24 ± 6.89, P = 0.030, respectively). There was no significant correlation between the expression and methylation status of NIS in PTC and FTC tissues. CONCLUSION: Perturbation in NIS promoter's methylation individually may have a potential utility in differentiating MNG and FTC tissues. The absence of a distinct methylation pattern implies the importance of other epigenetic processes, which may alter the production of NIS mRNA. In addition, according to the reversibility of DNA methylation, it is anticipated that the design of particular targeted demethylation medicines will lead to a novel cancer therapeutic strategy.

The Epigenetic Modification of SLC5A8 in Papillary Thyroid Carcinoma and its Effects on Clinic-Pathological Features.

Background: Epigenetic alterations such as DNA methylation are known as the main cause of different types of cancers through inactivation of tumor suppressor genes, especially thyroid cancer. Identification of novel and effective markers are important in diagnosis and prevention of thyroid cancer. In the present study, the expression and methylation of Solute carrier family 5 member 8 (SLC5A8) in Papillary Thyroid Carcinoma (PTC) in comparison to multinodular goiter (MNG) have been studied. Methods: Overall, 41 patients with PTC and 36 patients affected by MNG were recruited from four hospitals in Tehran and Qazvin, Iran in 2018. Thyroid tissues were obtained during thyroidectomy. RNA and DNA were extracted from thyroid tissues. Quantitative RT-PCR assay was performed for determining the mRNA level of SLC5A8 while Methylation-Sensitive High-Resolution Methylation was applied for assessing the methylation status. Results: Methylation status of three regions composed of 52 CpG islands in the promoter of SLC5A8 gene was studied by HRM assay. SLC5A8 level in PTC tissues was significantly downregulated in average 0.4 fold in comparison with MNG tissues (P=0.05). The aberrant methylation of SLC5A8 (b) region was remarkably different in PTC and MNG cases. The promoter methylation of SLC5A8 (c) was significantly related to BRAF mutations and vascular invasion in PTC patients. Conclusion: The aberrant promoter hyper methylation of SLC5A8 was related to aggressive PTC. Therefore, there is some evidence to support the hypothesis that SLC5A8 could be a paly important role in the development of PTC.

Sexual rehabilitation recommendations for prostate cancer survivors and their partners from a biopsychosocial Prostate Cancer Supportive Care Program.

PURPOSE: This study aimed to highlight the biopsychosocial recommendations provided to prostate cancer survivors and their partners during sexual rehabilitation. METHODS: Retrospective analysis of a prospectively maintained patient database was conducted for visits between 2013 and 2019. The sexual health rehabilitation action plan (SHRAP) is a standardized 29-item list of biopsychosocial recommendations. The frequency of biopsychosocial recommendations provided to patients via their SHRAPs was assessed. RESULTS: Among 913 patients, across 2671 appointments, nearly 74% of patients underwent radical prostatectomy. Other treatments included combination therapy (surgery, radiation, and/or androgen deprivation therapy (ADT)) (13%), radiation (external beam radiation or brachytherapy) (5%), and active surveillance (2%). Each patient had a median of 2 (SD 2.06) appointments and received a mean of 10.0 (SD 3.9) recommendations at each visit. Educational recommendations (penile rehabilitation, orgasmic guidelines, and climacturia management) were provided in 84% of visits followed by psychosexual recommendations (pleasure-focused, dedicated time, simmering, sexual aids, and sensate focus) in 71% of all appointments. The top recommendations (total n, frequency of recommendation) were penile rehabilitation (2253, 84%), pleasure-focus (1887, 71%), phosphodiesterase inhibitors (1655, 62%), clinical counselor (1603, 60%), vacuum erectile device (1418, 53%) and intracavernosal injections (1383, 52%). CONCLUSIONS: Biopsychosocial programs are evolving to be a key part of prostate cancer survivorship. This study's insight suggests that prostate cancer survivors require education around their sexual consequences and psychosexual counseling alongside proven biomedical strategies for erectile dysfunction. IMPLICATIONS FOR CANCER SURVIVORS: Cancer survivorship programs should integrate educational and psychosocial strategies alongside biological strategies for prostate cancer survivors and their partners.

