New-generation Sequencing Research Articles

Deep hematologic response to RD treatment in patients with multiple myeloma is associated with overexpression of IL-17R in CD138+ plasma cells

Lenalidomide (LEN) is widely used immunomodulatory drug (IMiD). Nonetheless, despite its efficacy, over time patients become resistant to LEN and relapse. Due to high clinical relevance, drug resistance in MM is being thoroughly investigated. However, less is known about predictors of good response to LEN-based treatment. The aim of this study was to identify molecular pathways associated with good and long response to LEN. The study included newly diagnosed MM patients (NDMM) and MM patients treated with first-line LEN and dexamethasone (RD) who achieved and least very good partial remission (VGPR). RNA was isolated from MM cells and new-generation sequencing was performed. Obtained results were validated with qRT-PCR. A global increase in gene expression was found in the RD group compared to NDMM, suggesting the involvement of epigenetic mechanisms. Moreover, upregulation of genes controlling the interaction within MM niche was detected. Next, genes controlling immune response were upregulated. In particular, the gene encoding the IL-17 receptor was overexpressed in the RD group which is a novel finding. This should be emphasized because IL-17-related signaling can potentially be targeted, providing the rationale for future research. Establishing the molecular background associated with long-lasting and profound response to LEN may improve LEN-based chemotherapy regimens and facilitate the development of adjuvant therapies to enhance its anti-MM activity.

Genetic etiology in patients diagnosed with congenital hypothyroidism with new-generation sequencing: A single-center experience

AimCongenital hypothyroidism (CH) is the most common endocrine disorder of the newborn; it is seen in every 3000–4000 births. Genetic features can guide treatment for patients with in situ glands. The present study aimed to contribute to the literature on CH variants and to show the benefit that genetic analysis can provide to patients in follow-up. MethodA total of 52 patients (47 families) diagnosed with CH were included in the study. Overall, 32 target genes involved in thyroid physiology were investigated by next-generation sequencing (NGS). ResultsIn total, 29 (55 %) of the patients were male, and the rate of dysgenesis was 19.2 %. In this study, 29 of 52 patients had at least one variant in one gene involved in CH (n = 29, 33 different variants) (Including likely benign variants and variants of unknown significance). There were 21 patients (40.3 %) with gland in situ. The most common variant was DUOX2 (20 %). The second most common variants were those in the TPO and TG genes (15 % and 15 %, respectively); 41.1 % of these were variants of uncertain significance (VUS), 26.4 % were pathogenic, 23.5 % were likely benign, and 11.7 % were likely pathogenic. On the basis of their zygosity, we identified 73.5 % heterozygous, 17.6 % homozygous, and 8.9 % combined heterozygous variants. There were mutant variants in two genes in six patients and three in one patient. ConclusionThis study found a variant in 55 % of the patients and shed light on the etiology of some cases of CH. The frequency of VUS was high. Although variants were identified in this study, their implication in the etiology of CH is not certain and, for most of the patients, it is also not sufficient for explaining the pathology with the current state of knowledge.

NK Cytotoxicity Mediated by NK-92 Cell Lines Expressing Combinations of Two Allelic Variants for FCGR3.

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by ligands of activating/inhibitory NK receptors, respectively. Allelic variants of the FCGR3A gene include the high-affinity single-nucleotide polymorphism (SNP) rs396991 (V176F), which is associated with the efficacy of monoclonal antibody (mAb) therapies, and the SNP rs10127939 (L66H/R). The contribution of FCGR3A SNPs to NK cell effector functions remains controversial; therefore, we generated a panel of eight NK-92 cell lines expressing specific combinations of these SNPs and tested their cytotoxicities. NK-92 cells were stably transfected with plasmids containing different combinations of FCGR3A SNPs. Messenger RNA and FcγRIIIa/CD16 cell surface expressions were detected using new generation sequencing (NGS) and flow cytometry, respectively. All FcγRIIIa/CD16-transfected NK-92 cell lines exhibited robust ADCC against three different target cell lines with minor differences. In addition, enhanced direct NK cytotoxicity against K562 target cells was observed, suggesting a mechanistic role of FcγRIIIa/CD16 in direct NK cytotoxicity. In conclusion, we generated eight FcγRIIIa/CD16-transfected NK-92 cell lines carrying different combinations of two of the most studied FCGR3A SNPs, representing the major genotypes described in the European population. The functional characterization of these cell lines revealed differences in ADCC and direct NK cytotoxicity that may have implications for the design of adoptive cancer immunotherapies using NK cells and tumor antigen-directed mAbs.

