Restriction Fragment Research Articles

Novel Insights into Hb Shaare Zedek Associated with β0-Thalassemia: Molecular Characteristics, Genetic Origin and Diagnostic Approaches.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Molecular typing methods to characterize Brucella spp. from animals: A review.

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-ladder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages.

Associations of ACE I/D and AGTR1 rs5182 polymorphisms with diabetes and their effects on lipids in an elderly Chinese population

BackgroundDiabetes mellitus is generally accompanied by dyslipidaemia, but inconsistent relationships between lipid profiles and diabetes are noted. Moreover, genetic variations in insertion/deletion (I/D) polymorphisms at angiotensin-converting enzyme gene (ACE) and T/C polymorphisms in the angiotensin type 1 receptor gene (AGTR1) are related to diabetes and lipid levels, but the associations are controversial. Thus, the current research aimed to explore the effects of ACE I/D, AGTR1 rs5182 and diabetes mellitus on serum lipid profiles in 385 Chinese participants with an average age of 75.01 years.MethodsThe ACE I/D variant was identified using the polymerase chain reaction (PCR) method, whereas the AGTR1 rs5182 polymorphism was identified using the PCR-based restriction fragment length polymorphism (PCR-RFLP) method and verified with DNA sequencing. Total cholesterol (TC), triglyceride (TG), apolipoprotein A (ApoA), apolipoprotein B (ApoB), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured using routine methods, and the lipid ratios were calculated.ResultsACE I/D, but not AGTR1 rs5182, was a predictor of TG/HDL-C for the whole study population. Both ACE I/D and AGTR1 rs5182 were predictors of HDL-C and LDL-C levels in females but not in males. Moreover, in females, diabetes mellitus and ACE I/D were identified as predictors of TG and TG/HDL-C, whereas AGTR1 rs5182 and diabetes mellitus were predictors of TG/HDL-C. Moreover, diabetes mellitus and the combination of ACE I/D and AGTR1 rs5182 variations were predictors of TG and TG/HDL-C exclusively in females.ConclusionsThe results demonstrated the potential for gender-dependent interactions of ACE I/D, AGTR1 rs5182, and diabetes on lipid profiles. These findings may serve as an additional explanation for the inconsistent changes of blood lipids in individuals with diabetes mellitus, thereby offering a novel perspective for the clinical management of blood lipid levels in diabetic patients.

A rapid quarantine approach for the pathogenic and invasive Phytophthora species associated with imported fruits in China.

Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor-saving techniques are necessary and anticipated. A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR-RFLP-CE) technology-based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR-RFLP-CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. We provide a quick, practical, and high-throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry.

Genetic diversity and antifungal susceptibilities of environmental Cryptococcus neoformans and Cryptococcus gattii species complexes

Cryptococcosis is an opportunistic systemic mycosis caused by Cryptococcus neoformans and C. gattii species complexes and is of increasing global importance. Maintaining continued surveillance of the antifungal susceptibility of environmental C. neoformans and C. gattii isolates is desirable for better managing cryptococcosis by identifying resistant isolates and revealing the emergence of intrinsically resistant species. Relevant research data from Egypt are scarce. Thus, this study aimed to report the genetic diversity of C. neoformans and C. gattii species complexes originating from different environmental sources in Egypt, antifungal susceptibility profiles, antifungal combinations, and correlations of susceptibility with genotypes. A total of 400 environmental samples were collected, 220 from birds and 180 from trees. Cryptococcus spp. were found in 58 (14.5%) of the samples, 44 (75.9%) of the isolates were recovered from birds and 14 (24.1%) from trees. These isolates were genotyped using M13 polymerase chain reaction-fingerprinting and URA5 gene restriction fragment length polymorphism analysis. Of the 31 C. neoformans isolates, 24 (77.4%), 6 (19.4%) and one (4.4%) belonged to VNI, VNII, and VNIII genotypes, respectively. The 27 C. gattii isolates belonged to VGI (70.4%), VGII (18.5%), and VGIII (11.1%) genotypes. Non-wild type C. neoformans and C. gattii isolates that may have acquired resistance to azoles, amphotericin B (AMB), and terbinafine (TRB) were observed. C. gattii VGIII was less susceptible to fluconazole (FCZ) and itraconazole (ITZ) than VGI and VGII. C. neoformans isolates showed higher minimum inhibitory concentrations (MICs) to FCZ, ITZ, and voriconazole (VRZ) than those of C. gattii VGI and VGII. Significant (P < 0.001) correlations were found between the MICs of VRZ and ITZ (r = 0.64) in both C. neoformans and C. gattii isolates, FCZ and TRB in C. neoformans isolates, and FCZ and TRB (r = 0.52) in C. gattii isolates.There is no significant differences in the MICs of TRB in combination with FCZ (P = 0.064) or in combination with AMB (P = 0.543) and that of TRB alone against C. gattii genotypes. By calculating the fractional inhibitory concentration (FIC) index, the combination of FCZ + AMB was synergistic against all tested genotypes. These findings expand our knowledge of ecological niches, genetic diversity, and resistance traits of C. neoformans and C. gattii genotypes in Egypt. Further investigations into how they are related to clinical isolates in the region are warranted.