New evidence on tumor suppressor activity of PTEN and KLLN in papillary thyroid carcinoma.

This study aimed to address the hypothesis that the expression of PTEN and KLLN tumor suppressor genes could diminish in papillary thyroid cancer (PTC) compared to paired normal tissue (PNT) and multinodular goiter (MNG). PTEN and KLLN expressions were assessed at both mRNA and protein levels in 82 tissue samples, including 30 PTC, 30 PNT, and 26 MNG using SYBR-Green Real-Time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Bioinformatics studies were performed to evaluate the genomic location and the genes promoter region. The mRNA expression of PTEN and KLLN in PTC was significantly lower than PNT (PTEN, P = 0.0033; KLLN, P = 0.0005). A significant decrease in the mRNA level of KLLN was also observed in PTC than MNG (P = 0.0304). Decreased level of PTEN mRNA (odds ratio=0.391; P = 0.013) or KLLN mRNA (odds ratio=0.023; P = 0.025) was associated with an increased risk of PTC tumorigenesis. Areas under the ROC curve for PTEN and KLLN were 0.69 and 0.78, respectively. PTEN and KLLN protein expressions in PTC compared to PNT or MNG were not significantly different. The bioinformatics studies revealed the sequence near the promoter region is lowly conserved across species. Four GC boxes were found upstream of the PTEN transcription start site (TSS), and one TATA box and one GC box were found upstream of KLLN TSS. The results suggest PTEN and KLLN are the two tumor suppressor genes that decreasing or loss of both of them occurs in sporadic PTC tumorigenesis. It appears they could have a promising application in both diagnostic and therapeutic areas.

Expression of miR-127, miR-154, and miR-183 in Medullary Thyroid Carcinoma Tumors.

BACKGROUND: Medullary thyroid cancer (MTC) accounts for 5%-10% of all thyroid cancers, but causes 13% of all thyroid cancer related deaths. MicroRNAs (miRs) have key functions in the development and progression of MTC. Altered expression of some miRs has been reported in many human cancers, including Thyroid cancer. Therefore, we aimed to analyze the expression of miR-154, miR-183 and miR-127 in MTC tumor tissues. METHODS: In this case-control study, 15 MTC Formalin-fixed, paraffin-embedded (FFPE) tissue samples and 15 adjacent normal thyroid FFPE tissues, as a control group, were collected from Taleghani, and Loghman Hakim Hospitals, Tehran, Iran since 2005 till 2015. After RNA extraction and cDNA synthesis, the expression of miR-127, miR-154 and miR-183 was measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). RESULTS: Our data showed a significant increase in the expression of miR-127 in MTC samples in comparison with the control group (P<0.05). Although miR-154 and miR-183 expression levels had increase expression in MTC tumors, this change was not statistically significant. CONCLUSION: The miR-127 could be considered as a prognostic, diagnostic and therapeutic marker for the management of MTC, and it is proposed for further investigation to fully establish the role of this miRNA in MTC.

Promoter Methylation of Tumor Suppressors in Thyroid Carcinoma: A Systematic Review.

