Molecular Genetic Approaches Research Articles

Identification and functional analysis of a novel de novo missense mutation located in the initiation codon of LAMP2 associated with early onset female Danon disease.

Danon disease is characterized by the failure of lysosomal biogenesis, maturation, and function due to a deficiency of lysosomal membrane structural protein (LAMP2). The current report describes a female patient with a sudden syncope and hypertrophic cardiomyopathy phenotype. We identified the pathogenic mutations in patients by whole-exon sequencing, followed by a series of molecular biology and genetic approaches to identify and functional analysis of the mutations. Suggestive findings by cardiac magnetic resonance (CMR), electrocardiogram (ECG), and laboratory examination suggested Danon disease which was confirmed by genetic testing. The patient carried a novel de novo mutation, LAMP2 c.2T>C located at the initiation codon. The quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis of peripheral blood leukocytes from the patients revealed evidence of LAMP2 haploinsufficiency. Labeling of the new initiation codon predicted by the software with green fluorescent protein followed by fluorescence microscopy and Western blotting showed that the first ATG downstream from the original initiation codon became the new translational initiation codon. The three-dimensional structure of the mutated protein predicted by alphafold2 revealed that it consisted of only six amino acids and failed to form a functional polypeptide or protein. Overexpression of the mutated LAMP2 c.2T>C showed a loss of function of the protein, as assessed by the dual-fluorescence autophagy indicator system. The mutation was confirmed to be null, AR experiments and sequencing results confirmed that 28% of the mutant X chromosome remained active. We propose possible mechanisms of mutations associated with haploinsufficiency of LAMP2: (1) The inactivation X chromosome carrying the mutation was not significantly skewed. However, it decreased in the mRNA level and the expression ratio of the mutant transcripts; (2) The identified mutation is null, and the active mutant transcript fails to translate into the normal LAMP2 proteins. The presence of haploinsufficiency in LAMP2 and the X chromosome inactivation pattern were crucial factors contributing to the early onset of Danon disease in this female patient.

What did COVID-19 really teach us about science, evidence and society?

What did COVID-19 really teach us about science, evidence and society?

Sharing biological information across generations: Parallels between indigenous knowledge and genetics for fisheries recovery in the Columbia River Basin

AbstractIndigenous tribes of the interior Columbia River have developed a mutual relationship with native fishes since time immemorial. However, extensive disruption to the natural ecosystem has occurred as European settlement of North America extended westward to utilize abundant natural resources in ways that conflicted with millennia of indigenous protection. This anthropogenic disturbance has led to dramatic declines in native fish species that are central to tribal cultures, but efforts are underway to enable these fishes and the people that rely upon them to persist for future generations. Here, we describe how pairing indigenous knowledge and western science have been applied to assist with fisheries recovery in the Columbia River. Parallel understanding of information passed across generations is central to this effort, from tribal elders with their historical grasp of the natural ecosystem and fisheries, to molecular genetic approaches that track DNA that is passed from parents to offspring and subsequent generations. Examples are provided that illustrate how both indigenous knowledge and genetic tools have been applied to support fisheries recovery in the Columbia River Basin.

Studying the Genetic Diversity of the Rice Break Cause <i>Pyricularia oryzae</i> Cav. on the Complex of Molecular and Morphological Features

On the basis of molecular genetic approaches, the study of the biodiversity of the phytopathogenic fungus Pyricularia oryzae Cav. in the south of Russia and a classification of the morphological and cultural characteristics of the pathogen was carried out. For 2020–2022 monitoring was carried out and 24 strains of the pathogen were isolated from the affected herbarium material collected from the fields of five ecological rice growing zones of the Krasnodar Territory (Krasnoarmeisky, Kalininsky, Abinsky, Slavyansky districts, Krasnodar). Using the multiplex PCR system developed by us based on fragment analysis, five genotypes were identified among the studied Pyricularia oryzae Cav. strains over the entire period of the project, each of which is characterized by a unique genetic profile. Compiled their “DNA passport”. The studied isolates of the causative agent of blast were characterized by morphological and cultural characteristics. Six morphotypes of the pathogen have been identified.