Interaction between microbiome and testicular tissue mastocytes in male infertility

Introduction. Male infertility is a complex condition with many potential causes, including hormonal imbalances, anatomical problems, genetic factors, lifestyle factors and more. But today, there is a fairly large group of infertile men with unknown causes of the disease.Objective. To analyze the taxonomic microbial diversity of testicular tissue and the urogenital tract of infertile men and to identify correlations between the microbiome and mastocytes in the testicular parenchyma.Materials & methods. The study was performed on testicular tissue samples from infertile patients with azoospermia (n = 33). All patients were divided into two groups based on the form of azoospermia: group 1 — infertile patients with non-obstructive azoospermia (NOA) (n=21); and group 2 — infertile with obstructive azoospermia (OA) patients (n=12). The bacterial diversity of testicular tissue was studied by the method of high-performance new generation sequencing (NGS). Immunohistochemical staining with anti-MCT (Anti-Mast Cell Tryptase) was used to determine the IHC expression of mastocyte markers.Results. The microbiome of patients with NOA differs markedly from the microbiome of patients with OA (p < 0.05). In group 1, representatives of the Enterobacteriaceae and Xanthomonadaceae families, the genera Finegoldia, Bifidobacterium, Porphiromonas, Prevotella, Peptoniphilus and Pseudomonas are significantly more often found. A distinctive feature of group 2 is the rare occurrence of the genus Prevotella. Histochemical analysis revealed mastocytosis in the in-between-canalicular stroma approximately in 83% of azoospermia cases. Mastocytes are found in tubule structures in 68% of cases and correlate with the microbiome of testicular tissue.Conclusions. Injuries caused by mastocytes in the stroma and tubular structures are interrelated with the taxonomic diversity of testicular tissue. Moreover, the testicles of NOA-patients have a qualitatively and quantitatively more diverse spectrum both at the level of families and genera, unlike OA-patients.

A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Clinical Significance of Genetic and Molecular Changes in Primary Myeloid Sarcoma

To investigate the clinical significance of genetic and molecular changes in primary myeloid sarcoma (MS). Fourteen patients with primary MS were selected in Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, The First People's Hospital of Lianyungang from September 2010 to December 2021. AML1-ETO fusion, PML-RARα fusion and CBFβ breakage were detected by fluorescence in situ hybridization (FISH), and the mutations of NPM1, CEBPA, FLT3, RUNX1, ASXL1, KIT and TP53 genes were detected by new generation sequencing (NGS). Among 14 patients, the MS occurred in bone, breast, epididymis, lung, chest wall, cervix, small intestine, ovary, lymph nodes and central nervous system. The tumor cells expressed MPO (13 cases), CD34 (7 cases), CD43 (8 cases), CD68 (7 cases), CD99 (8 cases) and CD117 (6 cases). Cytogenetic abnormalities were observed in 4 cases, including 3 cases of AML1-ETO fusion and 1 case of CBFβ breakage, while no PML-RARα fusion was detected. There were no significant differences in overall survival (OS) and leukemia-free survival (LFS) between patients with and without AML1-ETO fusion/CBFβ breakage (both P >0.05). Among the 14 patients, the number of NPM1, CEBPA, FLT3-ITD, RUNX1, ASXL1, KIT and TP53 gene mutations was 5, 3, 5, 3, 2, 2, 1, respectively, of which 7 cases had at least one mutation in FLT3-ITD, RUNX1, ASXL1 and TP53 gene. The OS and LFS of patients with FLT3-ITD, RUNX1, ASXL1 or TP53 mutation were shorter than those without mutations (both P <0.01). The genetic and molecular abnormalities of primary MS can be detected by FISH and NGS techniques. FLT3-ITD, RUNX1, ASXL1 or TP53 mutation indicates a worse prognosis, but further clinical studies are needed to confirm it.

Generation of major glutelin-deficient (GluA, GluB, and GluC) semi-dwarf Koshihikari rice line.

