Molecular Genetics Laboratory Research Articles

A Mitochondrial DNA-based Molecular Phylogenetics Study of the Mahalli Goat as a New Animal Genetic Resource in Southern Anatolia in Turkey

Background: The Karacadağ Mountains are of extinct volcanic origin and cover a large area in the triangle between Diyarbakır (Center, Nar and Ergani districts), Şanlılurfa (Siverek and Viranşehir districts) and Mardin (Derik district). Agricultural activities are limited due to the stony structure of the region; therefore, small animal husbandry is intensively practiced in the villages of the region. Mahalli goats, which are morphologically new domestic goats, are distinguished from other domestic goats by their spiral horn structure. They are not only resistant to cold weather conditions, but also resistant to diseases. Therefore, present study aimed to determine the genetic diversity and phylogenetic relationships of Mahalli goats in Diyarbakır region. Methods: The study was carried out at Şanlıurfa, Harran University of Agricultural Faculty, Laboratory of Animal Biotechnology and Molecular Genetics, in 2019-2020. Out of 24 Mahalli goats, forward and reverse primers were used to amplify the 598 bp region over the mtDNA D-loop region encompassing the 481 bp most variable region (HVR1). Analysis of goat D-loop sequences revealed 481 regions of 0.378 G+C, 97 polymorphic regions and 19 haplotypes. The phylogenetic analysis of Mahalli goats was carried out using molecular genetic techniques. Result: As a result of data analysis, both haplotype diversity (HD) and nucleotide diversity (ND) were calculated to be 1.000±0.0039 and 0.0375±0.00209, respectively. The genetic distance between the haplotypes of Mahalli goat ranged from 0.003 to 0.087 and the genetic distance between the haplotypes of Mahalli goat and other domestic goat breeds in Turkey ranged from 0.016 to 0.027. All 19 haplotypes used in this study were represented in the lineage A phylogenetic tree.

Expedited Exome Re-analysis following Deep Phenotyping and Muscle Biopsy in Suspected Mitochondrial Disorder

BackgroundExome sequencing (ES) is a useful tool in diagnosing suspected mitochondrial disease but can miss pathogenic variants for several reasons. Additional testing, such as muscle biopsy or biochemical testing, can be helpful in exome-negative cases. CaseWe report a patient who presented with repeated episodes of lactic acidosis and failure to thrive. ES and mitochondrial sequencing was initially negative but given clinical suspicion for mitochondrial disease was still high. After muscle biopsy showed evidence of mitochondrial dysfunction, the ES was re-analyzed and revealed novel variants in AARS2. ConclusionThis case demonstrates the importance of muscle biopsy and biochemical testing in evaluating patients with a high suspicion of mitochondrial disease, even in the genomics era. Closed-loop communication between molecular genetics laboratories and clinical geneticists an important step to help establish diagnosis in unsolved cases.

Phenotypic and Genotypic Characterization of Sheep Breeds in Diverse Habitats of Baluchistan Province through the Analysis of the vertnin Gene

This study was conducted to identify the vertnin gene in four different sheep breeds of Balochistan by studying their genetic characteristics and analyzing molecular markers. For this purpose, four different sheep breeds, Balochi, Rakhshani, Harnai, and Bibrik, were selected. A total of 10 animals from each breed were chosen to study the impact of breed and the vertnin gene on carcass features. The selection of animals was based on their phenotypic characteristics such as age and teeth. Blood samples of 10 ml were collected and transported to the Laboratory of Molecular Genetics at the Faculty of Veterinary and Animal Sciences, LUAWMS Uthal, Balochistan. Genomic DNA (gDNA) extraction was carried out using a modified inorganic method. The results showed that Balochi had a change in amino acid cytosine (C) > into thymine (T) mutation, Rakhshani had a change in guanine (G) > into adenine (A) mutation, Bibrik had a change in adenine (A) > into cytosine (C) mutation, and Harnai had a change in thymine (T) > into cytosine (C) mutation. The maximum divergence was 0.15715 bp in relation to the comparison in all studied sheep breeds. The phylogenetic results showed that the Balochi breed has 95–97% similarities with the other breeds when sequences were compared. The results revealed that the gel-electrophoresis bands of the Balochi breed were slightly higher, respectively. It was concluded that the vertnin gene exists in different breeds of sheep and might show a broad range of differences, even with similar DNA sequences. However, the purity based on the vertnin gene is at high risk, as a notable difference was found between the Balochi and Harnai sheep.