Molecular genetics and general management of androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is a rare genetic disorder that affects the development of the male reproductive system in individuals with a 46,XY karyotype. In addition to physical impacts, patients with AIS may face psychological distress and social challenges related to gender identity and acceptance. The major molecular etiology of AIS results from hormone resistance caused by mutations in the X-linked androgen receptor (AR) gene. Depending on the severity of androgen resistance, the wide spectrum of AIS can be divided into complete AIS (CAIS), partial AIS (PAIS), or mild AIS (MAIS). Open issues in the treatment and management of AIS include decisions about reconstructive surgery, genetic counseling, gender assignment, timing of gonadectomy, fertility and physiological outcomes. Although new genomic approaches have improved understanding of the molecular causes of AIS, identification of individuals with AIS can be challenging, and molecular genetic diagnosis is often not achievable. The relationship between AIS genotype and phenotype is not well established. Therefore, the optimal management remains uncertain. The objective of this review is to outline the recent progress and promote understanding of AIS related to the clinical manifestation, molecular genetics and expert multidisciplinary approach, with an emphasis on genetic etiology.

Construction of versatile yeast plasmid vectors transferable by Agrobacterium-mediated transformation and their application to bread-making yeast strains

Agrobacterium-mediated transformation (AMT) potentially has great advantages over other DNA introduction methods: e.g., long DNA and numerous recipient strains can be dealt with at a time merely by co-cultivation with donor Agrobacterium cells. However, AMT was applied only to several laboratory yeast strains, and has never been considered as a standard gene-introduction method for yeast species. To disseminate the AMT method in yeast species, it is necessary to develop versatile AMT plasmid vectors including shuttle type ones, which have been unavailable yet for yeasts. In this study, we constructed a series of AMT plasmid vectors that consist of replicative (shuttle)- and integrative-types and harbor a gene conferring resistance to either G418 or aureobasidin A for application to prototrophic yeast strains. The vectors were successfully applied to five industrial yeast strains belonging to Saccharomyces cerevisiae after a modification of a previous AMT protocol, i.e., simply inputting a smaller number of yeast cells to the co-cultivation than that in the previous protocol. The revised protocol enabled all five yeast strains to generate recombinant colonies not only at high efficiency using replicative-type vectors, but also readily at an efficiency around 10-5 using integrative one. Further modification of the protocol demonstrated AMT for multiple yeast strains at a time with less labor. Therefore, AMT would facilitate molecular genetic approaches to many yeast strains in basic and applied sciences.

Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala.

Females are more likely than males to develop post-traumatic stress disorder (PTSD). However, the neurobiological mechanisms responsible for these sex differences remain elusive. The ubiquitin proteasome system (UPS) is involved in fear memory formation and implicated in PTSD development. Despite this, proteasome-independent functions of the UPS have rarely been studied in the brain. Here, using a combination of molecular, biochemical, proteomic, behavioral, and novel genetic approaches, we investigated the role of proteasome-independent lysine-63 (K63)-polyubiquitination, the second most abundant ubiquitin modification in cells, in the amygdala during fear memory formation in male and female rats. Only females had increased levels of K63-polyubiquitination targeting in the amygdala following fear conditioning, which targeted proteins involved in ATP synthesis and proteasome function. CRISPR-dCas13b-mediated knockdown of K63-polyubiquitination in the amygdala via editing of the K63 codon in the major ubiquitin gene, Ubc, impaired fear memory in females, but not males, and caused a reduction in learning-related increases in ATP levels and proteasome activity in the female amygdala. These results suggest that proteasome-independent K63-polyubiquitination is selectively involved in fear memory formation in the female amygdala, where it is involved in the regulation of ATP synthesis and proteasome activity following learning. This indicates the first link between proteasome-independent and proteasome-dependent UPS functions in the brain during fear memory formation. Importantly, these data are congruent with reported sex differences in PTSD development and may contribute to our understanding of why females are more likely to develop PTSD than males.

