Novel pericentric inversion inv(9)(p23q22.3) in unrelated individuals with fertility problems in the Southeast European population.

Pericentric inversions are among the known polymorphisms detected in the general population at a frequency of 1-2%. Despite their generally benign nature, pericentric inversions affect the reproductive potential of carriers by increasing the risk for unbalanced live-born offspring, miscarriages, or other fertility problems. Here we present a novel large pericentric inversion of chromosome 9, inv(9)(p23q22.3), detected in 30 heterozygote carriers, 24 from seven apparently unrelated families and 6 isolated patients, where the probands were mainly referred for fertility and prenatal problems. The inversion carries a significant risk for recombinant abnormal chromosomes, as in two families one supernumerary rec(9)dup(9p) and one rec(9)dup(9q) were identified, leading to neonatal death and miscarriage, respectively. The inversion carriers were identified by three different laboratories in Greece, Cyprus and Germany respectively, however all carriers have Southeast European origin. The inversion appears to be more frequent in the Greek population, as the majority of the carriers were identified in Greece. We were able to determine that the inversion is identical in all individuals included in the study by applying a combination of several methodologies, such as karyotype, fluorescence in situ hybridization (FISH), chromosomal microarrays (CMA) and haplotype analysis. In addition, haplotype analysis supports that the present inversion is identical by descent (IBD) inherited from a single common ancestor. Our results are, therefore, highly indicative of a founder effect of this inversion, presumably reflecting an event that was present in a small number of individuals that migrated to the current Southeast Europe/Northern Greece from a larger population.

High miR-96 levels in colorectal adenocarcinoma predict poor prognosis, particularly in patients without distant metastasis at the time of initial diagnosis.

MicroRNA-96 (miR-96) is an oncomiR that facilitates the development of malignant tumors by promoting growth, proliferation, and survival of cancer cells. Previous studies using high-throughput techniques have shown that miR-96 is upregulated in colorectal cancer compared to adjacent normal colorectal tissue. The aim of this study was the investigation of the potential clinical value of miR-96 as a molecular prognostic biomarker in colorectal adenocarcinoma. For this purpose, total RNA was extracted from 108 primary colorectal adenocarcinoma samples and 54 paired non-cancerous colorectal tissue specimens. After polyadenylation and reverse transcription, miR-96 molecules were determined using an in-house developed real-time quantitative PCR based on SYBR Green chemistry. Calculations were carried out with the comparative CT method, using SNORD48 as endogenous reference gene. Finally, extensive biostatistical analysis was performed and showed that miR-96 is significantly upregulated in colorectal adenocarcinoma specimens compared to their non-cancerous counterparts (p < 0.001) as well as in tumors having invaded regional lymph nodes (p = 0.009) and those of advanced TNM stage (p = 0.008). miR-96 expression is an unfavorable prognostic marker in colorectal adenocarcinoma, predicting poor disease-free and overall survival (p = 0.041 and 0.028, respectively), independently of classical clinicopathological parameters. Most importantly, miR-96 expression stratifies patients without distant metastasis (M0) at the time of diagnosis into two groups with substantially different prognosis (p = 0.040). In conclusion, high tissue levels of miR-96 are associated with advanced stages of colorectal adenocarcinoma and predict an increased risk for disease recurrence and poor overall survival, especially in patients without distant metastasis at the time of diagnosis.

Enhanced miR-182 transcription is a predictor of poor overall survival in colorectal adenocarcinoma patients.

BACKGROUND: Colorectal cancer is the second most frequent cause of cancer-related death in the developed world. Recent studies have tried to associate colorectal cancer with the aberrant expression of several microRNAs. The aim of the present study was the development of a highly sensitive quantitative real-time PCR which can be used to evaluate the miR-182 expression levels in colorectal adenocarcinoma and adjacent non-cancerous tissue specimens and associate them with several clinicopathological characteristics, aiming to examine the prognostic potential of miR-182. METHODS: Total RNA was isolated from 116 malignant colorectal adenocarcinoma specimens and 60 paired non-cancerous tissues. Then, polyadenylation of 2 µg total RNA by poly(A) polymerase and reverse transcription with suitable oligo-dT-adapter followed. miR-182 levels were quantified by real-time PCR based on SYBR Green chemistry. The results were analyzed by the comparative quantification cycle method and by extensive biostatistical analysis. RESULTS: miR-182 was found to be significantly upregulated in colorectal adenocarcinoma specimens compared to their non-cancerous counterparts (p<0.001). miR-182 expression increases as the histological grade increases (p=0.013). miR-182 overexpression is associated with high depth of tumor invasion, positive regional lymph node status, and advanced TNM stage of patients. Therefore, miR-182 is an unfavorable prognostic marker in colorectal adenocarcinoma, predicting poor overall survival (p=0.007). Most importantly, miR-182 expression retained its unfavorable prognostic significance among patients with well- or moderately differentiated colorectal adenocarcinoma (p=0.006) and among metastasis-free patients (p=0.025). CONCLUSIONS: The increased levels of the oncogene-like miR-182 increase the risk for disease progression and predict poor overall survival for colorectal adenocarcinoma patients.

miR-34a overexpression predicts poor prognostic outcome in colorectal adenocarcinoma, independently of clinicopathological factors with established prognostic value.