ClinGen variant curation expert panel recommendations for classification of variants in GAMT, GATM and SLC6A8 for cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are inherited metabolic phenotypes of creatine synthesis and transport. There are two enzyme deficiencies, guanidinoacetate methyltransferase (GAMT), encoded by GAMT and arginine-glycine amidinotransferase (AGAT), encoded by GATM, which are involved in the synthesis of creatine. After synthesis, creatine is taken up by a sodium-dependent membrane bound creatine transporter (CRTR), encoded by SLC6A8, into all organs. Creatine uptake is very important especially in high energy demanding organs such as the brain, and muscle. To classify the pathogenicity of variants in GAMT, GATM, and SLC6A8, we developed the CCDS Variant Curation Expert Panel (VCEP) in 2018, supported by The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded resource. We developed disease-specific variant classification guidelines for GAMT-, GATM-, and SLC6A8-related CCDS, adapted from the American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant interpretation guidelines. We applied specific variant classification guidelines to 30 pilot variants in each of the three genes that have variants associated with CCDS. Our CCDS VCEP was approved by the ClinGen Sequence Variant Interpretation Working Group (SVI WG) and Clinical Domain Oversight Committee in July 2022. We curated 181 variants including 72 variants in GAMT, 45 variants in GATM, and 64 variants in SLC6A8 and submitted these classifications to ClinVar, a public variant database supported by the National Center for Biotechnology Information. Missense variants were the most common variant type in all three genes. We submitted 32 new variants and reclassified 34 variants with conflicting interpretations. We report specific phenotype (PP4) using a points system based on the urine and plasma guanidinoacetate and creatine levels, brain magnetic resonance spectroscopy (MRS) creatine level, and enzyme activity or creatine uptake in fibroblasts ranging from PP4, PP4_Moderate and PP4_Strong. Our CCDS VCEP is one of the first panels applying disease specific variant classification algorithms for an X-linked disease. The availability of these guidelines and classifications can guide molecular genetics and genomic laboratories and health care providers to assess the molecular diagnosis of individuals with a CCDS phenotype.

Analysis of 9 microsatellite markers in Kalmyk cattle

A genetic analysis of the Kalmyk cattle population for 9 microsatellite loci: BM1824, BM 2113, INRA023, SPS 115, TGLA 122, TGLA 126, TGLA 227ETH 10, ETH 225 was carried out on the basis of the youth scientific laboratory of molecular genetics of the Kalmyk State University. 60 animals of the Kalmyk breed of breeding reproducer “Plodovitoe” of the Republic of Kalmykia were subjected to genetic testing. The loci INRA 023, TGLA 122, and TGLA 227 turned out to be the most informative for the Kalmyk breed. A comparison of the observed and expected heterozygosity in the Kalmyk breed shows that all 9 microsatellite loci showed an excess of the expected heterozygosity value over the observed one. The number of possible genotypes (Ng) has the highest value at the TGLA 122 locus, the indicator is 171, a smaller number of possible genotypes is observed at the TGLA 126 locus. Thus, microsatellite analysis showed a high level of genetic diversity in the studied herd of Kalmyk cattle. The data obtained indicate the possibility of using microsatellite DNA analysis for genetic examination of breeding animals by origin and analysis of pedigrees in order to study the transmission of traits by inheritance.