OsMADS1 Regulates Grain Quality, Gene Expressions, and Regulatory Networks of Starch and Storage Protein Metabolisms in Rice.

OsMADS1 plays a vital role in regulating floret development and grain shape, but whether it regulates rice grain quality still remains largely unknown. Therefore, we used comprehensive molecular genetics, plant biotechnology, and functional omics approaches, including phenotyping, mapping-by-sequencing, target gene seed-specific RNAi, transgenic experiments, and transcriptomic profiling to answer this biological and molecular question. Here, we report the characterization of the 'Oat-like rice' mutant, with poor grain quality, including chalky endosperms, abnormal morphology and loose arrangement of starch granules, and lower starch content but higher protein content in grains. The poor grain quality of Oat-like rice was found to be caused by the mutated OsMADS1Olr allele through mapping-by-sequencing analysis and transgenic experiments. OsMADS1 protein is highly expressed in florets and developing seeds. Both OsMADS1-eGFP and OsMADS1Olr-eGFP fusion proteins are localized in the nucleus. Moreover, seed-specific RNAi of OsMADS1 also caused decreased grain quality in transgenic lines, such as the Oat-like rice. Further transcriptomic profiling between Oat-like rice and Nipponbare grains revealed that OsMADS1 regulates gene expressions and regulatory networks of starch and storage protein metabolisms in rice grains, hereafter regulating rice quality. In conclusion, our results not only reveal the crucial role and preliminary mechanism of OsMADS1 in regulating rice grain quality but also highlight the application potentials of OsMADS1 and the target gene seed-specific RNAi system in improving rice grain quality by molecular breeding.

Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Plants and Small Molecules: An Up-and-Coming Synergy.

The emergence of Arabidopsis thaliana as a model system has led to a rapid and wide improvement in molecular genetics techniques for studying gene function and regulation. However, there are still several drawbacks that cannot be easily solved with molecular genetic approaches, such as the study of unfriendly species, which are of increasing agronomic interest but are not easily transformed, thus are not prone to many molecular techniques. Chemical genetics represents a methodology able to fill this gap. Chemical genetics lies between chemistry and biology and relies on small molecules to phenocopy genetic mutations addressing specific targets. Advances in recent decades have greatly improved both target specificity and activity, expanding the application of this approach to any biological process. As for classical genetics, chemical genetics also proceeds with a forward or reverse approach depending on the nature of the study. In this review, we addressed this topic in the study of plant photomorphogenesis, stress responses and epigenetic processes. We have dealt with some cases of repurposing compounds whose activity has been previously proven in human cells and, conversely, studies where plants have been a tool for the characterization of small molecules. In addition, we delved into the chemical synthesis and improvement of some of the compounds described.

Eye Morphogenesis in Vertebrates.

Proper eye structure is essential for visual function: Multiple essential eye tissues must take shape and assemble into a precise three-dimensional configuration. Accordingly, alterations to eye structure can lead to pathological conditions of visual impairment. Changes in eye shape can also be adaptive over evolutionary time. Eye structure is first established during development with the formation of the optic cup, which contains the neural retina, retinal pigment epithelium, and lens. This crucial yet deceptively simple hemispherical structure lays the foundation for all later elaborations of the eye. Building on descriptions of the embryonic eye that started with hand drawings and micrographs, the field is beginning to identify mechanisms driving dynamic changes in three-dimensional cell and tissue shape. A combination of molecular genetics, imaging, and pharmacological approaches is defining connections among transcription factors, signaling pathways, and the intracellular machinery governing the emergence of this crucial structure.