&lt;i&gt;IGF1R&lt;/i&gt; rs907806 and &lt;i&gt;GHSR&lt;/i&gt; rs572169 genetic variants in fetal macrosomia

BACKGROUND: Being strongly affected by maternal and fetal genetic factors, intrauterine environment plays a critical role in fetal growth. Intrauterine conditions, in turn, are largely determined by the genetic factors. AIM: The aim of this study was to evaluate the effect of the IGF1R rs907806 and GHSR rs572169 genetic variants on the weight of newborns. MATERIALS AND METHODS: This prospective study included 221 mother-newborn pairs. The inclusion criteria were singleton pregnancy and informed consent to participate in the study. The exclusion criteria were severe somatic, oncological and acute illnesses three months prior or during pregnancy, gestational diabetes mellitus, refusal to participate in the study at any stage, and insufficient data. The study groups included patients with type 1 diabetes mellitus (group I), type 2 diabetes mellitus (group II), and no carbohydrate metabolism disorders (group III). Cord blood samples were obtained after delivery. The IGF1R rs907806 and GHSR rs572169 genotypes were determined by polymerase chain reaction with restriction fragment length polymorphism analysis. The main outcomes were delivery of a large for gestational age fetus (weight of more than the 90th percentile INTERGROWTH-21st). The secondary outcomes were diabetic fetopathy, diabetic cardiomyopathy, and neonatal hypoglycemia. RESULTS: In group III, the minor G allele of the IGF1R rs907806 variant was more frequent in large-for-gestational-age newborns (p = 0.017; odds ratio 3.039; 95% confidence interval 1.244–7.424) than in those with a weight of less than 90th percentile. For the GHSR rs572169 variant, no similar trend was observed. This pattern was pronounced in the male newborns only (rs907806 AA vs. AG+GG; p = 0.046; odds ratio 4.229, 95% confidence interval 1.181–15.139). In newborns with hypoglycemia, a higher frequency of the GG genotype of the GHSR rs572169 (GA) variant was observed in the total sample (p = 0.004) and the type 1 diabetes mellitus group (p = 0.0496). CONCLUSIONS: The IGF1R rs907806 variant may affect birthweight in the male newborns of mothers without carbohydrate metabolism disorders. The observed association of neonatal hypoglycemia with the GG genotype of the GHSR rs572169 variant requires further investigation. Genetic factors can contribute to prognostic models of perinatal complications, while improving their early diagnosis opportunities.

Methodological approaches to gene identification of tea raw materials and raw material composition of tea-based soft drinks

