Germline mutations in patients with oral mucosal leukoplakia and squamous cell carcinoma: a prospective observational study

Background. The number of studies devoted to the molecular genetics of oral mucosal leukoplakia and squamous cell carcinoma is small, while the obtained results are usually preliminary in nature. We can assume the existence of region-specific pathogenic genetic variants involved in the development of oral mucosal leukoplakia and squamous cell carcinoma. With the knowledge of such variants, it would become possible to develop PCR (polymerase chain reaction) and NGS (next-generation sequencing) test systems for the detection of clinically significant germline mutations.Objectives — to identify pathogenic germline genetic variants in patients with oral mucosal leukoplakia accompanied by grade 1 epithelial dysplasia, as well as oral mucosal squamous cell carcinoma, using new-generation sequencing.Methods. Study design: prospective, observational, cross-sectional, without a control group. The sample included patients (48 persons) of either sex (18 years of age or older) with the following proven and morphologically confirmed diagnoses: oral mucosal leukoplakia accompanied by grade 1 squamous intraepithelial neoplasia of epithelium (24 people) and oral mucosal squamous cell carcinoma (24 people), who sought medical care at the Vitebsk Regional Clinical Dental Center and Vitebsk Regional Clinical Oncological Center in 2019–2020. The identified pathogenic and presumably pathogenic genetic variants involved in the development of these diseases were quantitatively assessed. The study was conducted at the Shareable Core Facilities GENOME of the Institute of Genetics and Cytology of the National Academy of Sciences of Belarus. In order to isolate deoxyribonucleic acid (DNA) from blood samples, a QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) was used. The preparation of DNA libraries and sequencing were carried out by means of an Illumina NextSeq 550 sequencing system (Illumina, Inc., USA) using an Illumina Nextera DNA Exome kit (USA). Bioinformatic analysis was conducted using Illumina BaseSpace specialized software (USA) and Galaxy Project (Galaxy Community, an international non-profit project) in accordance with current guidelines. The obtained data were statistically processed employing specialized software packages Statistica 12 (StatSoft, Inc., USA) and MedCalc 18.9.1 (MedCalc Software, Ltd, Belgium).Results. Next-generation whole-exome sequencing of deoxyribonucleic acid samples isolated from the blood of patients with oral mucosal leukoplakia and squamous cell carcinoma has been conducted in the Republic of Belarus for the first time. The total number of unique germline genetic variants in the exome of both groups of patients was shown to be very high, yet most of them were not pathogenic. In the examined patients, the majority of germline mutations were found to be localized only in 19 exome genes: MAP2K3, DNAH5, HSPG2, OBSCN, SYNE1, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-A, HLA-B, PKD1L2, TTN, AHNAK2, PDE4DIP, MUC3A, MUC4, MUC12, MUC16, and MUC17. In both clinical groups, the greatest number of genetic variants (> 40% of the total number) was detected in MUC3A, MUC4, MUC12, and MUC16, responsible for the synthesis of the glycoprotein mucin family.Conclusion. Oral mucosal leukoplakia and squamous cell carcinoma can arise from the pathogenic variants of MUC3A, MUC4, MUC12, and MUC16

Clinical Significance of Pathogenicity of Somatic Mutations in Oral Leukoplakia: a Prospective Observational Study