Polymorphism of the myostatin gene exon 1 using PCR-RFLP technique in five beef cattle in Indonesia

This study aims to analyze the polymorphism of the myostatin (MSTN) gene in exon 1 at SNPc.111G>C (rs523392653) and SNPc.267G>A (rs383271508) on various beef cattle in Indonesia. A total of 136 DNA samples were analyzed in this study, consisting of 33 heads of Madura, 31 heads of PO, 36 heads of Bali, 18 heads of Simmental, and 18 heads of Limousin cattle. The DNA sample was collected from the Animal Molecular Genetic Laboratory, Faculty of Animal Science, IPB University. Along 608 bp of DNA sequence was amplified using PCR with forward primer was 5′-CAA GTT GTC TCT CAG ACT GG-3′ and reverse primer was 5′-CTC CTC CTT ACA TAC AAG CC-3′ and annealing temperature was 59°C. The amplified DNA was then digested with HaeIII and AluI restriction enzymes using the RFLP method to determine polymorphism. Genotype frequencies, allele frequencies, PIC, and heterozygosity values were calculated with the PopGen32 program. The HaeIII enzyme produces two genotypes, GG and CG, while the AluI enzyme produces three: GG, AG, and AA. SNPc.111G>C polymorphism was found in Madura and PO cattle, while SNPc.267G>A polymorphism was found in Madura, PO, and Bali cattle. Both Simmental and Limousin cattle were monomorphic.

CHRONIC ALCOHOL-INDUCED ENCEPHALOPATHY: PREDICTORS OF COGNITIVE IMPAIRMENT AND FUNCTIONAL DISABILITY

Introduction: Predicting the risk of cognitive dysfunction and disability in everyday life among chronic alcohol-induced encephalopathy (CAIE) patients allows timely treatment to prevent dementia.
 The aim: to develop a mathematical model for predicting the risk of developing cognitive disorders and functional disability in patients with CAIE based on the analysis of polymorphic variants of the genes ACE, AT2R1, eNOS, PON1, IL-1β, IL-10, TNF-α, as well as cofactors (gender, age group, the disease duration, and presence/absence of somatic comorbidity).
 Materials and methods: We examined the 102 patients with CAIE who were undergoing inpatient treatment in the communal non-profit enterprise «Ternopil Regional Clinical Psychoneurological Hospital» during 2021-2022 and were included in the retrospective analysis. The molecular-genetic testing was performed for 26 patients of the general group. The molecular-genetic differentiation of the studied polymorphic variants of genes was carried out in the molecular-genetic laboratory of the state institution «Reference Centre for Molecular Diagnostics of the Ministry of Health of Ukraine», Kyiv. Cognitive functions were assessed using the Montreal Cognitive Test (MoCA); activities of daily living were assessed using the Barthel index. A logistic regression analysis was performed to determine the risk of cognitive impairment and functional disability developing in patients with CAIE.
 Results: Analyzing polymorphic variants of the ACE, AT2R1, eNOS, PON1, IL-1β, IL-10, TNF-α genes, as well as such cofactors as gender, age group, history and presence/absence of somatic comorbidity in the context of the development of cognitive disorders in patients with CAIE it has been established that the C108T polymorphism of the PON1 gene has the most significant prognostic value (in the presence of the T/T genotype, the probability of cognitive impairment is 39.84 %). At the same time, carriage of both the T allele and the C allele of the PON1 gene is associated with a significant decrease in the MoCA score. Regarding the development of functional disability in patients with CAIE, the C108T polymorphism of the PON1 gene also has the most significant prognostic value (in the presence of the T/T genotype, the probability of functional disability is 48.08 %, respectively, C/C – 30.96 % and CT – 39.22 %, however, no statistically significant differences in the Barthel index values were found in carriers of the above genotypes).
 Conclusions: It was established that the C108T polymorphism of the PON1 gene is significantly associated with the development of cognitive impairment and functional disability in patients with CAIE, which indicates the feasibility of further studies involving a larger sample of patients.

Recommendations for laboratory workflow that better support centralised amalgamation of genomic variant data: findings from CanVIG-UK national molecular laboratory survey.