Tea or tea shrub is a plant of the Camellia sinensis species, the leaves of which, previously prepared in a special way, are the traditional raw material for the production of tea products. Varietal gene identification of tea allows us to increase the level of assessment of the authenticity of tea raw materials and tea products. It is predominantly based on DNA technologies for the detection and interpretation of SNP markers (Single Nucleotide Polymorphism), represented by a wide arsenal of both expensive high-tech methods and publicly available laboratory approaches. Species gene identification of the raw material composition of tea-based soft drinks is an equally important area of research due to the risk of falsification of this type of product. The purpose of this study was to find methodological approaches to the varietal gene identification of tea raw materials and to the species gene identification of the raw material composition of tea-based soft drinks. As a result of a bioinformatics study to identify polymorphic restriction sites in the nucleotide sequences of Camellia sinensis genome loci, diagnostically significant restriction enzymes were selected that were capable of detecting SNPs and identifying tea genotypes using the analyzed markers. At the same time, 16 loci had potential for practical application, of which 11 belonged to the group of the most informative SNP markers. A post-analytical assessment of tea varieties was carried out with them regarding their genotypic affiliation and identifiability as part of solving the first task of the study. To achieve the second task, a molecular genetic approach to the species identification of the raw composition of soft drinks based on green tea was tested. The study included the analysis of experimental drinks (with natural flavoring “Lemon” and synthetic flavoring “Peach 716”), as well as commercial concentrates “TIAKVA” (based on extracts from the coarse stems of green or black tea). The methods used in the work were PCR (Polymerase Chain Reaction), RFLP (Restriction Fragment Length Polymorphism) and direct sequencing of the amplified chloroplast DNA locus. The combination of two methods (PCR and sequencing) showed its effectiveness in establishing the belonging of the analyzed nucleic acid samples to the Camellia sinensis species, the raw material base of the studied drinks and concentrates. However, to unlock the authentication potential of PCR with primers #1 and #2 combined with RFLP analysis, it will be necessary to select diagnostically significant restriction enzymes suitable for generating species-specific combinations of PCR-RFLP profiles of marker sequence.

Clinico-mutational profile and the impact of splenectomy in children with glucose-6-phosphate isomerase deficiency

BackgroundThere is a lack of literature on the role of splenectomy in hemolytic anemias due to glucose-6-phosphate isomerase (GPI) deficiency. GPI deficiency is a rare red blood cell (RBC) enzymopathy of the glycolytic pathway. Most present with severe disease requiring frequent transfusions. In this study, we report the effect of splenectomy in the world's largest cohort of GPI deficiency patients. The study aimed to describe the clinical, mutational, and laboratory parameters of patients with GPI deficiency. A comparison of the transfusion requirement pre- and post-splenectomy is also included. Material and methodsThe ambispective study was performed from 2017 to 2023. Patients with congenital non-spherocytic hemolytic anemia (CNSHA) were screened for GPI deficiency. Detailed history, including demographic, clinical data, and transfusion details, were noted. Hematological parameters and RBC enzyme activity were estimated using spectrophotometry. The genetic study was done using restriction fragment length polymorphism, and confirmation was obtained through Sanger's sequencing. Patients were followed up after splenectomy. ResultsEighteen patients were diagnosed with GPI deficiency. About 3/4th (14/18; 77.7 %) had significant hepatosplenomegaly. Median serum ferritin levels were 890 ng/ml. Seven patients were on oral iron chelation. The nutritional status assessed as per the Indian Academy of Pediatrics growth charts revealed significant growth retardation. All the patients had severe anemia (mean Hb: 6.4 g/dl) and macrocytosis (mean MCV: 129.2 fl). Laboratory features of hemolysis were evident with reticulocytosis, raised serum lactate dehydrogenase, and indirect bilirubin. The mean GPI enzyme activity was 28.75 IU/g Hb.Ten (55.5 %) patients underwent splenectomy at a median age of 7 years. Five remain transfusion-free post-splenectomy at a median follow-up of 54 months. Other 5 had a significant reduction in transfusion requirement post-splenectomy (p < 0.05). Post-splenectomy, the mean hemoglobin levels increased from 6.2 to 8.1 gm%, and the mean reticulocyte counts reduced from 7.4 % to 4.8 %. Fifteen of 18 had the pathogenic c.1040G > A(p.Arg347His) homozygous mutation. ConclusionThe study demonstrates the benefit of splenectomy in patients with GPI deficiency.