Background. The vast majority of malignant neoplasms of the oral mucosa refer to squamous cell carcinomas. The development of squamous cell carcinoma of the oral mucosa is often promoted by previous potentially malignant diseases, with oral leukoplakia dominating among them.Objective. To determine the clinical significance of the pathogenicity of somatic mutations in oral mucosal leukoplakia.Methods. The study material included 24 samples of abnormal epithelium of the oral mucosa from leukoplakia patients. QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) was used for deoxyribonucleic acid (DNA) extraction from the samples. DNA sequencing was performed using IlluminaNextSeq 550 sequencer and TruSight™ Oncology 500 DNA Kit For Use with NextSeq (Illumina, USA). All DNA extractions from biological samples, preparation and sequencing of DNA libraries were performed step-by-step in strict accordance with the guidelines provided with the respective reagent kits. Bioinformatics analysis was carried out using specific software Illumina Base Space (Illumina, USA) and Galaxy Project (The Galaxy Community, a non-profit international project) according to current guidelines. The desired power of the study accounted for 90%. Two Proportions Z test was performed by means of The Sample Size Calculation of Statistica 12 (StatSoft, Inc.) with the set option “one-tailed hypothesis”, because it was initially assumed that pathogenic (oncogenic) genetic variants occur in the tissue of oral leukoplakia much more frequently than in the human reference genome used for sequence alignment.Results. The pathogenic somatic mutations in the TP53, KRAS, APC, NRAs and BRAF genes, identified in this study, alone or in combination, are highly likely (hazard ratio 3000-11000) to be associated with the development of oral mucosal leukoplakia and low-grade epithelial dysplasia. The multiplicity of pathogenic and likely pathogenic genetic variants associated with epithelial dysplasia, as well as the fact that a number of variants do not occur in all patients, suggests that the same histotype of oral mucosal dysplasia may develop under the influence of different mutations.Conclusion. The pathogenic and likely pathogenic variants of the TP53, KRAS, APC, NRAS and BRAF genes, identified in this study, alone or in combination, are highly likely (hazard ratio 3000–11000) to be associated with the development of leukoplakia and low-grade epithelial dysplasia

Генетический полиморфизм белков сурфактанта SP-B и SP-C у недоношенных новорожденных с дыхательными осложнениями

The respiratory distress syndrome (RDS) and the bronchopulmonary dysplasia (BPD) are the lung diseases that occur mainly in preterm infants. Polymorphic variants of surfactant protein genes are considered as candidates contributing to the pathogenesis of RDS and BPD. The association of 5 polymorphic variants of the SFTPB gene (rs2077079, rs1130866, D2S388, D2S2232, VNTR 4 introns) and 3 polymorphic substitutions of the SFTPC gene (rs4715, rs1124, rs2070687) in newborns with the development risk and severity of RDS and BPD was studied. 555 newborns were included in the study, among which 313 premature babies with a gestational age of 28–36 weeks. Genotyping was performed by the Sanger sequencing, the microsatellite analysis, and the real-time PCR. All premature newborns were characterized by the presence of RDS of different severity and BPD was detected in 36 newborns. The microsatellite marker D2S388 of the SFTPB gene contributes to the etiology of RDS and may serve as a gene for its predisposition. Allele 256 bp increases the risk of developing severe RDS. At the same time, the –18AA rs2077079 genotype of the SFTPB gene is associated with a reduced risk of developing severe RDS. The polymorphic variant c.413C>A p. T138N (rs4715) of the SFTPC gene is associated with BPD: the 413CC genotype increases, and the 413CA genotype reduces the risk of developing the disease. Синдром дыхательных расстройств (СДР) и бронхолегочная дисплазия (БЛД) являются заболеваниями легких, возникающими в основном у недоношенных новорожденных. Полиморфные варианты генов сурфактантных белков рассматриваются как кандидаты, вносящие вклад в патогенез СДР и БЛД. Изучена связь 5 полиморфных вариантов гена SFTPB (rs2077079, rs1130866, D2S388, D2S2232, VNTR 4 интрона) и 3 полиморфных замен гена SFTPС (rs4715, rs1124, rs2070687) у недоношенных новорожденных с СДР различной степени тяжести и БЛД. В исследование включены 555 новорожденных, среди которых 313 недоношенных младенцев со сроком гестации 28–36 недель. Генотипирование проводили секвенированием по Сэнгеру, микросателлитным анализом и ПЦР-РВ. Все недоношенные новорожденные характеризовались наличием СДР разной степени тяжести, у 36 новорожденных была выявлена БЛД. Микросателлитный маркер D2S388 гена SFTPB вносит вклад в этиологию СДР и может служить геном его предрасположенности. Аллель 256 п. н. увеличивает риск развития СДР тяжелой степени. В то же время генотип –18AА rs2077079 гена SFTPB ассоциирован с уменьшением риска развития СДР тяжелой степени. Полиморфный вариант c.413С>A p. T138N (rs4715) гена SFTPC ассоциирован с БЛД: генотип 413СС повышает, а генотип 413СА cнижает риск развития заболевания

