The present study aimed to determine frequencies of mutations in the phenylalanine hydroxylase gene (PAH) in unrelated children (n = 71) diagnosed with phenylketonuria, who presented to Morozovskaya Children’s City Clinical hospital (Moscow) over the period from 2015 to 2016. The patients were tested for the most common PAH mutations using the original real- time PCR-based technique for the identification of nucleotide variants; additionally, next generation sequencing (NGS) was performed on the unidentified genotypes. The original PCR-based technique allowed us to effectively identify 83 % of the pathogenic allelic variants in the sample. Using the combination approach (real-time PCR + NGS), we found mutations in both alleles of PAH in 66 of total 71 patients. Altogether, 26 pathogenic PAH mutations were identified, the most common being p.R408W (47.9 %) and p.R261Q (9.9 %). Frequencies of mutations common for the Russian population, such as IVS10nt546, IVS12+1G>A, p.R158Q, p.Y414C, and IVS4+5G>T, ranged from 4.2 to 2.8 %. Half of the identified variants accounted for the total frequency of < 10 %. Sequencing of PAH revealed a few functional mutations previously unreported for Moscow region residents, including p.D222Terfs, p.R111Ter, p.F161S, p.G188D, p.R270K, p.L311P, p.F55L, p.F55Leufs, IVS1+5G>T, and IVS8-7A>G. It could be reasonable to include mutations p.D222Terfs and p.R111Ter (carrier frequency of 2.1 %) in PCR testing panels. The data obtained in our study can also be used in the development of genetic tests for phenylketonuria.
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Colorectal cancer (CRC) is one of the most common cancer types in the world. Timely diagnosis of CRC and adenomatous polyps aided by effective screening techniques can considerably reduce mortality from this disease. MicroRNAs constitute a new class of promising biomarkers for a range of human diseases including cancer. The following article assesses the diagnostic significance of miR-146a concentrations in the blood plasma of patients with colorectal cancer. The main group included patients with stages I to III colorectal cancer (n = 102); the control group comprised patients with chronic colitis, nonspecific ulcerative colitis and Crohn’s disease (n = 58) and healthy individuals (n = 42). MicroRNA levels were quantified by reverse- transcription real-time PCR, revealing significantly higher miR-146a concentrations in the samples of patients with CRC than in the controls (p < 0.0001). The optimal diagnostic sensitivity determined by ROC analysis was 47.3 %, specificity was 91.5 %, with AUC = 0.79 ± 0.018. Our findings demonstrate that the studied approach does not have sufficient specificity, but still suggest that miR-146a can be included into screening tests based on quantification of other microRNAs with improved specificity.
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Screening for cell-free DNA usually referred to as liquid biopsy holds great promise in cancer diagnosis and treatment. This article presents the results of the analysis of somatic tumor-specific mutations in circulating free DNA (cfDNA) isolated from the blood plasma of patients with stages I–IV colorectal cancer, based on the use of wild-type blocking allele-specific real-time polymerase chain reaction. This technique was specially designed for the analysis of biological specimens containing small amounts of mutant circulating tumor DNA. The study included 46 patients (18 female and 28 male participants) between 48 and 86 years of age (mean age was 67.1 ± 8.8 years). All patients underwent surgical treatment (radical surgery was performed on 85 % of the participants). Besides the molecular genetic analysis of cfDNA isolated from the blood plasma, standard histological staining was performed. Patients’ blood samples were collected before the surgery and on day 5 after it to test for KRAS and BRAF mutations. The applied PCR technique proved to be effective in detecting mutations in the RAS genes in stages II–IV of the disease, its sensitivity threshold being 0.1 %. Analysis of cfDNA before and after surgery may provide additional information on the surgical treatment outcome, development of new metastases, or presence of those previously overlooked. Wild-type blocking allele-specific real-time PCR is awaiting further validation in different clinical situations.
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