Role of Telomere Length in Radiation Response of Hematopoietic Stem & Progenitor Cells in Newborns

Objective Wide inter-individual variations in ionizing radiation (IR) responses of neonatal hematopoietic system calls for identifying reliable biomarkers to effectively estimate radiation exposure damages in neonates. Methods Association between telomere length (TL) at birth and radiation sensitivity of cord blood hematopoietic stem cells (HSC) from 166 healthy newborns were investigated by assessing their clonogenic differentiation. TL was determined as terminal restriction fragment (TRF) by Southern blot method. Results TL correlated with surviving fractions of total progenitor colony forming cell (CFC) content at 0.75 Gy (p < 0.05), granulo-macrophagic lineage colony forming units (CFU-GM) at 0.75 Gy (p < 0.05) and erythroid burst forming unit (BFU-E) at 0.75 Gy (p < 0.05) & at 3 Gy (p < 0.05) of newborns. Conclusion Our results indicate risks for HSC clonogenic survival in neonates with shorter telomeres after IR exposure. These observations might aid in considering TL at birth as an assessment factor for radiation related hematopoietic challenges in children.

Association of the -c.108C>T and c.192Q>R polymorphisms of the PON1 gene with preeclampsia among Chinese women

To assess the association of -c.108C>T and c.192Q>R polymorphisms of paraoxonase 1 (PON1) gene with preeclampsia (PE) and the influence of genotypes on the metabolic and oxidative stress indexes among Chinese women. This case-control study has included 334 patients with PE and 1337 healthy pregnant women. The -c.108C>T and c.192Q>R genotypes were determined by PCR and restriction fragment length polymorphism method. Metabolic and oxidative stress parameters were also analyzed. No statistical difference in the genotypic and allelic frequencies for the -c.108C>T and c.192Q>R polymorphisms of the PON1 gene was found between the PE patients and the healthy controls (P > 0.05). Nevertheless, the 192Q-108T haplotype of these polymorphisms was associated with an increased risk of PE (P = 0.007). Total antioxidant capacity (TAC) and atherosderosis index were higher in patients with the -108TT genotype compared with those with a CT genotype (P < 0.05); whilst total oxidant status was lower in patients with a CT genotype compared with those with a CC genotype (P = 0.036). Malondialdehyde level was higher in patients with a 192RR genotype compared with those with a QQ genotype (P = 0.019). TAC level was higher in patients with a RR genotype compared with those with a QR genotype (P = 0.015). The 192Q-108T haplotype of the PON1 gene is associated with the risk for PE. These polymorphisms may be associated with abnormal lipid metabolism and oxidative stress among Chinese PE patients.

ACE I/D and A2350G Polymorphisms are Correlated with Body Mass Index, but Not with Body Weight and Essential Hypertension: Study in Javanese Postmenopausal Women

Background: Genetics was one of the risk factors for essential hypertension (EH). Research on ACE I/D and A2350G polymorphisms associated with risk factors for hypertension in Indonesia has never been done. Therefore, this study was conducted to analyze the relationship between the genotype and alleles of this gene with EH, body weight, and body mass index (BMI) in Javanese postmenopausal women.Materials and methods: This cross-sectional study involved 69 postmenopausal Javanese women according to several criteria related with hypertension risk factors. The data were obtained from the measurement and questionnaire results, along with Towards Health Card Records. The polymerase chain reaction (PCR) genotyping method used was the restriction fragment length polymorphism and allele-specific.Results: The prevalence of hypertension, prehypertension, and normotension in Javanese postmenopausal women were 0.246, 0.13, and 0.623, respectively. The frequency of BMI classification as underweight, normal, overweight, or obese were 0.029, 0.42, 0.261, and 0.29, respectively. The ACE I/D and A2350G polymorphism variant genotypes and frequencies found were II (0.464), ID (0.522), DD (0.014), and AA (1). Meanwhile, the alleles and their frequencies at ACE I/D gene polymorphism were I (0.725) and D (0.275). The II and ID genotype was mostly found in normotension subjects. The DD genotype was only available in hypertension subjects. There was no association between genotypes and alleles of ACE I/D, hypertension, body weight, and BMI classification (p&gt;0.05). There was an association between these genotypes, alleles, and BMI (p&lt;0.05).Conclusion: ACE I/D polymorphism is susceptible for BMI in Javanese postmenopausal women.Keywords: Javanese postmenopausal, essential hypertension, ACE I/D, ACE A2350G