OBJECTIVES: MicroRNA-34a (miR-34a) is regulated by TP53 and, in response, downregulates the expression of a gamut of protein-coding genes, including apoptosis regulators, transcription factors, cyclins, and cyclin-dependent kinases. Its upregulation initiates a reprogramming of gene expression and promotes apoptosis. The purpose of this study was the investigation of the potential clinical significance of miR-34a as a molecular prognostic biomarker in colorectal adenocarcinoma using an in-house real-time quantitative PCR (qPCR) methodology. DESIGN AND METHODS: Total RNA was extracted from 113 primary colorectal adenocarcinoma specimens and 61 paired non-cancerous colorectal tissue samples. After polyadenylation and reverse transcription, miR-34a molecules were determined using qPCR based on SYBR Green chemistry. Calculations were performed using the comparative CT method. Finally, extensive biostatistical analysis was performed. RESULTS: miR-34a expression does not significantly differ between colorectal adenocarcinoma tissue specimens and adjacent non-cancerous mucosae. However, miR-34a expression increases progressively as colorectal adenocarcinoma loses its differentiation, being highest in grade III tumors (P=0.010). Moreover, miR-34a expression is a potential unfavorable prognostic biomarker in colorectal adenocarcinoma, predicting poor disease-free and overall survival (P=0.002 and P=0.019, respectively), independently of classical clinicopathological parameters. Most importantly, miR-34a expression stratifies patients without local (N0) and/or distant metastasis (M0) at the time of diagnosis into two groups with substantially different prognosis (P=0.013 and P=0.002, respectively). CONCLUSIONS: High miR-34a levels in colorectal adenocarcinoma predict a rather increased risk for disease recurrence and poor overall survival, particularly in patients at an early TNM stage. The unfavorable prognostic potential of miR-34a expression is independent of established prognostic features of colorectal adenocarcinoma.

miR-224 overexpression is a strong and independent prognosticator of short-term relapse and poor overall survival in colorectal adenocarcinoma.

Colorectal adenocarcinoma constitutes the most frequent form of colorectal cancer and a serious cause of cancer-related deaths. The expression of multiple miRNAs, including miR-224, is deregulated in colorectal adenocarcinoma. The aim of this study was the investigation of the prognostic value of miR-224 in colorectal adenocarcinoma. For this purpose, total RNA was isolated from 115 colorectal adenocarcinomas and 66 adjacent non-cancer mucosae. Total RNA (2 µg) was polyadenylated and reverse transcribed. A quantitative PCR method based on SYBR-Green chemistry was developed and applied for the quantification of miR-224 levels, followed by extensive biostatistical analysis. miR-224 levels in malignant colorectal adenocarcinomas ranged between 1.81 and 187.75 RQU (miR-224 copies/1,000 SNORD48 copies) with a median of 34.27, and were significantly elevated, compared to miR-224 levels in adjacent non-cancer mucosae (p<0.001). Enhanced miR-224 expression constitutes a rather strong prognosticator in colorectal adenocarcinoma, predicting short-term relapse and poor overall survival in these patients (p=0.012 and p=0.005, respectively), independent of established clinicopathological parameters. In conclusion, miR-224 is significantly upregulated in malignant colorectal tumors compared to adjacent non-cancer mucosae, and its enhanced expression constitutes an independent predictor of short-term relapse and poor overall survival in colorectal adenocarcinoma patients.

The use of array-CGH in a cohort of Greek children with developmental delay.

BACKGROUND: The genetic diagnosis of mental retardation (MR) is difficult to establish and at present many cases remain undiagnosed and unexplained. Standard karyotyping has been used as one of the routine techniques for the last decades. The implementation of Array Comparative Genomic Hybridization (array-CGH) has enabled the analysis of copy number variants (CNVs) with high resolution. Major cohort studies attribute 11% of patients with unexplained mental retardation to clinically significant CNVs. Here we report the use of array-CGH for the first time in a Greek cohort. A total of 82 children of Greek origin with mean age 4.9 years were analysed in the present study. Patients with visible cytogenetic abnormalities ascertained by standard karyotyping as well as those with subtelomeric abnormalities determined by Multiplex Ligation-dependent Probe Amplification (MLPA) or subtelomeric FISH had been excluded. RESULTS: Fourteen CNVs were detected in the studied patients. In nine patients (11%) the chromosomal aberrations were inherited from one of the parents. One patients showed two duplications, a 550 kb duplication in 3p14.1 inherited from the father and a ~1.1 Mb duplication in (22)(q13.1q13.2) inherited from the mother. Although both parents were phenotypically normal, it cannot be excluded that the dual duplication is causative for the patient's clinical profile including dysmorphic features and severe developmental delay. Furthermore, three de novo clinically significant CNVs were detected (3.7%). There was a ~6 Mb triplication of 18q21.1 in a girl 5 years of age with moderate MR and mild dysmorphic features and a ~4.8 Mb duplication at (10)(q11.1q11.21) in a 2 years old boy with severe MR, multiple congenital anomalies, severe central hypotonia, and ataxia. Finally, in a 3 year-old girl with microcephaly and severe hypotonia a deletion in (2)(q31.2q31.3) of about ~3.9 Mb was discovered. All CNVs were confirmed by Fluorescence in situ hybridization (FISH). For the remaining 9 patients the detected CNVs (inherited duplications or deletions of 80 kb to 800 kb in size) were probably not associated with the clinical findings. CONCLUSIONS: Genomic microarrays have within the recent years proven to be a highly useful tool in the investigation of unexplained MR. The cohorts reported so far agree on an around 11% diagnostic yield of clinically significant CNVs in patients with unexplained MR. Various publicly available databases have been created for the interpretation of identified CNVs and parents are analyzed in case a rare CNV is identified in the child. We have conducted a study of Greek patients with unexplained MR and confirmed the high diagnostic value of the previous studies. It is important that the technique becomes available also in less developed countries when the cost of consumables will be reduced.