National and international amalgamation of genomic data offers opportunity for research and audit, including analyses enabling improved classification of variants of uncertain significance. Review of individual-level data from National Health Service (NHS) testing of cancer susceptibility genes (2002-2023) submitted to the National Disease Registration Service revealed heterogeneity across participating laboratories regarding (1) the structure, quality and completeness of submitted data, and (2) the ease with which that data could be assembled locally for submission. In May 2023, we undertook a closed online survey of 51 clinical scientists who provided consensus responses representing all 17 of 17 NHS molecular genetic laboratories in England and Wales which undertake NHS diagnostic analyses of cancer susceptibility genes. The survey included 18 questions relating to 'next-generation sequencing workflow' (11), 'variant classification' (3) and 'phenotypical context' (4). Widely differing processes were reported for transfer of variant data into their local LIMS (Laboratory Information Management System), for the formatting in which the variants are stored in the LIMS and which classes of variants are retained in the local LIMS. Differing local provisions and workflow for variant classifications were also reported, including the resources provided and the mechanisms by which classifications are stored. The survey responses illustrate heterogeneous laboratory workflow for preparation of genomic variant data from local LIMS for centralised submission. Workflow is often labour-intensive and inefficient, involving multiple manual steps which introduce opportunities for error. These survey findings and adoption of the concomitant recommendations may support improvement in laboratory dataflows, better facilitating submission of data for central amalgamation.

THE STUDY OF ASSOCIATIONS BETWEEN IL1Β C3953T GENE POLYMORPHISM AND CLINICAL-NEUROLOGICAL, NEUROIMAGING, HEMODYNAMIC CHARACTERISTICS AND COGNITIVE DYSFUNCTION IN PATIENTS WITH POST-INFECTIOUS ENCEPHALOPATHY

The aim: To investigate potential associations between the C3953T polymorphic variant of the interleukin-one beta (IL1β) gene and clinical-neurological, neuroimaging, hemodynamic characteristics, as well as cognitive dysfunction in patients with post-infectious encephalopathy (PIE).
 Materials and methods: A total of 128 patients with post-infectious encephalopathy (PIE) who were receiving inpatient treatment in the neurological departments of the Communal Non-commercial Enterprise “Ternopil Regional Clinical Psychoneurological Hospital” of Ternopil Regional Council,” Ternopil, Ukraine, were examined and included in the retrospective analysis in 2021–2022. The molecular-genetic testing was performed for 26 patients in the molecular genetics laboratory of the State Institution “Reference Centre for Molecular Diagnostics of the Ministry of Health of Ukraine,” Kyiv. The control group consisted of 12 people, who were representative in age and gender. Statistical processing of the results was performed using the STATISTICA 10.0 software.
 Results: Analysis of the dependence of neuroimaging changes on the frequency of genotypes of the C3953T polymorphic variant of the IL1β gene in patients with PIE showed a significant relationship between their frequency distribution and the presence/absence of gliosis phenomena (p=0.009). Thus, gliosis was detected in all carriers of the T/T genotype C3953T polymorphic variant of the IL1β gene. Analyzing the dependence of changes obtained during the transcranial Doppler ultrasound scanning of cerebral vessels on the polymorphic variant C3953T of the IL1β gene in patients with PIE, all carriers of the T/T genotype were diagnosed with angiospasm (p=0.038) and vertebrobasilar insufficiency (p=0.010).
 Conclusions: Results suggest the reasonability of further researching the interaction between IL1β and glial cells and changes in the cognitive functioning of cytokine genotypes with larger sample sizes that may help explain the pathophysiological mechanisms leading to cognitive impairment in patients with PIE.