Association of ET1 and APE1 Genes Polymorphism with Coronary Artery Disease

AbstractCoronary artery disease (CAD) is one of the most common cardiovascular diseases (CVDs), being the foremost reason for mortality and disability globally. It is a cascade of polygenic architecture of various disorders as a resultant of complexities between various genetic factors and environmental factors. The aim of the present study was to investigate whether the ET1 and APE1 gene polymorphism is associated with the susceptibility to CAD. This study was approved by the Ethical Review Committee of Era Medical College and Hospital. Three milliliters of venous blood sample was collected in ethylenediaminetetraacetic acid-coated vials, and genomic DNA was isolated by using the standard phenol-chloroform extraction method for restriction fragment length polymorphism-polymerase chain reaction study. The APE1 gene AA, AG, GG, and AG + GG genotypes frequencies were 13.91, 40.87, 45.22, and 86.99% in CAD cases and 13, 50, 37, and 87% in controls, respectively. The ET1 gene GG, GT, TT, GT + TT genotypes frequencies were 6.67, 37.5, 55.8, and 93.34% in CAD cases and 6.67, 25.33, 68, and 93.34% in controls. The ET1 and APE1 gene polymorphisms were not significantly associated with the risk of CAD.

Exploration of IL-32 (rs45499297) gene variations in EBV and MS patients

Interleukin-32 is a multifunctional cytokine linked to a variety of illnesses and inflammatory disorders, the central nervous system is affected by the chronic inflammatory disease known as multiple sclerosis (MS). Studies about found relationship between them are rare and inconclusive specially when MS patients with Epstein-Barr virus (EBV) positive. Recently, IL-32 identified increase and considers proinflammatory following EBV infection. A case-control study (79 MS patients and 108 controls) for the purpose of investigating T/C genotype of (rs45499297) in all participant then identify their role in MS and EBV patients by Restriction fragment length polymorphism (RFLP) technique. The results appeared that alleles and genotype (rs45499297) was no significant differences between MS patients and healthy controls, also between groups of expanded disability status scale in MS patients but with EBV infection was results appeared that allele C of( rs45499297) more effector in MS patients than controls. The mean of EBV load among multiple sclerosis patients according to sex, EDSS, and therapy line the results revealed that elevated viral load in male, EDSS ≥ 3.0 and in second line therapy compare to opposite groups. In conclusion, the study indicated the role of sex and MS activity in susceptibility to EBV reactivation. However, analysis of the IL-32 gene variants (rs45499297) may influence susceptibility to EBV along with MS associated with the allele C occurrence.

Detection of different groups of phytoplasma in palm species of Puerto Rico

Palm trees play important cultural, ecological, and economic roles in the tropics. Native palm species such as the royal palm, Roystonea borinquena O.F. Cook, are extensively planted in landscapes and urban areas of Hispaniola, Puerto Rico and the Virgin Islands. Worldwide, palms are affected by a disease commonly known as lethal yellowing, linked to the presence of phytoplasmas. Coconut Lethal Yellowing (CLY) is the most devastating disease of palms, associated with the 16SrIV phytoplasma group in the Americas. In Puerto Rico, palms displayed symptoms such as leaf chlorosis or yellowing, inflorescence and fruit necrosis, and eventual death. The objective of this research was to identify and characterize phytoplasmas in palms and their potential insect vectors, focusing on a native palm, R. borinquena. To fulfill this objective, 69 palms belonging to the species: Cocos nucifera (n=15), Gaussia attendata (n=1), Leucothrinax morrisii (n=1), Pseudophenix sargentii (n=1), Roystonea borinquena (n=50) and Washingtonia robusta (n=1) were sampled within three transects across the island. Ninety percent of palms sampled displayed typical lethal yellowing symptoms. In addition, 12 different insect species (Auchenorrhyncha: Fulgoroidea) that could act as potential phytoplasma vectors were sweep-collected from palms and grasses near the study area. The 16S ribosomal region of phytoplasmas was amplified using nested polymerase chain reaction (PCR) and subjected to restriction fragment length polymorphism (RFLP) analyses to allow their ribosomal grouping. Overall, 17 palm samples were positive to phytoplasmas. These are C. nucifera (n = 1), L. morrisii (n = 1) and R. borinquena (n = 15). Phytoplasmas detected belong to six ribosomal groups: 16SrII, 16SrIII, 16SrIV, 16SrVI, 16SrIX and 16SrX. The most frequent phytoplasma detected (59%) is enclosed in the 16SrII group. Samples from nine royal palms (R. borinquena) and one key thatch palm (L. morrisii) were positive to the 16SrII-related group. Only one insect specimen, the cixiid Haplaxius crudus collected from a phytoplasma positive R. borinquena palm, was positive for a 16SrII-related group phytoplasma. Another significant epidemiological finding was the detection of a phytoplasma related to CLY in the 16SrIV group in one individual of R. borinquena. This native species is the most abundant palm on the island. To our knowledge this is the first report of a phytoplasma related to the 16SrII group in R. borinquena in Puerto Rico and the world. Detection for the first time of other phytoplasmas related to 16Sr groups -III, -IX and -X in palms deserves further studies.