Strigolactones in Sugarcane Growth and Development

Sugarcane is a complex polyploid aneuploid cash crop, and transgenic varieties are important for molecular genetic and traditional breeding approaches. Herein, the sugarcane variety ROC22 served as the receptor, the Bar gene served as a screening marker, and positive and negative fragments of the ScD27.2 gene, upstream of strigolactones (SLs) biosynthesis genes driven by the 35S promoter, were introduced by Agrobacterium tumefaciens-mediated transformation. Regenerated plants were obtained by co-culture, screening culture, and differentiation induction, and 27 sense and antisense ScD27.2 transgenic seedlings were obtained by herbicide screening. PCR detection and 1% Basta (Glufosinate) application on leaves revealed Bar in all lines, with all testing positive for herbicide application and 23 containing the target gene (positive resistance screening rate = 87.5%). q-PCR and phenotypic analyses showed that ScD27.2 expression, plant height, tiller number, root length, stem diameter, and fresh weight were decreased in transgenic (ScD27.2R-9) compared with non-transgenic (NT and ScD27.2F-2) lines. ScD27.2 expression was downregulated, and growth potential was inhibited. Under 20% PEG treatment, malondialdehyde (MDA) content in ScD27.2R-9 was higher than in NT, while proline content was lower. Under drought stress, ScD27.2 expression, MDA levels, and proline content in ScD27.2F-2 and NT were higher than in non-treated controls, ScD27.2 expression increased with time, and MDA and proline levels also increased. ScD27.2 expression in ScD27.2R-9 decreased under 20% PEG treatment, MDA and proline increased (but not to NT levels), and growth was lower than NT. The 20% PEG treatment also increased the levels of (±)-2′-epi-5-deoxystrigol and (+)-abscisic acid in the rooting culture media of ScD27.2F-2, ScD27.2R-9, and NT lines, but the levels of (+)-abscisic acid content in ScD27.2R-9 was lower than in NT. Thus, interfering with ScD27.2 expression decreased resistance to 20% PEG treatment. ScD27.2 encodes a β-carotene isomerase involved in SLs biosynthesis that might function in sugarcane resistance to drought stress. It explains the role of SLs in sugarcane growth and development and responses to drought stress.

Primera evidencia de actividad enzimática nitrilasa en Xylaria sp., y su relación con la biosíntesis de ácido indol-3-acético

Endophytic fungi inhabit plant tissues internally and asymptomatically, and many of them are involved in the synthesis of bioactive metabolites of antifungal and therapeutic nature, as well as other compounds of biotechnological importance including indole derivatives, among many others. Ecologically, they provide some benefits to plants including protection against phytopathogens and promotion of root growth. In this sense, Xylaria sp. is a cellulose-decomposing fungus with biotechnological potential. It is worth mentioning that indole-3-acetic acid (IAA) also plays an extremely important role in plant–micro-organism interactions, as it is essential for physiology and proper plant morphological development. It is known that nitrile-hydrolytic enzymes (nitrilases) are involved in the synthesis of plant indole compounds; however, relatively little information is available concerning the nature of these enzymes in the fungal kingdom. In view of the above, through a biochemical and molecular-genetic approach, it has been demonstrated for the first time that Xylaria sp. carries out nitrile-hydrolytic enzyme activity using nitrogen and carbon-rich compounds as substrate. The studied strain increased its relative gene expression levels and showed mycelial growth, both in the presence of chemical compounds such as cyanobenzene and KCN. Thus, the results of this work suggest that the micro-organism is capable of degrading complex nitrogenous molecules. On the other hand, through fungal biofertilization, it was observed that Xylaria sp. promotes the development of the root system of Arabidopsis thaliana seedlings, in addition to synthesizing IAA.

Something old, something new - can adding genomic data to family studies advance our understanding of the impact of nature and nurture on mental health? Commentary on McAdams et al. (2023).

In their annual research review, McAdams, Cheesman, and Ahmadzadeh (2023) provide a thorough overview of how the use of novel genetically informative approaches can increase our knowledge about the intergenerational transmission of psychopathology. Many JCPP readers will already be familiar with genetically sensitive family-based designs, such as twin and adoption studies, as well as with newer molecular-genetic approaches, such as polygenic-score studies. McAdams et al.'s (2023) review discusses the innovative combination of family-based and molecular-genetic methods, and what this combination can reveal about developmental psychopathology.