Development and validation of a test system for determining the polymorphism in the DGKH and PPP1R1C genes associated with body weight of sheep

Relevance. National sheep breeding is lagging behind other branches of animal husbandry considering using of modern DNA technologies. The search for new genes — potential candidates associated with economically significant traits — is relevant for a more complete disclosure of the genetic potential of sheep. An earlier genome-wide association study showed that the DGKH and PPP1R1C genes had a certain effect on body weight of sheep at birth and at the age of 90 days. In this regard, a more detailed study of polymorphism in the DGKH and PPP1R1C genes can deepen understanding of growth and development processes in domestic sheep, so we have chosen these genes as targets for the experiment.Methods. Primers and probes were selected for amplification of a fragment with targeted SNPs in the DGKH and PPP1R1C genes with a length of 68 nucleotide pairs based on the reference DNA sequence on the 10th (NC_056063.1) and the 2nd chromosome (NC_056055.1) sheep represented in NCBI. To determine polymorphism, real-time PCR-based test systems were developed. Genotypes were determined using a multiparametric graph. The test systems were tested on 147 sheep of the southern meat breed.Results. The developed test systems for promising DGKH and PPP1R1C genes allowed to clearly determine sheep genotypes based on using the PCR-RT. All three genotypes (T/T, C/T, and C/C) were identified in the DGKH gene. No homozygous genotype (T/T) was identified in the PPP1R1C gene in the studied population. The test systems are suitable for routine DNA analysis in molecular genetic laboratories.

Implementing Next-Generation Sequencing Process Changes to Increase Capacity and Improve Timeliness of Molecular Biomarker Profiling for Lung Cancer Patients.

Faced with expansion of molecular tumor biomarker profiling, the molecular genetics laboratory at Kingston Health Science Centre experienced significant pressures to maintain the provincially mandated 2-week turnaround time (TAT) for lung cancer (LC) patients. We used quality improvement methodology to identify opportunities for improved efficiencies and report the impact of the initiative. We set a target of reducing average TAT from accessioning to clinical molecular lab report for LC patients. Process measures included percentage of cases reaching TAT within target and number of cases. We developed a value stream map and used lean methodology to identify baseline inefficiencies. Plan-Do-Study-Act cycles were implemented to streamline, standardize, and automate laboratory workflows. Statistical process control (SPC) charts assessed for significance by special cause variation. A total of 257 LC cases were included (39 baseline January-May 2021; 218 post-expansion of testing June 2021). The average time for baseline TAT was 12.8 days, peaking at 23.4 days after expansion of testing, and improved to 13.9 days following improvement interventions, demonstrating statistical significance by special cause variation (nonrandom variation) on SPC charts. The implementation of standardized manual and automated laboratory processes improved timeliness of biomarker reporting despite the increasing volume of testing at our center.

Appetite- and Weight-Regulating Neuroendocrine Circuitry in Hypothalamic Obesity.

Since hypothalamic obesity (HyOb) was first described over 120 years ago by Joseph Babinski and Alfred Fröhlich, advances in molecular genetic laboratory techniques have allowed us to elucidate various components of the intricate neurocircuitry governing appetite and weight regulation connecting the hypothalamus, pituitary gland, brainstem, adipose tissue, pancreas, and gastrointestinal tract. On a background of an increasing prevalence of population-level common obesity, the number of survivors of congenital (eg, septo-optic dysplasia, Prader-Willi syndrome) and acquired (eg, central nervous system tumors) hypothalamic disorders is increasing, thanks to earlier diagnosis and management as well as better oncological therapies. Although to date the discovery of several appetite-regulating peptides has led to the development of a range of targeted molecular therapies for monogenic obesity syndromes, outside of these disorders these discoveries have not translated into the development of efficacious treatments for other forms of HyOb. This review aims to summarize our current understanding of the neuroendocrine physiology of appetite and weight regulation, and explore our current understanding of the pathophysiology of HyOb.