Background: The tumor suppressor genes play a critical role in cellular and molecular mechanisms such as cell cycle processes, cell differentiation and apoptosis. Aberrant DNA methylation of tumor suppressor genes and subsequent gene expression changes have shown to be involved in the initiation and progression of various malignancies including thyroid malignancies. In this review, we investigated what is known about the impact of promoter hypermethylation on the key tumor suppressor genes known to be involved in cell growth and/or apoptosis of thyroid cancer. Methods: The most important databases were searched for research articles until June 2020 to identify reported tumor suppressor genes that are modulated by methylation modulation changes in thyroid carcinoma. Following the inclusion and exclusion criteria, 26 studies were reviewed using the full text to meet the inclusion and exclusion criteria. Results: The tumor suppressor genes reviewed here are suggestive biomarkers and potential targetable drugs. Inactivation of RASSF1A, DAPK1, SLCFA8, and TSHR through aberrant epigenetic methylation could activate BRAF/MEK/ERK kinase pathways with potential clinical implications in thyroid cancer patients. RARß2 and RUNX3 could suppress cell cycle and induce apoptosis in malignant cells. TIMP3 and PTEN could prevent angiogenesis and invasion through PIP3 pathway and arrest VEFG activity. Conclusion: The methylation status of key genes in various types of thyroid malignancies could be used in early diagnosis as well as differentiation of malignant and benign thyroid. This is valuable in drug repurposing and discovering alternative treatments or preventions in thyroid cancer.

Clinic Utilization and Characteristics of Patients Accessing a Prostate Cancer Supportive Care Program's Sexual Rehabilitation Clinic.

Prostate cancer (PC) treatment leads to impairment of sexual function. The Prostate Cancer Supportive Care (PCSC) Program's Sexual Rehabilitation clinic (SRC) assists patients and their partners with sexual recovery using a biopsychosocial approach to rehabilitation. This study characterizes patients seen in the SRC between July 2013-1 July 2019. Data was retrospectively abstracted from clinic records. In total, 965 patients were seen over 3391 appointments during the study period. Median age (standard deviation (SD)) was 66 years (SD = 7.1), 82.0% were partnered, yet 81.7% attended appointments alone. 88.0% were treated with surgery, 5.1% with brachytherapy, 3.7% with external beam radiation (EBRT), 1.8% with combined brachytherapy and EBRT, and 1.4% with androgen deprivation therapy. In total, 708 patients (73.4%) attended ≥1 follow-up appointment. Median time (SD) between end of prostate cancer treatment to first SRC appointment was 270 days (range 0-7766). The mean (SD) self-reported overall sexual satisfaction (extracted from International Index of Erectile Function-5 (IIEF-5)) significantly increased both with erectile aids (1.69 (SD = 1.52) to 2.26 (SD = 1.66), p < 0.001, n = 148) and without erectile aids (1.71 (SD = 1.44) to 2.35 (SD = 1.57), p < 0.001, n = 235). This study provides guidance for further investigation to refine treatment, wait-times, support, and/or resource offerings in this type of program.

Overexpression of mir-129-1, miR-146b, mir-183, and mir-197 in follicular thyroid carcinoma and adenoma tissues.

Follicular thyroid carcinoma (FTC) is responsible for approximately 10% of thyroid malignancies. Since this type of malignancy indicates no capsular and vascular invasions, adenoma and follicular carcinoma of thyroid are not distinguishable. It has been proved that microRNAs, which regulate approximately 30% of coding proteins, have an association with follicular thyroid adenoma (FTA) and carcinoma of the thyroid. Therefore, the aim of this study was to assess the expression of some miRNAs for detecting the most appropriate miRNA as potential biomarker in the diagnosis of FTA and FTC patients. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was applied to determine the expression levels of miR-129-1, miR-146b,-183 and miR-197 in 48 cases (16 FTC, 16 FTA and 16 hyperplasia/multinodular goiter (MNG) cases). The significance of miRNA differential expression levels among groups were assessed using Multivariate test by Statistical Package for Science Software (SPSS v.20) and Graph Pad Prism v.8. Results indicated that all of the miRNAs had significant overexpression in FTC and FTA versus MNG cases, and also increased expression level in FTC in comparison with FTA, however it was not significant. The results of ROC curve analysis determined the significant overexpression and prognostic value of miR-129-1 in FTA cases and miR-146b in both FTA and FTC cases compared to MNG group. Although all of the earlier mentioned microRNAs were overexpressed in FTC and FTA cases, the ROC curve results demonstrated that miR-129-1 had agreeable AUC for FTA cases. Therefore, it seems that it's cut-off point could be helpful in distinguishing between FTA and multinodular goiter cases. On the other hand, although miR-146b has excellent diagnostic value in both FTA and FTC groups, it seems that this microRNA is unable to act as a specific biomarker to discriminate between FTA and FTC cases. This data need to be confirmed in a large cohort study and other biological samples such as plasma.

