Genomic DNA Samples Research Articles

Quantitative PCR from human genomic DNA: The determination of gene copy numbers for congenital adrenal hyperplasia and RCCX copy number variation.

Quantitative PCR (qPCR) is used for the determination of gene copy number (GCN). GCNs contribute to human disorders, and characterize copy number variation (CNV). The single laboratory method validations of duplex qPCR assays with hydrolysis probes on CYP21A1P and CYP21A2 genes, residing a CNV (RCCX CNV) and related to congenital adrenal hyperplasia, were performed using 46 human genomic DNA samples. We also performed the verifications on 5 qPCR assays for the genetic elements of RCCX CNV; C4A, C4B, CNV breakpoint, HERV-K(C4) CNV deletion and insertion alleles. Precision of each qPCR assay was under 1.01 CV%. Accuracy (relative error) ranged from 4.96±4.08% to 9.91±8.93%. Accuracy was not tightly linked to precision, but was significantly correlated with the efficiency of normalization using the RPPH1 internal reference gene (Spearman's ρ: 0.793-0.940, p>0.0001), ambiguity (ρ = 0.671, p = 0.029) and misclassification (ρ = 0.769, p = 0.009). A strong genomic matrix effect was observed, and target-singleplex (one target gene in one assay) qPCR was able to appropriately differentiate 2 GCN from 3 GCN at best. The analysis of all GCNs from the 7 qPCR assays using a multiplex approach increased the resolution of differentiation, and produced 98% of GCNs unambiguously, and all of which were in 100% concordance with GCNs measured by Southern blot, MLPA and aCGH. We conclude that the use of an internal (in one assay with the target gene) reference gene, the use of allele-specific primers or probes, and the multiplex approach (in one assay or different assays) are crucial for GCN determination using qPCR or other methods.

A Metabarcoding Approach to Investigate Fungal and Oomycete Communities Associated with Kiwifruit Vine Decline Syndrome in Italy

Since 2012, kiwifruit vine decline syndrome (KVDS) has severely compromised all major kiwifruit-growing areas in Italy. Thus far, etiological studies were mainly focused on waterlogging effects or on the isolation of microorganisms from diseased plants; therefore, an all-encompassing picture of KVDS microbiota is still missing. This work aims to describe oomycete and fungal communities associated with KVDS and to identify key taxa potentially involved in the disease through a metabarcoding approach on root endosphere and rhizosphere samples. Two nearby fields with similar pedoclimatic conditions were identified based on KVDS spreading during a 4-year survey (2016 to 2019). In total, four sampling areas were selected, one from the control field with no sign of KVDS (asymptomatic site) and three from the KVDS-affected field (diseased site): (i) asymptomatic until the sampling date in 2018, (ii) symptomatic since 2018, and (iii) symptomatic since 2017. Total genomic DNA samples were subjected to a nested PCR approach separately targeting the internal transcribes spacer 2 regions of fungal and oomycete communities. The communities were compared in terms of α- and β-diversities, and key taxa were identified using univariate differential abundance tests. Major differences in taxa distribution were observed between samples from the different sites (asymptomatic and diseased) and were mostly linked to the oomycete community. Phytophthora sojae was the main taxa characterizing the diseased site and supposed to be involved in the disease and Phytopythium spp. were found related to the different plant health status. Finally, Dactylonectria macrodidyma, Phytopythium citrinum, and Thielaviopsis basicola were also proposed as new KVDS-related pathogens.

Homozygous mutation in CSF1R causes brain abnormalities, neurodegeneration, and dysosteosclerosis (BANDDOS).

The CSF1R gene encodes the receptor for colony-stimulating factor-1, the macrophage, and monocyte-specific growth factor. Mutations in this gene cause hereditary diffuse leukoencephalopathy with spheroids (HDLS) with autosomal dominant inheritance and BANDDOS (Brain Abnormalities, Neurodegeneration, and Dysosteosclerosis) with autosomal recessive inheritance. Targeted gene sequencing was performed on the genomic DNA samples of the deceased patient and a fetus along with ten healthy members of his family to identify the disease-causing mutation. Bioinformatics tools were used to study the mutation effect on protein function and structure. To predict the effect of the mutation on the protein, various bioinformatics tools were applied. A novel homozygous variant was identified in the gene CSF1R, c.2498C>T; p.T833M in exon 19, in the index patient and the fetus. Furthermore, some family members were heterozygous for this variant, while they had not any symptoms of the disease. In silico analysis indicated this variant has a detrimental effect on CSF1R. It is conserved among humans and other similar species. The variant is located within the functionally essential PTK domain of the receptor. However, no structural damage was introduced by this substitution. In conclusion, regarding the inheritance pattern in the family and clinical manifestations in the index patient, we propose that the mentioned variant in the CSF1R gene may cause BANDDOS.