We generated a new Koshihikari rice line with a drastically reduced content of glutelin proteins and higher lodging resistance by using new and conventional plant breeding techniques. Using CRISPR/Cas9-mediated genome editing, we generated mutant rice with drastically decreased contents of major glutelins. A Koshihikari rice mutant line, a123, lacking four glutelins (GluA1, GluA2, GluB4, and GluB5) was used as a host, and another five major glutelin genes (GluA3, GluB1a, GluB1b, GluB2, and GluC) were knocked out through two iterations of Agrobacterium-mediated transformation. Mutant seeds were deficient in the GluA family, GluB family, and GluC, and the line obtained was named GluABC KO. Glutelin content was much lower in GluABC KO than in the existing low-glutelin rice mutant LGC-1. A null segregant of GluABC KO was selected using new-generation sequencing and backcrossing, and the sd-1 allele for the semi-dwarf trait was introduced to increase lodging resistance.

Evaluation of New Generation Sequencing (NGS)-Based Somatic Gene Variations and Real-Time Polymerase Chain Reaction (PCR)-Based Gene Fusions in Elderly and Young Acute Leukemia Patients: A Retrospective View.

Malignant diseases occurring in elderly patients follow a different course from younger patients and show different genetic structures. Therefore, in this retrospective study, the somatic gene variant profile and fusion gene profiles of elderly and young acute leukemia patients were determined to draw attention to the existing genetic difference, and the results were compared. In this study, the records of 204 acute leukemia patients aged 18+ who were referred to the Molecular Pathology Laboratory from the Hematology Clinic between 2018 and 2022 were reviewed retrospectively. Fusion gene detection in patients was performed with the HemaVision®-28Q Panel. The NGS Myeloid Neoplasms Panel was conducted using the MiniSEQ NGS platform according to the manufacturer's protocol. When all cases are evaluated together, the most frequently diagnosed acute leukemia is acute myeloid leukemia (85.8%). Both groups had a similar fusion gene profile; however, the fusion burden was higher in the elderly group. When the groups were evaluated in terms of somatic gene variations, there were differences between the groups, and the variation load was higher in the elderly group. Considering the different somatic gene variation profiles, it is understood that the genetic structure of tumor cells is different in elderly patients compared to young cases.

An Overview of Advances in Rare Cancer Diagnosis and Treatment.

Cancer stands as the leading global cause of mortality, with rare cancer comprising 230 distinct subtypes characterized by infrequent incidence. Despite the inherent challenges in addressing the diagnosis and treatment of rare cancers due to their low occurrence rates, several biomedical breakthroughs have led to significant advancement in both areas. This review provides a comprehensive overview of state-of-the-art diagnostic techniques that encompass new-generation sequencing and multi-omics, coupled with the integration of artificial intelligence and machine learning, that have revolutionized rare cancer diagnosis. In addition, this review highlights the latest innovations in rare cancer therapeutic options, comprising immunotherapy, targeted therapy, transplantation, and drug combination therapy, that have undergone clinical trials and significantly contribute to the tumor remission and overall survival of rare cancer patients. In this review, we summarize recent breakthroughs and insights in the understanding of rare cancer pathophysiology, diagnosis, and therapeutic modalities, as well as the challenges faced in the development of rare cancer diagnosis data interpretation and drug development.

Liver Bacterial Colonization in Patients with Obesity and Gut Dysbiosis.

Recently, the link between gut microbiota, liver inflammation, and obesity has become an interesting focus of research. The aim of this study is to show the possible relation between gut microbiota dysbiosis in patients with obesity and the presence of bacterial genomes in their liver biopsies. A prospective study on patients undergoing bariatric surgery was carried out. Anthropometric and metabolic data, comorbidities, stool samples, and hepatic biopsies were collected and analyzed at the time of surgery. The V3-16S rRNA region was sequenced using the Ion Torrent new-generation sequencing platform. In each of the 23 patients enrolled, the bacterial population was analyzed both in the stools and liver. In eight patients (34.7%), Prevotella (62.5%), Bacteroides (50%), Streptococcus (12.5%), and Dalister (12.5%) were found in both samples, simultaneously; in 15 cases, the liver was free from colonization. The statistically significant difference between groups was a Roseburia intestinalis reduction in fecal samples of patients with liver biopsies colonized by bacteria (1% vs 3%; p = 0.0339). To the best of our knowledge, this is the first study reporting the presence of bacterial genome in a liver biopsy on bariatric patients, instead of the microbe-associated molecular patterns. Notably, in literature, the presence of Roseburia intestinalis in stool samples has been shown to prevent intestinal inflammation playing its role in the gut barrier integrity. In our population, the Roseburia reduction was associated with the presence of bacterial genome in the liver, probably related to a greater permeability of the gut and vascular barriers.