NOL4 is Downregulated and Hyper-Methylated in Papillary Thyroid Carcinoma Suggesting Its Role as a Tumor Suppressor Gene.

BACKGROUND: Thyroid cancer is the fourth most common cancer in the world. Papillary thyroid carcinoma (PTC) accounts for 80% of all types of thyroid neoplasm. Epigenetic alterations such as DNA methylation are known as the main cause of different types of cancers through inactivation of tumor suppressor genes. OBJECTIVES: In the present study, the expression and methylation of suggested gene namely nucleolar protein 4 (NOL4) in PTC in comparison to multi nodular goiter (MNG) have been studied. METHODS: Forty-one patients with PTC and 38 patients affected by MNG were recruited. Thyroid tissues were obtained during thyroidectomy. RNA and DNA were extracted from thyroid tissues. Quantitative RT-PCR assay was performed for determining the mRNA level of NOL4 while methylation-sensitive high resolution methylation was applied for assessing the methylation status with designing six pairs primers for six regions on gene promoter which were named from NOL4 (a) to NOL4 (f). RESULTS: Methylation assessment of 81 CpG islands in the promoter region of NOL4 gene revealed that NOL4 (f), the nearest region to the start codon, was significantly hypermethylated in PTC cases compared to MNG cases. NOL4 level in PTC cases in comparison with MNG cases were downregulated. The methylation status and mRNA level of NOL4 (f) were associated with age of diagnosis (Age of the patient at the time of diagnosis), lymph node metastasis, and advanced stages of disease. CONCLUSIONS: These data suggested an aberrant promoter hyper-methylation of NOL4 in PTC cases may be linked with its downregulation. Therefore, NOL4 gene can be proposed as a potential tumor suppressor gene in PTC tissues.

An investigation on the expression of miRNAs including miR-144 and miR-34a in plasma samples of RET-positive and RET-negative medullar thyroid carcinoma patients.

Medullary thyroid carcinoma (MTC) is a scarce cancerous disease, originating from parafollicular C cells of the thyroid gland. MTC can be manifested as an aggressive carcinoma with metastasis, especially in sporadic forms. Mutations of the rearranged during transfection (RET) proto-oncogene occurs in all hereditary and a few somatic MTCs, so detection of RET mutations is needed for prompt and appropriate treatment. MicroRNAs (miRNAs) are noncoding regulatory RNAs. Extensive studies have done in progress or suppression of several types of cancers such as MTCs with the remarkable application as prognostic markers. Of the effective miRNAs in cancers, miR-144 and miR-34 were evaluated in our study. Blood samples of 25 RET-positive and 25 RET-negative blood samples of patients with MTC were evaluated for these miRNAs, using quantitative real-time polymerase chain reaction (RT-qPCR). Analysis of the results was performed by the 2 -ΔΔCt method, showing that miR-144 and miR-34a expression had a relative increase in patients with MTC compared with normal control samples and also in RET positives versus RET negatives. We recruited 50 out of 350 MTC plasma samples (27 female and 23 male) which were selected based on RET mutation in exon 11 (25 RET-positive and 25 RET-negative), with a mean ± SD age of 37.04 ± 1.74 years. Receiver operating characteristic (ROC) curve analysis was done to investigate the prognostic value of these miRNAs; although, they showed no significant prognostic value as MTC biomarkers in plasma samples. In conclusion, miRNAs can be used as biomarkers of cancers such as MTC; however, more studies are needed to find the best candidate miRNAs for the diagnosis of cancers.

The Impact of Physical Activity on Non-communicable Diseases: Findings from 20 Years of the Tehran Lipid and Glucose Study.