Establishment of molecular diagnostics targeting the 23S ribosomal RNA gene for the detection of Mycoplasma suis infection in Thai domestic pigs

Mycoplasma (M.) suis is a pathogenic hemotropic Mycoplasma sp. that causes acute hemolytic anemia or chronic infection in pigs. M. suis infection can be diagnosed using several methods, including molecular diagnosis such as conventional PCR (cPCR) and quantitative PCR (qPCR). In these cases, the common target is the 16S rRNA gene; however, this genetic marker cannot distinguish hemoplasma at the species level owing to high sequence identity. Therefore, the 23S rRNA gene has emerged as another target gene. Other than PCR, the loop-mediated isothermal amplification (LAMP) method can be applied for M. suis. The objective of the present study was to establish cPCR, TaqMan qPCR, and LAMP assays in which the 23S rRNA gene is used to detect M. suis infection in Thai domestic pigs. The analytical sensitivity of cPCR was determined as 7.46 × 104 copies/μl of plasmid DNA, whereas those of qPCR and LAMP were 7.46 × 102 copies/μl. There was no cross reaction with other pathogens in any of the assays. To evaluate the diagnostic performance of the assays, they were tested using 173 samples of genomic DNA. The detection percentage of M. suis infection was 24.86% (43/173; 95% CI: 18.61%–31.89%), 28.32% (49/173; 95% CI: 21.75%–35.66%), and 29.48% (51/173; 95% CI: 22.80%–36.88%) using cPCR, qPCR, and LAMP, respectively. Using qPCR as a reference assay, cPCR showed 81.63% sensitivity, 97.58% specificity, and an almost perfect level of agreement (kappa = 0.823). In comparison, LAMP showed 77.55% sensitivity, 89.52% specificity, and a substantial level of agreement (kappa = 0.662). All assays tested here could be applied in veterinary diagnostic laboratories for monitoring porcine health in the herds. Furthermore, the LAMP assay could be used as a screening test in farm practice without the need for any special equipment.

Associations of molecular genetic predictors of arterial hypertension in adults with the development and course of arterial hypotension in premature newborns

Purpose. This study aims at assessing the association of the development and clinical course of arterial hypotension in premature newborns, depending on the polymorphism of the genes of predisposition to hypertension.Material and methods. The study design is prospective, controlled, single-center, and non-randomized. Genomic DNA samples were studied in premature newborns (n = 199), as well as a population sample of adults (n = 100). Newborns with arterial hypotension (n = 23) formed the study group, patients without it (n = 100) formed the comparison group. Loci with already known association with the development of arterial hypertension and coronary heart disease were selected for analysis: AGT (rs4762), AGTR1 (rs5186), ACE (Ins\Del), ADRB1 (rs1801253), ADD1 (rs4961), CYP11B2 (rs1799998), eNOS (rs1799983), eNOS (rs1549758), eNOS (rs2070744). The distribution of allele frequencies between the studied groups of individuals was compared.Results. Premature babies are significantly more likely to be carriers of the allele with the AGT gene. In newborns with arterial hypotension, we additionally revealed a more frequent occurrence of dominant alleles of the eNOS gene, and children with the SS genotype of the eNOS gene required longer vasopressor therapy. Conclusion. The carriage of risky alleles of genes predisposing to arterial hypertension, not only does not increase the risk of arterial hypotension in premature newborns, but also may, in the case of carriage of mutant alleles of the eNOS rs1549758 gene, contribute to less prolonged vasopressor therapy.