Association of NRAMP1 Gene Polymorphism with Hemoglobin and Iron Parameters in Patients with β-Thalassemia Major

Abstract BACKGROUND: Thalassemia is an inherited disorder that is also called mediterranean anemia and is described by a lack of hemoglobin synthesis. β-thalassemia is more common in people of certain origins, particularly those from the Mediterranean region and Arabian Peninsula. The NRAMP1 protein in membranes catalyzes the exit of divalent metal ions from phagolysosomes and their entry into the cytoplasm, including iron and manganese. AIMS: To investigate the possible link between beta-thalassemia major (β-TM) illness and the single-nucleotide polymorphism (3′ untranslated region) rs3 of the NRAMP1 gene. MATERIALS AND METHODS: To investigate the relationship between the condition β-TM and the NRAMP1 gene, the study used restriction fragment length polymorphisms-polymerase chain reaction. The enzyme-linked fluorescent assay was used to quantify the ferritin level. A hematology analyzer was used to quantify hemoglobin concentration. A spectrophotometer technique was used to assess the serum iron, serum total iron-binding capacity (TIBC), and serum unsaturated iron-binding capacity. RESULTS: The results showed a difference in hemoglobin and serum iron levels, serum unbound iron-binding capacity, serum TIBC, and ferritin concentration between individuals suffering from β-thalassemia and healthy groups. In NRAMP1 gene polymorphism, a highly significant difference was observed between the distribution of two alleles (TG− 244 bp, and TG+ 211 bp). CONCLUSION: During an investigation of NRAMP1 gene polymorphism, a significant variance was observed between β-thalassemia and control groups. Those who carried the (244 bp −TG) allele were found to have a 3.6 times higher risk of developing complications of β-TM than those who carried the (211 bp −TG+) allele.

Detection the Genetic Polymorphisms of the Programmed Cell Death Gene PD1.3 G/A (rs11568821) in Coronavirus Disease Patients

Abstract Background: Programmed cell death-1 protein (PD-1, Pcd1), a receptor from the CD28/CTLA-4 group, inhibits antigen receptor signaling by attracting protein tyrosine phosphatase in response to an interaction with any of the two ligands, PD-L1 or PD-L2. Human phenotypic variations can be attributed to both genetic and environmental influences. Objectives: Because SNPs (single nucleotide polymorphisms) are not absolute indicators of disease progression, in this work, the PD-1 gene polymorphism in coronavirus disease (COVID-19) patients was examined to assess the presence of SNPs in viral infections, in particular COVID-19 is the main goal of this investigation. Materials and Methods: Sixty confirmed COVID-19 patients were recruited to this study; 30 of them had severe COVID-19, whereas the other patients showed moderate sickness, who were admitted to the COVID-19 specialized ward in Salahuddin province, and all of them were over the age of 18 years. Also 30 healthy subjects were recruited for the purpose of the comparison. Blood was drawn from all the subjects for polymerase chain reaction (PCR) using the restriction fragment length (RFLP)-PCR for the assessment of the G/A SNP genotype of PD1.3 and ELISA test for the estimation of PD-1 and PD-L-1 serum level. Results: The examined PD-1 SNPs did not correlate with the incidence of COVID-19, according to a data analysis. Both the dominant and recessive models used in the research failed to detect a connection with the risk of COVID-19 severity. The PD1.3 genotypes frequency between the two groups did not show significant differences (P &gt; 0.05). Only AG was substantially and mainly linked to COVID-19 susceptibility. This study compared the concentrations of immune check point inhibitors PD-1, PD-L-1 to find possible association with genotype frequency, and approved that PD-1 did not have any significant differences in the three groups of genotypes, whereas the difference was highly significant (0.048) in PDL-1 and AA genotype. Conclusion: Among all calculated haplotypes were unrelated to the disease’s prognosis (P &gt; 0.05) concluded that the frequency of AA genotype in patients group decreed the expiation of PD-L-1, leads to immune inhibitions. Future research may clarify the relationship between some immune checkpoint molecule polymorphisms.