CONTEXT: Low physical activity is one of the major risk factors for non-communicable diseases (NCD) such as cardiovascular disease and type 2 diabetes. The current paper reviews the main findings from Tehran lipid and glucose study (TLGS) that focus on physical activity and its association with cardiometabolic risk factors over the past two decades. EVIDENCE ACQUISITION: We conducted a literature search for articles from 1999 to December 2017 using the search terms: (Physical activity, leisure time physical activity, non-communicable disease, and TLGS). RESULTS: The prevalence of low physical activity was 69.8% during phase ΙΙ of TLGS (2000 - 2004). During 6.5 years of follow up, the prevalence of low physical activity in the total population decreased significantly between phases II (2000 - 2004) and IV of TLGS (2008 - 2010) (P < 0.05). Overweight individuals with sedentary lifestyles had a higher risk of metabolic syndrome, compared to those who had high levels of leisure-time physical activity in phase IV of TLGS (2008 - 2010); in the obese group, systolic blood pressure, and triglyceride levels differed significantly between different leisure-time physical activity categories (106.9 ± 14.3 vs. 119.1 ± 17.2 mmHg, P = 0.03) and (111.4 ± 1.6 vs. 147.1 ± 1.6 mg/dL, P = 0.01), respectively. CONCLUSIONS: The present review highlights the impact of low physical activity on the health of the TLGS community from adolescence to adulthood. The decreased prevalence of low physical activity from phase ΙΙ to phase ΙV of TLGS indicates the necessity for lifestyle interventions as a potentially effective strategy, which could have a positive impact on various risk factors and indicators of non-communicable diseases such as body mass index, waist circumference, systolic blood pressure, and lipid profiles.

Biochemical Assessment: Findings from 20 Years of the Tehran Lipid and Glucose Study.

CONTEXT: The Tehran Lipid and Glucose Study (TLGS) is a community-based study to reveal the frequency of non-communicable diseases (NCDs) in Tehran's population. This research consists of two main parts, a cross-sectional study on the prevalence of cardiovascular risk factors and a 20-year-ongoing prospective cohort study, which was initiated in 1999 in several phases with an approximate duration of 3.6 years, and is still ongoing. The aim of the present study is review the 20 year biochemical findings of the TLGS related to the NCDs in a large sample. METHODS: All articles on biochemical assessments derived from the TLGS from the earliest publications (2002) until 30 January 2018 were reviewed for their findings on different risk factors of NCDs. RESULTS: According to the TLGS findings high sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), homocysteine (Hcy), age, smoking, hypertension, and obesity were the most important risk factors of cardiovascular diseases (CVD). It was illustrated that in subjects with abdominal obesity, the hs-CRP and IL-6 serum levels were higher than in normal subjects. The most appropriate prognostic indexes and associations were for hs-CRP, IL-6, and Hcy with abdominal obesity, waist circumference, WHtR, and wrist circumference, respectively. Previous studies have demonstrated a direct relationship between obesity and serum levels of inflammatory factors. CONCLUSIONS: According to the results of TLGS, serum levels of biochemical risk factors such as hs-CRP, IL-6, and Hcy could be beneficial in early diagnosis and effective treatment of cardiovascular, obesity and other metabolic diseases.

Plasma Calcitonin Levels and miRNA323 Expression in Medullary Thyroid Carcinoma Patients with or without RET Mutation