Myeloproliferative Neoplasms: Contemporary Review and Molecular Landscape.

Myelofibrosis (MF), Myeloproliferative neoplasms (MPNs), and MDS/MPN overlap syndromes have a broad range of clinical presentations and molecular abnormalities, making their diagnosis and classification complex. This paper reviews molecular aberration, epigenetic modifications, chromosomal anomalies, and their interactions with cellular and other immune mechanisms in the manifestations of these disease spectra, clinical features, classification, and treatment modalities. The advent of new-generation sequencing has broadened the understanding of the genetic factors involved. However, while great strides have been made in the pharmacological treatment of these diseases, treatment of advanced disease remains hematopoietic stem cell transplant.

Cardiac magnetic resonance imaging in athletes with suspected structural myocardial disease: results of a high volume single-center study

Abstract Introduction Structural heart disease is one of the leading causes of sudden cardiac death in athletes. Cardiac magnetic resonance imaging (CMR) has a key role in the diagnosis, providing accurate functional and morphological information. Aim To investigate the prevalence of structural heart disease and the etiology of sudden cardiac death (SCD) in athletes. Methods We enrolled athletes (training ≥6 hours/week) who underwent CMR due to suspected structural myocardial disease at our Heart and Vascular Center between 2009 and 2022. We also performed cardiogenetic testing with new-generation sequencing (174 genes) in athletes with confirmed structural heart disease and/or malignant arrhythmia who gave consent. Results CMR was performed on a total of 556 athletes (445 male, 24±10 age). The indications for CMR were as follows: aborted sudden cardiac death/sustained ventricular tachycardia (3%), ECG (46%) or echocardiographic abnormality (36%), positive family history of SCD or cardiomyopathy (CMP) (3%), or athletes' complaints (31%). Structural myocardial abnormalities were confirmed in 99 athletes: hypertrophic (HCM) in 27, arrhythmogenic (AC) in 11, dilated (DCM) CMP in 7, and left ventricular non-compaction (LVNC) in 9 cases. MR images consistent with Fabry disease were found in 5 patients. We have discovered post-myocardial infarction scars in 10 cases, and atypical non-ischemic scarring (excluding hinge point fibrosis) was observed in 32 athletes. Besides pathological abnormalities, minor alterations were identified in 76 patients. Among the athletes examined after aborted sudden cardiac death/sustained ventricular tachycardia (n=18), we found structural heart disease in 14 patients, AC was confirmed in 7, HCM in two, non-ischemic fibrosis in three cases and LVNC in one athlete. Cardiogenetic test was performed in 26 athletes, we found pathogenic or likely pathogenic mutations in 5 cases, typical of HCM, AC, DCM (MYBPC3, PKP2, LMNA, TTN). The genetic test showed VUS (variant of unknown significance) in 15 additional athletes. Conclusions HCM was identified as the most frequent CMP, whereas AC and nonischaemic scar was the leading cause of SCD or SVT in our athletes.Figure A)Short axis late gadolinium ehancement CMR image of a 26-year-old female tennis player with hypertrophic cardiomyopathy and nonischemic fibrosis (arrow) and MYBPC3 pathogenic variant.Figure B)Short axis late gadolinium enhancement CMR image of a 40-year-old male elite discus thrower with sustained ventricular tacchycardia and nonischaemic scar formation (arrows). LMNA and titin pathogenic variants could be detected.

Epitachophoresis – A Method for the Extraction of Biomacromolecules from Complex Biological Matrices

Epitachophoresis is a new separation preparative technique based on discontinuous electrolyte systems enabling the preconcentration of biomacromolecules from complex biological matrices. Moreover, it enables the transfer of these compounds from biological liquids, such as urine or growth medium, to a simpler solution, suitable for subsequent analyses. In our recently published papers, we focused on the use of epitachophoresis to pretreat the samples containing proteins and DNA. The technique enabled more than seventyfold preconcentration with recoveries reaching more than 90%. The preparative nature of epitachophoresis enables one to subsequently analyse the isolated biomacromolecules by various techniques including mass spectrometry, capillary electrophoresis, gel electrophoresis, ELISA analysis, or new-generation sequencing. Full text English translation is available in the on-line version.