Polymorphism of angiogenesis regulation factor genes (VEGF/VEGFR), and extracellular matrix remodeling genes (MMP/TIMP), and the levels of their products in extracellular tissues of patients with primary and secondary lymphedema.

Cells of various organs and systems perform their functions and intercellular interactions not in an inert environment, but in the microenvironment of tissue fluids. Violations of the normal drainage of tissue fluids accompany lymphedema. An important mechanism of angiogenesis and vasculogenesis regulation in tissue fluids is the production and reception of vascular endothelial growth factors in combination with the regulation of matrix metalloproteinases. The aim of the work was to perform: a comparative analysis of some polymorphisms of vascular endothelial growth factor and their receptors and the genes encoding matrix metalloproteinases in two forms of lymphedema; an analysis of the relationship of these genes' polymorphisms with the levels of vascular endothelial growth factor and matrix metalloproteinases and their inhibitors in serum and affected tissues. Polymorphism of VEGF (rs699947, rs3025039), KDR (rs10020464, rs11133360), NRP2 (rs849530, rs849563, rs16837641), matrix metalloproteinases MMP2 (rs2438650), MMP3 (rs3025058), MMP9 (rs3918242), Timp1 (rs6609533) and their combinations were analyzed by the Restriction Fragment Length Polymorphism method and TaqMan RT-PCR. The serum and tissue fluid levels were determined using the ELISA test system. Changes in the frequency distribution of MMP2 genotypes in primary and MMP3 in secondary lymphedema are shown. Significant frequency differences in NRP2 genotypes were revealed by comparing primary and secondary lymphedema. Features of the distribution of complex genotypes in primary and secondary lymphedema were revealed. The correlation analysis revealed the interdependence of the concentrations of the MMP, TIMP and VEGF products and differences in the structure of the correlation matrices of patients with both forms of lymphedema. It was shown that, in primary lymphedema, genotypes associated with low MMP2 and TIMP2 in serum and tissue fluid are detected, while in secondary lymphedema, other associations of the production levels with combined genetic traits are observed.

The Association of MMP-2, MMP-9, and MMP-13 Gene Polymorphisms With Knee Osteoarthritis in the Greek Population.

Introduction Primary knee osteoarthritis (OA) is a multifactorial degenerative joint disorder characterized by articular cartilage degradation. Matrix metalloproteinases (MMPs) have been reported to play a vital role in OA pathogenesis, significantly contributing to extracellular matrix (ECM) catabolism. The purpose of this study is to investigate the association of MMP-2 -1575G/A (rs243866), MMP-9 836A/G (rs17576), and MMP-13 -77A/G (rs2252070) gene polymorphisms with knee OA in the Greek population. Methods One hundred patients (24% males, mean age: 68.3 years) with primary knee OA were included in the study along with 100 controls (47% males, mean age: 65.2 years). Genotypes were identified through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) technique. Allelic and genotypic frequencies were compared between patients and controls. Results The MMP-13 -77A/G polymorphism was significantly associated with knee OA in the crude analysis (P = 0.008). After binary logistic regression analysis, the dominant model of the MMP-13-77A/G (AG + GG versus AA) was found to be associated with increased risk for knee OA (odds ratio (OR) = 2.290, 95% confidence interval (95%CI) = 1.059-4.949, P= 0.035).Compared to the A allele, the G allele in the MMP-13rs2252070 locus was a predictive factor for knee OA (OR = 2.351, 95%CI = 1.134-4.874, P= 0.022). No significant associations were detected for the MMP-2 -1575G/A and MMP-9 836A/G polymorphisms (P > 0.05). Conclusions The present study shows that the MMP-2 -1575G/A and MMP-9 836A/G polymorphisms are not significantly associated with primary knee OA in the Greek population. The MMP-13 -77A/G was found to be a significant risk factor for knee OA in the Greek population. Additional research is needed to verify this association in larger and different populations, in different joints, to elucidate the role of this single nucleotide polymorphism (SNP) in OA pathogenesis.