Molecular Genetic Approach in Diagnosing Childhood Primary Immunodeficiency Disease (PID) Attending Six Major Hospitals in Bangladesh

Astract not available The Journal of Ad-din Women's Medical College; Vol. 11 (1), Jan 2023; p 67-69

Cultural and morphological features of <i>Fusarium oxysporum</i> and <i>Fusarium brachygibbosum</i> species associated with garden strawberry plants

This study is focused on two micromycetes of the genus Fusarium: the complex species F. oxysporum and F. brachygibbosum Padwick isolated from strawberry roots from different regions of Russia. Studies were performed in 2021-2022 on samples of symptomatic plants collected in the Republic of Crimea and Voronezh Oblast. The micromycetes were studied by means of a combination of classical biological methods and molecular genetic approaches with decoding of nucleotide sequences. The subject of the work was to study the biological features of F. oxysporum and F. brachygibbosum Padwick. The work included the study of macro- and micro features of the target objects. The following cultural features were determined: structure and coloring of aerial and substrate mycelium, character of colony surface, growth rate, sporulation of fungi, pigmentation of structures and nutrient media on the studied media. The species F. brachygibbosum from planting material of strawberry originating from the Republic of Crimea was isolated and identified for the first time. As a result of studies of different composition of nutrient media, it was found that potato-glucose agar (PGA) 2 % is the most optimal nutrient medium for growth and sporulation of target isolates. It was determined that the addition of citric acid to PGA altered the macro features of the mycelium of F. brachygibbosum. Universal sites of several genes (internal transcribed spacer, actin gene, elongation translation factor gene) were studied for species identification of F. oxysporum and F. brachygibbosum. Variable gene sites for identification of the species identity of Fusarium isolates were determined. It was revealed that ITS1-5.8S-ITS2 and TEF sites could be used for interspecific differentiation of Fusarium wilts.

ANALYSIS OF THE TAXONOMIC STATUS OF LIMONIUM CASPIUM (WILLD.) P. FOURN. USING THE ITS1-5.8S-ITS2 REGION

There are 15 representatives of the genus Limonium Mill in Ukraine. The taxonomic status of some of them requires clarification. In particular, some taxonomists consider L. caspium (Willd.) P. Fourn. to be a separate species, while others tend to consider this name as a synonym for L. bellidifolium Dumort. Until now, only one sample of L. caspium from eastern Iran has been analyzed using molecular genetic approaches: for this accession, the ITS1-5.8S-ITS2 of 35S rDNA region was sequenced. For both species, L. caspium and L. bellidifolium, samples from the territory of Ukraine have not yet been examined using molecular taxonomy methods. In this work, we amplified and sequenced ITS1-5.8S-ITS2 region for two L. caspium and two L. bellidifolium samples from southern Ukraine. All four samples appeared to be identical in this region. They also do not differ from previously sequenced samples of L. bellidifolium from France, Turkey and Greece. On the other hand, the sequence of L. caspium from Iran has five nucleotide substitu-tions and four indels compared to the mentioned samples. The phylogenetic analysis revealed that representatives of the so-called "L. bellidifolium complex", which, in addition to the two species mentioned, also includes L. iconium and L. tamaricoides analyzed in this work, are grouped together with representatives of the Nephrophyllum section. A similar result was obtained in the studies of other authors using the ITS1-5.8S-ITS2 sequence and regions of chloro-plast DNA. However, according to the IGS 5S rDNA sequence, representatives of the "L. bellidifolium complex" in our previous work were grouped together with the species of the Limonium section, to which they were previously as-signed based on morphological characters. Such a discrepancy between phylogenies using different genomic regions indicates the reticular nature of the evolution of the genomes of representatives of this group and indicates probable hybridization events in their past. Within the clade of the "L. bellidifolium complex" that we obtained in our analysis, the sample of L. caspium from Iran forms a sister branch to all its other representatives. Thus, the comparison of the ITS1-5.8S-ITS2 region shows that the Ukrainian samples identified as L. caspium do not differ from L. bellidifolium. However, to finally clarify the taxonomic status of L. caspium, it is necessary to analyze more samples from different geographic origins.