Technical comparison of MinIon and Illumina technologies for genotyping Chikungunya virus in clinical samples

New-generation sequencing (NGS) techniques have brought the opportunity for genomic monitoring of several microorganisms potentially relevant to public health. The establishment of different methods with different mechanisms provides a wide choice, taking into account several aspects. With that in mind, the present aim of the study was to compare basic genomic sequencing metrics that could potentially impact genotyping by nanopores from Oxford Nanopore Technologies and by synthesis from Illumina in clinical samples positive for Chikungunya (CHIKV). Among the metrics studied, running time, read production, and Q score were better represented in Illumina sequencing, while the MinIOn platform showed better response time and greater diversity of generated files. That said, it was possible to establish differences between the studied metrics in addition to verifying that the distinctions in the methods did not impact the identification of the CHIKV virus genotype.

A systematic review of real-world applications of genome sequencing for newborn screening

Aim: With the costs of genomic sequencing falling quickly and an ever-increasing number of clinical laboratories equipped with new-generation sequencing machines, healthcare systems around the world are getting ready to enter the era of genomic newborn screening (NBS). However, the adoption of Genomic Sequencing (GS), encompassing whole-exome sequencing (WES) and whole-genome sequencing (WGS), in NBS programs raises a number of clinical, ethical, and legal questions as well as organizational and economic challenges. This systematic review is part of a feasibility study to assess the introduction of WGS for NBS in Lombardy region with the specific aim of gathering evidence from existing pilots in the field whose results have been published. Methods: Three different sources were identified for the selection of articles in order to obtain a various and unbiased set of publications. 33 articles were retained for analysis to answer the following questions: 1. Clinical: Does genomic sequencing demonstrate clinical utility in the context of NBS? What are the limitations of these kind of programs? 2. Societal: What are the social, ethical and psychological implications of using GS for NBS? 3. Governance: What are the legal, economic, and organizational challenges for GS-based NBS programs? Results: There is a general consensus in the literature on the key principles that should guide the adoption of GS in NBS, such as the inclusion of actionable genes only, the need for informed consent from the parents, the right of the newborn to an open future, which means the exclusion of late-onset diseases even when those are considered treatable. However, there are still several differences in how these principles are detailed and applied. Conclusion: Real-world evidence from a handful of pilot projects (namely BabySeq and NC-Nexus, both carried out in the USA) have been published recently; however, this evidence is not yet sufficient to put an end to the broad and animated debate on the use of GS for NBS. Ethical, legal, and social issues still constitute great challenges and major barriers to wide and uniform adoption of GS in NBS. On the clinical side, a number of issues remain unaddressed, such as the benefits and limitations of the different approaches (targeted sequencing, GS only versus GS+standard NBS), the genes/diseases to include and the frequency of incidental findings, identification of carrier status, and variants of uncertain significance (VUS). Further pilots and consultations with involved stakeholders will be necessary before GS-based NBS can be accepted and systematically implemented in national healthcare programs.

Principles of the differential diagnosis of achondroplasia and pseudoachondroplasia