Background: Medullary thyroid cancer (MTC) is an endocrine tumor featuring parafollicular or C-cell differentiation, with calcitonin as a specific biomarker in MTC diagnosis. Germline mutations in the RET proto-oncogene are considered responsible for its familial occurrence and somatic mutations can cause sporadic lesions. MicroRNAs can act as oncogenes or tumor suppressors by inhibiting the expression of target genes.. The aim of this study was to investigate relationships between plasma levels of calcitonin and miRNA323 expression in MTC patients with or without RET mutation. Methods: In this cross-sectional study, MTC lesions (based on pathological confirmation) were investigated. Genomic DNA was extracted and Exons 10 and 11 of RET were genotyped using PCR-sequencing. Division was into two groups of 43 cases each with or without mutation. Plasma levels of calcitonin were determined in both. Results: miRNA323 was measured using real-time-PCR. After performing normality tests, independent T-tests and Mann Whitney tests were used for the statistical comparison of parametric and nonparametric data, respectively. Plasma levels of calcitonin were significantly higher in MTC cases without a RET mutation compared to those with a mutation. Conclusion: There was no significant difference between the two groups regarding the expression of miRNA323 so that this parameter could not be used as a bio-index germ line mutations in MTCs. However, determination of calcitonin levels in plasma might be helpful in this regard.

Diversity of mutations in the RET proto-oncogene and its oncogenic mechanism in medullary thyroid cancer.

Thyroid cancer is the most common endocrine malignancy and accounts for nearly 1% of all of human cancer. Thyroid cancer has four main histological types: papillary, follicular, medullary, and anaplastic. Papillary, follicular, and anaplastic thyroid carcinomas are derived from follicular thyroid cells, whereas medullary thyroid carcinoma (MTC) originates from the neural crest parafollicular cells or C-cells of the thyroid gland. MTC represents a neuroendocrine tumor and differs considerably from differentiated thyroid carcinoma. MTC is one of the aggressive types of thyroid cancer, which represents 3-10% of all thyroid cancers. It occurs in hereditary (25%) and sporadic (75%) forms. The hereditary form of MTC has an autosomal dominant mode of inheritance. According to the present classification, hereditary MTC is classified as a multiple endocrine neoplasi type 2 A & B (MEN2A & MEN2B) and familial MTC (FMTC). The RET proto-oncogene is located on chromosome 10q11.21. It is composed of 21 exons and encodes a transmembrane receptor tyrosine kinase. RET regulates a complex network of signal transduction pathways during development, survival, proliferation, differentiation, and migration of the enteric nervous system progenitor cells. Gain of function mutations in RET have been well demonstrated in MTC development. Variants of MTC result from different RET mutations, and they have a good genotype-phenotype correlation. Various MTC related mutations have been reported in different exons of the RET gene. We proposed that RET genetic mutations may be different in distinct populations. Therefore, the aim of this study was to find a geographical pattern of RET mutations in different populations.

Expression Analysis of Aurora-C and Survivin, Two Testis-Specific Genes, in Patients with Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The high frequency of positive families shows the importance of public awareness and screening strategies in those families. Cancer/testis (CT) antigens such as Aurora-C and Survivin are a group of antigens expressed in various tumor types of human cancers. Therefore, the aim of this study was to investigate the expression of Aurora-C and Survivin genes in malignant and normal tissues and their correlation to clinicopathological characteristics. METHODS: Tumor samples were obtained from 33 patients and adjacent non-tumorous tissues from 7 patients were also used as control. Patients were diagnosed with various stages of colorectal cancer. The level of Aurora-C and Survivin genes were evaluated by using real-time quantitative Polymerase Chain Reaction. RESULTS: The expression pattern of Survivin and Aurora-C revealed significant changes in tumor tissues when compared with normal tissues (p < 0.05). Also, these expressions were associated with the grade of disease and tumor size. There was no significant relationship between the expression of Survivin and Aurora-C genes (p > 0.05). CONCLUSIONS: In conclusion, the overexpression of Aurora-C and Survivin genes may play an important role in the development of colorectal cancer and may play a potential role in cancer therapy.

RET proto oncogene mutation detection and medullary thyroid carcinoma prevention.