Research of the New Biomarkers of Mammary Gland Tumors in Domestic Cats

This article presents the results of the clinical significance study of changes in the activity of adenosine deaminase and the content of polyamines in the blood plasma of cats with mammary gland tumors. In our work, we investigated the activity of the adenosine deaminase gene in the tissues of mammary gland tumors in cats and compared the activity of this gene with normal mammary gland tissue. In the course of this work, we also found out the frequency of occurrence of mammary gland tumors in the population of domestic cats in the Rostov region and evaluated the prognosis of survival after surgical treatment of breast cancer. The incidence of mammary gland tumors was detected, which amounted to 0.4% of the domestic cats who have applied to veterinary clinics in Rostov region, where the cats with mammary gland neoplasms amounted to approximately 400 individuals for every 100,000 animals. These data reflect the importance of the problem of mammary gland tumors and emphasize the need to study the pathophysiological foundations of carcinogenesis in order to create molecular genetic approaches for early diagnosis of mammary gland tumors. The results of the studies showed that the level of expression of the adenosine deaminase gene in the tissues of invasive nonspecific carcinoma and in the tissues of fibroepithelial hyperplasia sharply increases compared to the tissue of a healthy mammary gland, 36 times in the tissues of invasive carcinomas and 131 times in the tissues of fibroepithelial hyperplasia. At the same time, there were no significant differences between the activity of adenosine deaminase in the blood plasma of healthy animals and in the blood plasma of animals with malignant neoplasms of the mammary gland. Measurement of the level of polyamines in the blood of animals showed that the content of putrescine in the erythrocytes of the blood of cats with benign and tumor-like neoplasms of the mammary fibroepithelial hyperplasia significantly increased by 5 times compared with the indicators of the control group. In malignant neoplasms of the mammary gland invasive nonspecific carcinoma, the content of putrescine and spermin in the blood significantly exceeded the control values by 6 and 10 times, respectively.

Advances in Physiological, Transcriptomic, Proteomic, Metabolomic, and Molecular Genetic Approaches for Enhancing Mango Fruit Quality.

Mango (Mangifera indica L.) is a nutritionally important fruit of high nutritive value, delicious in taste with an attractive aroma. Due to their antioxidant and therapeutic potential, mango fruits are receiving special attention in biochemical and pharmacognosy-based studies. Fruit quality determines consumer's acceptance, and hence, understanding the physiological, biochemical, and molecular basis of fruit development, maturity, ripening, and storage is essential. Transcriptomic, metabolomic, proteomic, and molecular genetic approaches have led to the identification of key genes, metabolites, protein candidates, and quantitative trait loci that are associated with enhanced mango fruit quality. The major pathways that determine the fruit quality include amino acid metabolism, plant hormone signaling, carbohydrate metabolism and transport, cell wall biosynthesis and degradation, flavonoid and anthocyanin biosynthesis, and carotenoid metabolism. Expression of the polygalacturonase, cutin synthase, pectin methyl esterase, pectate lyase, β-galactosidase, and ethylene biosynthesis enzymes are related to mango fruit ripening, flavor, firmness, softening, and other quality processes, while genes involved in the MAPK signaling pathway, heat shock proteins, hormone signaling, and phenylpropanoid biosynthesis are associated with diseases. Metabolomics identified volatiles, organic acids, amino acids, and various other compounds that determine the characteristic flavor and aroma of the mango fruit. Molecular markers differentiate the mango cultivars based on their geographical origins. Genetic linkage maps and quantitative trait loci studies identified regions in the genome that are associated with economically important traits. The review summarizes the applications of omics techniques and their potential applications toward understanding mango fruit physiology and their usefulness in future mango breeding.