STUDY OF THE NUCLEOTIDE POLYMORPHISM IL-4 RS2243250 (C-589T) FREQUENCY IN PATIENTS WITH ALLERGIC RESPIRATORY

<h3>Introduction</h3> Increased serum levels of some interleukines (IL) especially IL-4 and IL-13 are associated with allergy. The frequency of polymorphism IL-4 rs2243250 (C-589T) among patients with year-round and seasonal allergic sensitization was investigated. <h3>Methods</h3> Blood samples from 21 healthy volunteers and 15 sensitized patients were collected in the 6th Minsk Clinical Hospital after informed consent from patients 18–60 years old. Total and allergenspecific immunoglobulin E was obtained. Diagnosis of asthma was verified by GINA2021criteria, and allergic rhinitis by ARIA2020 criteria. Genomic DNA samples were extracted from peripheral blood using "NK-extra" kit, RB. The polymorphic part of the gene encoding the cytokine IL-4 was amplified by using PCR specific primers: (F) 5′-TAAACTTGGGAGAACATGGT-3′; 5′-TGGGGAAAGATAGAGTAATA-3′. The method of polymorphic length of restrictive fragments was used to detect SNP IL-4 rs2243250 (C-589T). Restriction was carried out according to the protocol for AVA II restriction enzyme (Thermo Fisher Scientific). <h3>Results</h3> 12 (80%) sensitized patients of study group were found to be homozygous with "wild" type C allele versus the control group with 12 patients (57.2%). The mutant T allele in the homozygous state was found only in a group of healthy volunteers - 2 samples (9.5%). Heterozygous alleles C and T in the group of sensitized individuals were found in 3 patients (20%), versus the control group in 7 (33.3%). <h3>Conclusion</h3> This preliminary study showed that polymorphism IL-4 rs2243250 (C-589T) occurs in 10% of cases with the predominant allele "wild" type C, in 90%.

Genetic analyses provide new insight on the mating strategies of the American Black Swift (Cypseloides niger)

Avian mating strategies play a vital role in the demographic and genetic dynamics of a species and understanding avian reproductive tactics is important to conservation, population management and restoration. Classifications of avian mating strategies have historically been based on direct physical observations and tend to be rigid population‐level generalizations that overlook the variations inherent in most ecological systems. Based on limited empirical field observations, the American Black Swift Cypseloides niger borealis is considered to be a socially monogamous species with pair bonds lasting for many years. To test this hypothesis, we collected genomic DNA samples from banded swifts from six American Black Swift colonies in the western United States from 2004 to 2019 and isolated and developed primers for highly polymorphic microsatellite loci and used them to genotype our samples. Our parentage analysis revealed that sampled females never mated with the same male in subsequent years, suggesting that they are not sexually monogamous with a single partner for many years as previously hypothesized.

Novel mutations in TUBB8 and ZP3 cause human oocyte maturation arrest and female infertility

PurposeVariations in many genes may lead to the occurrence of oocyte maturation defectsand female infertility. The objective was to describe newly discovered mutations in TUBB8 and ZP3, and to characterise the accompanying spectrum of phenotypes and modes of inheritance. MethodsTUBB8 and ZP3 were sequenced from genomic DNA samples extracted from peripheral blood of patients and their family members by the whole-exome sequencing. The TUBB8 and ZP3 sequences are then aligned with cryptographic software to identify rare variations. Sanger sequencing and mass spectrometry were used to validate mutations. ExAC database was used to retrieve the frequency of corresponding mutations. PolyPhen-2 and PROVEAN were analyzed for mutations using silicon. ResultsWe identified Three novel mutations and two known variant in TUBB8 and ZP3 associated with maturation in five families, and fertilization and developmental arrest are in these patients. These mutations include four heterozygous mutations in TUBB8 (c.730G > A, p.Gly244Ser, c.124C > G, p.Leu42Val, c.1172G > T, p.Arg391Leu and c.178G > A, p.Val60Met), and a heterozygous mutation in ZP3 (c.400G > A, p.Ala134Thr). Among them, these variants of TUBB8 were highly conserved among primates. ConclusionAs far as we know, the TUBB8 mutations detected in our study at four sites have not been reported before, and the variant of ZP3 has been published as pathogenic. Our findings extend the known mutant spectrum of TUBB8 and ZP3, and provide insights into the etiology of infertility in human women. The exact molecular mechanism has not been analyzed and should be further investigated in the future.