Определение статуса метилирования промоторных областей генов MARCH11, HOXA9, PTGDR и UNCX у пациентов с немелкоклеточным раком легкого

The aim of this study was to determine the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue in patients with non-small cell lung cancer (NSCLC). A relative level of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes was determined by the quantitative methylation-specific PCR in 73 patients with NSCLC. The quantitative methylation-specific reaction was performed both for tumor tissue samples and non-tumor tissue samples of the same patient. For each of the samples, a reaction was set both by the investigated genes (MARCH11, UNCX, HOXA9, and PTGDR) and by the reference beta-actin gene (β-actin). Positive levels of methylation of the HOXA9 gene were established for 83.5 % patients; the MARCH11 gene – for 80.8 % patients; the PTGDR gene – for 68.4 % patients; the UNCX gene – for 84.9 % patients. In the study group of patients with NSCLC, significant differences were found in the relative levels of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue. The data suggest that hypermethylation of MARCH11, HOXA9, PTGDR, and UNCX genes may play a role in NSCLC tumor progression.Целью данного исследования было определение статуса метилирования промоторных областей генов MARCH11, HOXA9, PTGDR и UNCX в опухолевой и неопухолевой ткани легкого у пациентов с немелкоклеточным раком легкого (НМРЛ). Относительный уровень метилирования промоторных областей генов MARCH11, HOXA9, PTGDR, UNCX определяли методом количественной метилспецифической ПЦР у 73 пациентов с НМРЛ. Количественная метилспецифическая реакция проводилась как для образцов опухолевой ткани, так и для образцов неопухолевой ткани одного и того же пациента. Для каждого из образцов ставили реакцию по изучаемым генам (MARCH11, UNCX, HOXA9, PTGDR) и по референсному гену бета-актина (β-actin). Установлены положительные уровни метилирования гена HOXA9 у 83,5 % пациентов, гена MARCH11 – у 80,8 % пациентов, гена PTGDR – у 68,4 % пациентов, гена UNCX – у 84,9 % пациентов. В исследуемой группе пациентов с НМРЛ выявлены достоверные различия относительных уровней метилирования промоторных регионов генов MARCH11, HOXA9, PTGDR и UNCX в опухолевой и неопухолевой ткани легкого. Эти данные позволяют предположить, что гиперметилирование генов MARCH11, HOXA9, PTGDR и UNCX может играть роль в опухолевой прогрессии НМРЛ

ГЕНЕТИЧЕСКИЙ ПОЛИМОРФИЗМ АРОМАТАЗЫ У ПАЦИЕНТОК С СЕРОЗНЫМ РАКОМ ЯИЧНИКОВ В РЕСПУБЛИКЕ БЕЛАРУСЬ

The aim of the present research was to study the frequency of the distribution of variants of gene polymorphism of aromatase (СYP19A1) in Belarusian patients with serous ovarian cancer. This population established the influence of gene polymorphism rs10046 with the risk of serous ovarian cancer in women older than 50 years: CT genotype in a group of patients with serous ovarian cancer was met significantly less than in the control group (OR = 0.58; 95 % CI: 0.37–0.91; p = 0.023).В работе изучена частота распределения полиморфных вариантов гена ароматазы у белорусских пациенток с серозным раком яичников. В данной популяции пациентов установлена связь генетического полиморфизма rs10046 с риском развития серозного рака яичников у женщин старше 50 лет: генотип СТ в группе пациенток встречался достоверно реже (OR = 0,58; 95 % CI: 0,37–0,91; р = 0,023)