Relationship between the SLC27 gene's polymorphism and some broiler carcass and economic factors

This study was conducted at the poultry field, College of Agriculture and the Marshes, University of Thi-Qar from 10/11/2021 to 26/12/2021, and the Molecular Genetics Laboratories at the Marsh Research Center to determine the FATP gene polymorphism on some productive and physiological traits of broilers of ROSS 308. A total of 150 birds were used. The results of this study showed the following. Three genotypes were identified in the sequence of nitrogenous bases in the presence of the mutation G237A: GG, GA, and AA. It was noted that there were significant differences in the distribution ratios of the genotypes of the FATP gene according to the mutation G237A, where the genotype GA recorded the highest percentage, followed by genotype GG and then genotype AA. The G allele frequency is superior to the A. It was noted that there were no significant differences for the genotypes of the G237A mutation on the body weight and no significant differences between the GA, GG and AA genotypes of the FATP gene on the body weight, a significant difference in the genotypes of the G237A mutation on carcass weight, AA genotype outperformed the GG and GA genotypes, the AA genotype was superior to the GA and GG genotypes on wings relative weight. Keywords: Polymorphism SLC27 gene, economic, carcass, broiler.

SSR marker based profiling and population structure analysis in peach (Prunus persica) germplasm

For breeding programmes to be successful and for germplasm conservation, it is essential to characterize and analyze the genetic diversity of available germplasm. The present experiment was conducted at Molecular Biology and Genetic Engineering Laboratory of Uttarakhand Council for Biotechnology, Haldi, Uttarakhand during 2022 to study the molecular profile of 41 peach [Prunus persica (L.) Stokes] accessions using 23 polymorphic SSR markers. The number of alleles detected ranged from 3 to 8 with an average of 4.65 alleles per locus (Na) and a total of 107 alleles were amplified. The average effective number of alleles (Ne) were 2.89 per marker. The SSR marker MA015a produced maximum number of 8 alleles followed by BPPCT 015 and CPPCT14 which produced 7 alleles each. The polymorphic information content (PIC) varied between 0.317–0.836 with a mean value of 0.563. The observed heterozygosity examined was lower (Ho = 0.02) and the expected heterozygosity (He = 0.61) ranged between 0.34 to 0.85. The presence of a higher Shannon’s information index (I) of 1.17 indicates higher diversity in the given set of peach genotypes. Jaccard’s similarity coefficient ranging from 0.533 to 1, indicated a pair-wise relationship among the peach accessions. The cluster dendrogram partitioned the accessions into two main clusters. However, the total accessions were stratified into 3 groups (K=3) based on population structure analysis which was further confirmed by Principal Coordinate Analysis (PCoA). The information generated in the study may have great implications in molecular characterization, fingerprinting and documentation of accessions in the peach improvement programme.

Assessment of genetic divergence in wheat lines (Triticum aestivum L.) involving biochemical and protein markers in rainfed conditions

Wheat (Triticum aestivum L.) is the important and strategic cereal crop for the majority of world’s populations. It is significant staple food of about two billion people. In next few years, world demand for wheat is expected to be 40 percent higher than that of its level today. Keeping in view the importance of the crop research work was conducted in the laboratory of Plant Breeding and Molecular Genetics, University of Poonch Rawalakot, AJK, Pakistan. The aim of the research was to find out the genetic diversity of different wheat lines. In the experiment, 50 different lines of wheat species (Triticum aestivum L.) was used to detect genetic diversity by utilizing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and biochemical analysis. On the basis of biochemical analysis lines 3111 and 3123 was diverse among 50 lines for antioxidant activity by using DPPH radical and 3135 and 3139 for phenolic contents and for flavonoid 3148 and 3107 was found more promising. Molecular characterization by SDS PAGE showed diversity in three wheat lines 3136, 3138 and 3110. These wheat lines could be our potential lines for future wheat improvement program as they were also promising regarding to the high yields.