BACKGROUND: Achondroplasia and pseudoachondroplasia are hereditary systemic skeletal dysplasias characterized by a certain similarity of clinical manifestations; however, they have different etiopathogenetic mechanisms and confirmation methods for molecular genetic diagnosis. Their common phenotypic features often make differential diagnosis difficult during the clinical examination of patients, planning DNA diagnostics, and appropriate time detection of neurosurgical and orthopedic complications.&#x0D; AIM: This study aimed to identify differential diagnostic criteria for achondroplasia and pseudoachondroplasia and optimize the strategy for their molecular genetic diagnosis.&#x0D; MATERIALS AND METHODS: A comprehensive examination of 76 children from 74 unrelated families aged 1 month to 18 years with phenotypic signs of achondroplasia and pseudoachondroplasia was conducted. To clarify the diagnosis through genealogical and amnestic analysis, clinical and neurological examination data according to the standard method and radiographic data were used. Molecular genetic confirmation of diseases was conducted by searching for hotspot mutations in the FGFR3 gene, assessing the number of GAC repeats located in exon 13 of the COMP gene, and new-generation sequencing of the target panel consisting of 166 genes responsible for hereditary skeletal pathology.&#x0D; RESULTS: Based on a comparative analysis of the specific phenotypic characteristics, the criteria for the differential diagnosis of achondroplasia and pseudoachondroplasia were identified. The leading signs of achondroplasia are disproportionate nanism from birth, macrocrania, and facial dysmorphism, which are not specific to pseudoachondroplasia. Certain radiological features are essential in the differential diagnosis of pseudoachondroplasia, which should be considered when referring to patients for molecular genetic analysis. A deletion of the GAC repeat c.1417_1419del in the COMP gene was identified in 27% of patients with pseudoachondroplasia. Thus, the analyses of these two mutations in FGFR3 and COMP were conducted first. In the absence of target mutations, further diagnostic search should be continued with a target panel consisting of 166 genes responsible for hereditary skeletal pathology or whole-exome sequencing.&#x0D; CONCLUSIONS: The analysis of the clinical, radiological, and molecular genetic characteristics of patients with achondroplasia and pseudoachondroplasia, together with the literature data analysis, made it possible to clarify the differential diagnostic criteria for these diseases and optimize the algorithm for their molecular genetic diagnosis.

The launch of satellite: DNA repeats as a cytogenetic tool in discovering the chromosomal universe of wild Triticeae.

Fluorescence in situ hybridization is a powerful tool that enables plant researchers to perform systematic, evolutionary, and population studies of wheat wild relatives as well as to characterize alien introgression into the wheat genome. This retrospective review reflects on progress made in the development of methods for creating new chromosomal markers since the launch of this cytogenetic satellite instrument to the present day. DNA probes based on satellite repeats have been widely used for chromosome analysis, especially for "classical" wheat probes (pSc119.2 and Afa family) and "universal" repeats (45S rDNA, 5S rDNA, and microsatellites). The rapid development of new-generation sequencing and bioinformatical tools, and the application of oligo- and multioligonucleotides has resulted in an explosion in the discovery of new genome- and chromosome-specific chromosome markers. Owing to modern technologies, new chromosomal markers are appearing at an unprecedented velocity. The present review describes the specifics of localization when employing commonly used vs. newly developed probes for chromosomes in J, E, V, St, Y, and P genomes and their diploid and polyploid carriers Agropyron, Dasypyrum, Thinopyrum, Pseudoroegneria, Elymus, Roegneria, and Kengyilia. Particular attention is paid to the specificity of probes, which determines their applicability for the detection of alien introgression to enhance the genetic diversity of wheat through wide hybridization. The information from the reviewed articles is summarized into the TRepeT database, which may be useful for studying the cytogenetics of Triticeae. The review describes the trends in the development of technology used in establishing chromosomal markers that can be used for prediction and foresight in the field of molecular biology and in methods of cytogenetic analysis.

Genetic diversity and population structure of a Peruvian cattle herd using SNP data.

New-generation sequencing technologies, among them SNP chips for massive genotyping, are useful for the effective management of genetic resources. To date, molecular studies in Peruvian cattle are still scarce. For the first time, the genetic diversity and population structure of a reproductive nucleus cattle herd of four commercial breeds from a Peruvian institution were determined. This nucleus comprises Brahman (N = 9), Braunvieh (N = 9), Gyr (N = 5), and Simmental (N = 15) breeds. Additionally, samples from a locally adapted creole cattle, the Arequipa Fighting Bull (AFB, N = 9), were incorporated. Female individuals were genotyped with the GGPBovine100K and males with the BovineHD. Quality control, and the proportion of polymorphic SNPs, minor allele frequency, expected heterozygosity, observed heterozygosity, and inbreeding coefficient were estimated for the five breeds. Admixture, principal component analysis (PCA), and discriminant analysis of principal components (DAPC) were performed. Also, a dendrogram was constructed using the Neighbor-Joining clustering algorithm. The genetic diversity indices in all breeds showed a high proportion of polymorphic SNPs, varying from 51.42% in Gyr to 97.58% in AFB. Also, AFB showed the highest expected heterozygosity estimate (0.41 ± 0.01), while Brahman the lowest (0.33 ± 0.01). Besides, Braunvieh possessed the highest observed heterozygosity (0.43 ± 0.01), while Brahman the lowest (0.37 ± 0.02), indicating that Brahman was less diverse. According to the molecular variance analysis, 75.71% of the variance occurs within individuals, whereas 24.29% occurs among populations. The pairwise genetic differentiation estimates (FST) between breeds showed values that ranged from 0.08 (Braunvieh vs. AFB) to 0.37 (Brahman vs. Braunvieh). Similarly, pairwise Reynold's distance ranged from 0.09 (Braunvieh vs. AFB) to 0.46 (Brahman vs. Braunvieh). The dendrogram, similar to the PCA, identified two groups, showing a clear separation between Bos indicus (Brahman and Gyr) and B. taurus breeds (Braunvieh, Simmental, and AFB). Simmental and Braunvieh grouped closely with the AFB cattle. Similar results were obtained for the population structure analysis with K = 2. The results from this study would contribute to the appropriate management, avoiding loss of genetic variability in these breeds and for future improvements in this nucleus. Additional work is needed to speed up the breeding process in the Peruvian cattle system.