Comparative genetic profiling of neuroblastoma at the onset vs recurrence or progression of the tumor and determination of the prognostic role of genetic alterations

Neuroblastoma (NB) is the most prevalent extracranial solid embryonal tumor in children, capable of both spontaneous regression and extremely aggressive course. The clinical heterogeneity of NB feeds the continual interest in its molecular genetic profiles as a potential key to the variable tumor behaviors and treatment personalization. To date, a number of studies indicate unfavorable prognostic roles of p53 and RAS–MAPK pathway activation, as well as that of telomere maintenance mechanisms. The increase in mutational load observed in recurrent NB has been shown to reduce the degree of subclonal heterogeneity, leading to pronounced distinction between genetic profiles of the tumor as recorded at primary and recurrent manifestations. This study aimed to analyze the alterations in genetic profiles of NB over time from the initial verification of the diagnosis till a disease-related adverse event (relapse or progression), as well as the potential prognostic significance of such alterations. The retrospective-prospective study enrolled 46 patients with morphologically verified peripheral neurogenic tumors, receiving treatment entirely or partially at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology from July 2013 till December 2021 in accordance with the modified NB-2004 protocol. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The cohort included an observation group of 25 patients, 1 intermediate-risk case and 20 high-risk cases. The main inclusion criterion concerned the availability of tumor tissue suitable for molecular examination, obtained both at the onset of the disease and at relapse/progression. We used the multiplex ligation-dependent probe amplification assay (MLPA) for the assessment of segmental and copy number chromosome aberrations, next-generation sequencing (NGS) to identify nucleotide variants, and reverse transcription polymerase chain reaction to assess the TERT gene expression levels. The statistical analysis used data available by April 01, 2022. In the studied cohort, the analysis confirmed prognostic contribution of MYCN amplifications (p < 0.001) detected at the onset of the disease or at its progression, as well as of 1p deletions (p = 0.01) detected in primary tumors. The expression of TERT in primary NBs was shown to correlate with the clinical course of the disease in the intermediate-risk and observation group, including local recurrence/progression (n = 3; –5 (–10.5…–3.5)), systemic relapses (n = 3; –2 (–2.5…–1)) and the lack of second adverse event (n = 20; –7 (–9…–5.5)), р = 0.037. Pairwise comparison of mutational profiles at the onset of the disease and at relapse revealed extreme degree of instability and significant variations: 38.5% (10/26) of the patients had no clinically significant genetic variants in the analyzed genomic region of interest in paired samples, 19.2% (5/26) of the patients had identical oncogenic variants in primary and relapsed tumor tissues, albeit with altered variant allele fraction (2/5), loss of one of the variants identified in the primary tumor (1/5), or acquisition of additional mutations upon recurrence (2/5), in 19.2% (5/26) of the patients, oncogenic genetic variants detected in primary NB were subsequently lost, in 7.7% (2/26) of patients, a loss of an oncogenic genetic variant identified at the onset on the disease was accompanied by the emergence of new clinically significant variants, and 15.4% (4/26) of the patients presented with new mutations in relapsed tumor tissue. Genetic variants leading to RAS–MAPK pathway activation and р53 pathway inhibition in primary tumors were identified in 31% (9/29) and 20.7% (6/29) of primary tumors and 29% (9/31) and 6.5% (2/31) of relapsed tumors, respectively. Risk group stratification based solely on cytogenetic aberrations fails to account for all cases of aggressive course of NB. Advanced molecular genetic approaches including MLPA and NGS, as well as the TERT mRNA quantitation, provide expanded characterization of the tumor biology and aggressiveness. Although prognostic markers are typically found in primary tumors, for NB, the use of such markers can be hampered by extremely high intratumoral heterogeneity. The data on acquisition or loss of prognostically significant molecular genetic markers in the course of the disease is useful for long-term prognosis and selection of targets for specific therapies.