Effect of Moringa Leave Ethanol Extract on Accelerating Wound Healing Process

TGF-β and VEGF are vital in cell proliferation and regeneration, as evidenced in processes like wound healing. The leaves of Moringa oleifera Lam exhibit anti-inflammatory and cell regenerative properties that may facilitate the transition from the inflammatory to the proliferative phase, enhancing wound repair. This research sought to discern the potential of orally administered moringa leaf extract in augmenting systemic wound healing, focusing on TGF-β and VEGF serum as in vivo molecular markers. This research was conducted at the Animal Laboratory of the Faculty of Medicine, Universitas Padjadjaran, and the Laboratory of Molecular Genetics, Universitas Padjadjaran, from January to March 2022. We divided thirty Swiss Webster mice into two categories: healthy and wound-treated. Wounded mice received 100 mg/kgBW Na CMC as a negative control, 500 mg/kgBW zinc syrup as a positive control, and M. oleifera leaves ethanol extract (MOLE) in doses of 280 and 560 mg/kgBW orally from day 3 to day 6. Wound progression was documented and measured on days 0, -1, -3, and -6. Day-6 blood samples were obtained, and TGF-β and VEGF serum levels were gauged using ELISA. Results from day 6 revealed that wound coverage in the 280 and 560 mg/kgBW MOLE groups was 13.76±5% and 13.38±4%, respectively. These percentages notably surpassed that of the negative control group (p=0.005). However, the TGF-β and VEGF serum levels in the MOLE-treated groups did not differ significantly from the negative control (p=0.081 and p=0.149, respectively). Thus, the study concludes that while MOLE expedites wound closure, it does so without the systemic involvement of TGF-β and VEGF in vivo.

The study of Associations between TNFα GENE G308A polymorphism and clinical-neurological, neuroimaging, hemodynamic characteristics and cognitive dysfunction in patients with post-infectious encephalopathy

Introduction. Infectious diseases can affect brain function and cause the development of encephalopathy, even if the pathogen does not directly affect the central nervous system. Infections caused by viruses, bacteria, or parasites can lead to a secondary inflammatory response in the brain, commonly known as neuroinflammation, through the action of inflammatory mediators that affect the brain endothelium and parenchyma, and the response of brain cells to these mediators. Neurological consequences associated with infectious diseases are poorly understood. Nowadays, there is no established strategy for the treatment or prevention of neurological damage associated with peripheral infections. Aim of study was: to establish probable associations of the G308A polymorphic variant of the TNFα gene with clinical-neurological, neuroimaging, hemodynamic characteristics and cognitive dysfunction in patients with post-infectious encephalopathy. Material and methods. 128 patients with PIE who were undergoing treatment in the neurological departments of the communal non-profit enterprise "Ternopil Regional Clinical Psychoneurological Hospital" during 2021-2022 were examined. 26 patients underwent molecular genetic analysis. The control group consisted of 12 practically healthy persons, representative in terms of age and sex. All patients met the inclusion criteria for the study. Neuroimaging was performed using multispiral computed tomography (CT) or magnetic resonance imaging (MRI). The state of cerebral blood flow was studied using transcranial duplex scanning (TCI) of intracranial vessels and extracranial brachiocephalic vessels on a Philips HDI device. Research in the cognitive sphere was carried out using the Montreal Cognitive Test (The Montreal Cognitive Assessment, MoCA). The molecular genetic study of the G308A polymorphic variant of the TNFα gene was carried out according to standard protocols developed in the molecular genetic laboratory of the state institution "Reference Center for Molecular Diagnostics of the Ministry of Health of Ukraine". The results. Analyzing the dependence of clinical-neurological syndromes, neuroimaging, hemodynamic characteristics, and cognitive dysfunction on the polymorphic variant G308A of the TNFα gene in patients with PIE, probable differences in the distribution of genotype frequencies were established only for clinical-neurological syndromes (cephalic syndrome, p=0.005 and movement disorder syndrome, p =0.038) and neuroimaging changes (gliosis phenomenon, p=0.026). Regarding the frequency distribution of alleles of the G308A polymorphic variant of the TNFα gene in patients with PIE, a probable predominance of carriers of the A allele among persons with cephalic syndrome compared to persons without cephalic syndrome was found (91.67% vs. 8.33%). Conclusions. Thus, the allelic polymorphism of the TNFα gene affects the course of PIE, which determines the expediency of further research.