Evaluation of Association between PPARGC1A Gene Polymorphism and Competitive Performance of Elite Athletes

Limited number of researches exist on the relationship between PPARGC1A gene polymorphism (rs8192678) and affects the athletic performance. Thus, the present study aims to decipher any possible association of the rs8192678 polymorphism in the PPARGC1A gene with the competitive performances of Turkish elite track and field athletes. A total of 60 elite athletes (31 sprint/power and 29 endurance) and 20 control/sedentary with the ages of 18-35 voluntarily participated in the study. The International Association of Athletics Federations (IAAF) score scale was used to determine the performance levels of the personal best (PB) of the athletes. Whole exome sequencing (WES) was performed by the genomic DNA sample isolated from blood of the participants. The association between rs8192678 polymorphism and PB was examined by one-way analysis of covariance (ANCOVA) with the adjustment of sex and sport experience. According to the results, there were not any significant deviation between the wild-type (G/G), heterozygote (G/A) and homozygote (A/A) genotypes within and between the groups (p&gt;0.05). Our results underlined that there were not any significances for association of rs8192678 polymorphism with PBs within the groups of the sprint/power and endurance athletes. However, it is recommended that similar studies be conducted with more participants to provide clearer information about the research.

Whole exome sequencing in Brugada and long QT syndromes revealed novel rare and potential pathogenic mutations related to the dysfunction of the cardiac sodium channel

BackgroundBrugada syndrome (Brs) and long QT syndrome (LQTs) are the most observed “inherited primary arrhythmia syndromes” and “channelopathies”, which lead to sudden cardiac death.MethodsDetailed clinical information of Brs and LQTs patients was collected. Genomic DNA samples of peripheral blood were conducted for whole-exome sequencing on the Illumina HiSeq 2000 platform. Then, we performed bioinformatics analysis for 200 genes susceptible to arrhythmias and cardiomyopathies. Protein interaction and transcriptomic co-expression were analyzed using the online website and GTEx database.ResultsAll sixteen cases of Brs and six cases of LQTs were enrolled in the current study. Four Brs carried known pathogenic or likely pathogenic of single-point mutations, including SCN5A p.R661W, SCN5A p.R965C, and KCNH2 p.R692Q. One Brs carried the heterozygous compound mutations of DSG2 p.F531C and SCN5A p.A1374S. Two Brs carried the novel heterozygous truncated mutations (MAF < 0.001) of NEBL (p.R882X) and NPPA (p.R107X), respectively. Except for the indirect interaction between NEBL and SCN5A, NPPA directly interacts with SCN5A. These gene expressions had a specific and significant positive correlation in myocardial tissue, with high degrees of co-expression and synergy. Two Brs carried MYH7 p.E1902Q and MYH6 p.R1820Q, which were predicted as "damaging/possibly damaging" and "damaging/damaging" by Polyphen and SIFT algorithm. Two LQTs elicited the pathogenic single splicing mutation of KCNQ1 (c.922-1G > C). Three LQTs carried a single pathogenic mutation of SCN5A p.R1880H, KCNH2 p.D161N, and KCNQ1 p.R243S, respectively. One patient of LQTs carried a frameshift mutation of KCNH2 p. A188Gfs*143.ConclusionsThe truncated mutations of NEBL (p.R882X) and NPPA (p.R107X) may induce Brugada syndrome by abnormally affecting cardiac sodium channel. SCN5A (p.R661W, p.R965C and p.A1374S) and KCNH2 (p.R692Q) may cause Brugada syndrome, while SCN5A (p.R1880H), KCNQ1 (c.922-1G > C and p.R243S) and KCNH2 (p.D161N and p.A188Gfs*143) may lead to long QT syndrome.

A feasibility study of noninvasive prenatal diagnosis in facioscapulohumeral muscular dystrophy type 1 in a Chinese family.