Molecular Markers in Plant Breeding

Molecular markers are an important tool for plant breeding. Since the 1980s, in response to the technology development, molecular marker approaches have been further diversified. The establishment of new-generation sequencing and high-throughput plant phenotyping has greatly decreased the time to genotype large numbers of individuals. For breeders who are not very familiar with molecular techniques and want to catch up with the advances in the field, this review offers basic knowledge. Each molecular marker technology has specific advantages as well as limitations. Molecular marker types, diversity studies, QTL mapping, associative mapping, marker-assisted backcrossing and genomic selection are explored. Marker application in plant breeding is also described. In the genome, molecular markers can detect the genetic architecture of a trait, but also identify candidate genes with an important role in plant breeding programs.

Molecular genetic sequencing of neuroendocrine tumors of the gastrointestinal tract.

651 Background: The objective of this work was to evaluate the molecular and genetic features of the NET gastrointestinal tract. Genomic sequencing of 30 tumor samples of the latest generation was carried out and the data obtained were evaluated. Methods: In total, 30 tumor blocks of patients with gastrointestinal NET were tested by the new generation sequencing (NGS) method. The following genes were analyzed: ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CHEK1, CHEK2, EPCAM, FANCL, MLH1, MSH2, NBN, NF1, PALB2, PMS2, RAD51B, RAD51C, RAD51D, RAD54L, STK11, TP53, POLE, KRAS, NRAS, BRAF, EGFR, ERBB2, PIK3CA, MET ex14, BAT25, BAT26, NR21, NR24, MONO27, KIT, PDGFRA, Pi3Ca. Preparation of libraries for sequencing was carried out using NimblGen SepCapEZ Choice. Sequencing was performed on the Illumina MiSeq device. Results: In the study cohort of patients whose tumor blocks were sent for examination by the NGS method, there were more women – 19 (63.3%) than men – 11 (36.7%). When analyzing the age of patients, it should be noted that among all age groups, elderly patients predominated (60-74 years according to the WHO classification, 2016) – 12 (40%). In the majority of patients – 10 (33.3%) – the primary tumor focus was localized in the pancreas, in 6 (20%) patients it was primarily affected stomach, 7 (23.3%) – small intestine. Other parts of the gastrointestinal tract were affected much less frequently: the colon – in 1 (3.3%) patients. According to the division according to the degree of differentiation of gastrointestinal NET10 tumors had a degree of differentiation G1 (33.3%), 16 -G3 (54.3%), 4– G3 (13.3%). In the tumor sample of a patient with gastric NET G1 (Ki 67 = 2%), POLE mutations were found: c.2276G &gt; A, p.R759H and CHEK2: c.470T &gt; C, p.I157T. Also, mutations MLH1: c.199G &gt; A, p.G67R BRCA1: c.1881 were found in the tumor sample of another patient with gastric NET G2 (Ki 67 = 7%). Conclusions: To improve the survival rates of patients, it is worth paying special attention to the molecular genetic study of the NET gastrointestinal tract. It is molecular genetic profiling that can become the basis for building more individualized algorithms for treating patients. The results of this study allow us to count on the fact that when conducting a study on a larger cohort of patients with gastrointestinal NET, it is possible to obtain results that will expand our knowledge of this nosology and allow us to supplement the already available information about prognosis factors and predictors of response to treatment.