Thyroid cancer is the most common endocrine neoplasia. The medullary thyroid carcinoma (MTC) is one of the most aggressive forms of thyroid malignancy,accounting for up to 10% of all types of this disease. The mode of inheritance of MTC is autosomal dominantly and gain of function mutations in the RET proto-oncogene are well known to contribute to its development. MTC occurs as hereditary (25%) and sporadic (75%) forms. Hereditary MTC has syndromic (multiple endocrine neoplasia type 2A, B; MEN2A, MEN2B) and non-syndromic (Familial MTC, FMTC) types. Over the last two decades, elucidation of the genetic basis of tumorigenesis has provided useful screening tools for affected families. Advances in genetic screening of the RET have enabled early detection of hereditary MTCs and prophylactic thyroidectomy for relatives who may not show any symptom sof the disease. In this review we emphasize the main RET mutations in syndromic and non syndromic forms of MTC, and focus on the importance of RET genetic screening for early diagnosis and management of MTC patients, based on American Thyroid Association guidelines and genotype-phenotype correlation.

Skewed mutational spectrum of RET proto-oncogene Exon10 in Iranian patients with medullary thyroid carcinoma.

Thyroid cancer is the most common endocrine malignant tumor. Medullary thyroid carcinoma (MTC) is an aggressive tumor arising from calcitonin-producing parafollicular cells. MTC has autosomal dominant inheritance and accounts for 5-10 % of all thyroid cancers. It occurs in hereditary (25 %, hMTC) and sporadic (75 %, sMTC) forms. Gain-of-function mutations in the REarranged during transfection (RET) proto-oncogene have been identified in 98 % of hMTC and 50 % of sMTC. The aim of this investigation was to identify mutation(s) in the much conserved RET exon10 in Iranian MTC patients. We started screening patients with MTC for RET in 2001. This study included 347 individuals (154 with sMTC, 38 with FMTC, 8 with multiple endocrine neoplasia type 2A [MEN2A], 3 with MEN2B, and 3 with pheochromocytoma; 207 index cases and 140 relatives). Germline mutation screening of RET exon10 was performed with PCR-DNA sequencing. A total of 14 missense mutations (10 mutations in men and 4 in women) were identified in cysteine codons 611, 618, and 620 (exon10) in 11 patients and three first-degree relatives as follows: four C611Y (three with FMTC and one relative), one C618R (FMTC), one C618S (sMTC), one C620G (sMTC), four C620R (one with FMTC and three with sMTC), and three C620F (one with FMTC and two relatives). In the present study, six different mutations were identified in exon10 of RET in 14 patients with sMTC and FMTC that were restricted to codons 611, 618, and 620, but not in codon 609. This data showed a skewed pattern of RET exon10 mutation compared to other populations. No mutation was found for MEN2A, MEN2B, and pheochromocytoma in exon10 in this population. In the most common mutations in exon10, the FMTC and sMTC patients were C611Y and C620R, respectively.

Haplotype Frequency of G691S/S904S in the RET Proto-Onco-gene in Patients with Medullary Thyroid Carcinoma.

BACKGROUND: Medullary thyroid carcinoma (MTC) occurs in both sporadic (75%) and hereditary (25%) forms. The missense mutations of the REarranged during Transfection (RET) proto-oncogene in MTC development have been well demonstrated. The aim of this study was to investigate frequency of G691S/S904S haplotype in MTC patients and their relatives. METHODS: In this research 293 participants were studied, including 181 patients (102 female, 79 male) and 112 their relatives (58 female, 54 male). Genomic DNA was extracted from peripheral blood leucocytes using the standard Salting Out/Proteinase K method. Nucleotide change detection was performed using PCR and direct DNA sequencing methods. RESULTS: According to DNA sequencing results, 159 individuals (104 patients, 55 relatives) had both G691S (rs1799939) missense mutation in exon11 and S904S (rs1800863) synonymous mutation in exon 15 of RET proto-oncogene. The allele frequency of G691S/S904S haplotype was 21.15% in patients and 10.75% in their relatives. CONCLUSION: The obtained data showed the frequency of G691S/S904S RET gene haplotype among Iranian MTC patients and their relatives. The G691S and S904S nucleotide changes were in complete linkage disequilibrium, so the results were grouped together and referred to as G691S/S904S haplotype. Further analysis is need to demonstrate the association between this haplotype and MTC development.