The establishment of a molecular diagnostic platform for mitochondrial diseases: from conventional to next-generation sequencing

BackgroundThe aim of this study was to create a molecular diagnostic platform and establish a diagnostic pipeline for patients highly suspected of mitochondrial disorders. The effectiveness of three methods, namely, traditional restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), Sanger sequencing for hotspot detection and whole mitochondrial DNA (mtDNA), and third-generation (Nanopore) whole mtDNA sequencing, will be compared in diagnosing patients with suspected primary mitochondrial diseases (PMDs). The strengths and limitations of different methods are also discussed. Material and methodsA single-center prospective cohort study was conducted to validate the diagnostic pipeline for suspected mitochondrial diseases. In the first stage, a PCR-based method with five sets of primers was used to screen for eight hotspots (m.3243A>G, m.3460G>A, m.8344A>G, m.8993T>G, m.9185T>C, m.11778G>A, m.13513G>A, and m.4977deletion) using either RFLP or direct Sanger sequencing. Sanger sequencing was also used to confirm the RFLP-positive samples. In the second stage, for samples with negative screening results for the eight hotspots, mitochondrial whole-genome sequencing was performed using Sanger sequencing or third-generation nanopore sequencing. ResultsBetween June 2020 and May 2023, 30 patients from ages 0 to 63 with clinically suspected mitochondrial disease were enrolled. The positive yield for the diagnosis of PMDs was 8/30=26.7%, and the sensitivity of the heteroplasmy level for the RFLP-based method was approximately 5%. The remaining 22 patients who tested negative at the first stage were tested using Sanger sequencing or the third-generation sequencing Nanopore, and all tested negative for pathological mtDNA mutations. Compared to the Sanger sequencing method, the results of RFLP-PCR were compromised by the limitations of incomplete RFLP enzyme digestion. For whole-genome sequencing of mtDNA, Sanger sequencing, instead of nanopore sequencing, is preferred at our institution because of its cost-effectiveness. Conclusions: In our highly selective cohort, most tested positive in the first stage of the 8 hot spots screen. Sanger sequencing is a conventional and accurate method for mitochondrial disease screening, at least for the most common hot spots in the region. The results revealed that Sanger sequencing is an accurate method with the benefit of being more cost-effective. This integral platform of molecular diagnosis bears the advantages of being relatively low cost and having a shorter reporting time, facilitating crucial identification of patients with clinical evidence of such disorders. This diagnostic flowchart has also been translated into routine clinical use in the tertiary hospital.

ZALEŻNOŚCI POMIĘDZY POLIMORFIZMAMI GENÓW RBP4, FSHB I EGF A CECHAMI ROZRODCZYMI U ŚWIŃ

Genes encoding retinol binding protein 4 (RBP4), follicle stimulating hormone subunit beta (FSHB) and epidermal growth factor (EGF) have been proposed as candidate genes for reproduction traits in pigs. The study presented in this work aimed to find associations between variants of these genes and reproduction traits in pigs reared in Poland. Investigation included Polish Large White x Polish Landrace (n = 288) crossbreed and Yorkshire (n = 195) breed sows. Insertion-deletion polymorphism (indel) in EGF gene was determined by means of PCR (polymerase chain reaction) method, however single nucleotide polymorphism (SNP) in RBP4 and FSHB genes by means of PCR-RFLP (restriction fragment length polymorphism) method. Obtained results showed that total number born (TNB) and number born alive (NBA) in 1st, 2nd and 5th parities were associated with RBP4 gene variants, TNB and NBA in first two parities with FSHB variants, however both traits in all parities with EGF variants (p ≤ 0.05 or p ≤ 0.01). In case of RBP4 gene, heterozygous genotype (AB) was favorable for analyzed traits, however in relation to FSHB and EGF genes homozygous genotypes, AA and BB respectively. Obtained results indicate that polymorphism in three analyzed genes is associated with important reproductive traits of pigs kept in Poland.