Molecular genetic characterization of sudden deaths due to thoracic aortic dissection or rupture

BackgroundSudden deaths due to thoracic aortic dissection or rupture (TADR) are often investigated by forensic pathologists in the United States. Up to a quarter of reported TADR result from a highly penetrant autosomal dominant single gene variant. Testing genes associated with familial TADR provides an underlying etiology for the cause of death and informs effective sudden death prevention for at-risk family members. At the New York City Office of Chief Medical Examiner (NYC-OCME), TADR cases are routinely tested by the in-house, CAP-accredited Molecular Genetics Laboratory. In this retrospective study, TADR and cardiovascular cases were reviewed to understand the burden of TADR in sudden deaths, value of molecular diagnostic testing in TADR, and genotype-phenotype correlations in a demographically diverse TADR cohort. MethodsBetween July 2019 and June 2022, cases with in-house cardiovascular genetic testing at NYC-OCME were retrospectively reviewed. Twenty genes associated with familial TADR were analyzed using high throughput massive parallel sequencing on postmortem tissues or bloodspot cards. Variant interpretation was conducted according to ACMG/AMP guidelines. ResultsA total of 1078 cases were tested for cardiovascular genetic conditions, of which 34 (3%) had TADR. Eight of those TADR cases had a pathogenic or likely pathogenic variant (P/LPV), 4 had a variant of uncertain significance (VUS), and 22 cases were negative for variants in TADR genes. The molecular diagnostic yield using the TADR subpanel was 23.5%. The genes with the greatest prevalence of P/LPV were FBN1 (6), followed by TGFBR2 (2), TGFBR1 (1), and MYLK (1). Highly penetrant P/LPV in TGFBR2, FBN1, and TGFBR1 were found in TADR individuals who died younger than 34 years old. Two P/LPV in FBN1 were secondary findings unrelated to cause of death. P/LPV in FBN1 included five truncating variants located in the N-terminal domains and one missense variant involved in the disulfide bonds of the EGF-like domain. All P/LPV in TGFBR1 and TGFBR2 were missense or in-frame deletion variants located in the protein kinase catalytic domain. Three variants were first reported in this study. ConclusionsMolecular testing of familial TADR-associated genes is a highly effective tool to identify the genetic cause of TADR sudden deaths and benefits surviving at-risk families.

Актуальность внедрения референсного подхода в патологоанатомическую практику

The project to establish the reference center for addressing scientific-practice, organizational and methodological challenges of immunohistochemical, pathomorphological and molecular genetic laboratories and centers of data analysis and interpretation of radiological methods started in 2020 within the Department of fundamental pathology of Endocrinology research center. The present review focuses on the prospects of a concept of reference center with an emphasis on the current state of Russian pathology service.

Impact of Arg72Pro polymorphism in P53 gene in cancer susceptibility in Algerian population

TP53 is a tumor suppressor gene involved in the development of several types of cancer. It has been reported that single nucleotide polymorphism in codon 72 of this gene, causing substitution of a Proline for an Arginine, may increase the risk of carcinogenesis. The objective of this study was to assess the frequency of the Arg72Pro polymorphism and to look for a possible association between this polymorphism and the occurrence of cancer. Our study concerned 82 DNA samples collected from the DNA bank of the Laboratory of Biology and Molecular Genetics of the Constantine University 3, and grouped 2 populations: 41 controls and 41 patients with one of three types of cancer: breast cancer, cervical cancer and lung cancer. This polymorphism was detected with PCR / RFLP. The results obtained in our study revealed a significant difference between the two patient and control groups for the heterozygous CG genotype (OR = 0.13, P = 0.001), which makes it possible to conclude that the CG genotype constitutes a risk factor in the occurrence cancer. However, the study showed that there is no difference between the Pro allele and the Arg allele in the patients compared to the controls (OR = 0.95, P = 0.87), which could rule out the possible association between the Arg72Pro polymorphism and the onset of cancer.