Objective: To demonstrate the feasibility of haplotype-based noninvasive prenatal diagnosis of Facioscapulohumeral Muscular Dystrophy type 1 (FSHD1). Methods: Bionano optical mapping was used to identify the D4Z4 structural variation of the genomic DNA sample from the proband affected with FSHD1. In addition, based on the technique of next generation sequencing, the pathogenic haplotype was determined by using trio strategy through genotyping his parents, and also fetal inheritance of paternal haplotypes was then deduced using the Hidden Markov Model. Results: Bionano optical mapping analysis revealed that the proband has only three D4Z4 repeats left in the 4q35 chromosomal region and a disease-permitting 4qA haplotype. The other normal allele of the proband contains 29 D4Z4 repeats and also a 4qA haplotype. The noninvasive cell-free fetal DNA (cffDNA)-based haplotype analysis suggested that the fetus inherited the pathogenic allele from his father and thus was predicted to be affected by FSHD1. In addition, Bionano optical mapping also demonstrated the presence of the pathogenic allele in the fetus by interrogating the genomic DNA from the amniotic fluid cells. Conclusion: Our study showed the cffDNA-based haplotyping was feasible for the noninvasive prenatal diagnosis of FSHD1, which is able to provide earlier testing results with a lower risk of miscarriage and infection than invasive techniques.

Agarose gel microcapsules enable easy-to-prepare, picolitre-scale, single-cell genomics, yielding high-coverage genome sequences

A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality, single-cell, amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density, which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli, a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNA samples were prepared with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification and showed an average genome completeness that was much higher than that obtained using a conventional amplification method on the microlitre scale, regardless of the genomic guanine–cytosine content. Our novel method using AGM will allow many researchers to perform single-cell genomics easily and effectively, and can accelerate genomic analysis of yet-uncultured microorganisms.

Next Generation Sequencing of Genotype Variants and Genetic Association between Heat Shock Proteins HSPA1B Single Nucleotide Polymorphism at the g.31829044 Locus and Heat Tolerance: A Pilot Quasi-Experimental Study.

Heat tolerance and exertional heat stroke (EHS) are rare health conditions that have been described and characterised but have never been genetically solved. Knowledge of the role of single nucleotide polymorphisms (SNPs) in heat shock proteins (HSPs) genes and their associations with heat tolerance and EHS is limited. This pilot study aimed to identify SNP in HSPA1B, HSP90AA2 and DNAJA1 genes and their associations with heat tolerance and EHS history in a quasi-experimental design. Participants comprised Australian Defence Force members (ADF) who had a history of EHS and the general population. Genomic DNA samples were extracted from the venous blood samples of 48 participants, sequenced and analysed for SNP. Forty-four per cent (44%) of the participants were heat intolerant, and 29% had a history of EHS. Among participants with a history of EHS, there was an association between heat tolerance and HSPA1B SNP at the g.31829044 locus. However, there were no associations between HSPA1B and HSP90AA2 SNP and heat tolerance. All participants had the same distribution for the DNAJA1 SNP. In conclusion, the findings indicate an association between the HSPA1B genetic variant at the g.31829044 locus and heat tolerance among ADF participants with a history of EHS. Further research with a larger number of military participants will shed more light on the associations between HSP genes and heat tolerance.

Defective EMC1 drives abnormal retinal angiogenesis via Wnt/β-catenin signaling and may be associated with the pathogenesis of familial exudative vitreoretinopathy

Endoplasmic reticulum (ER) membrane protein complex (EMC) is required for the co-translational insertion of newly synthesized multi-transmembrane proteins. Compromised EMC function in different cell types has been implicated in multiple diseases. Using inducible genetic mouse models, we revealed defects in retinal vascularization upon endothelial cell (EC) specific deletion of Emc1, the largest subunit of EMC. Loss of Emc1 in ECs led to reduced vascular progression and vascular density, diminished tip cell sprouts, and vascular leakage. We then performed an unbiased transcriptomic analysis on human retinal microvascular endothelial cells (HRECs) and revealed a pivotal role of EMC1 in the β-catenin signaling pathway. Further in-vitro and in-vivo experiments proved that loss of EMC1 led to compromised β-catenin signaling activity through reduced expression of Wnt receptor FZD4, which could be restored by lithium chloride (LiCl) treatment. Driven by these findings, we screened genomic DNA samples from familial exudative vitreoretinopathy (FEVR) patients and identified one heterozygous variant in EMC1 that co-segregated with FEVR phenotype in the family. In-vitro expression experiments revealed that this variant allele failed to facilitate the expression of FZD4 on the plasma membrane and activate the β-catenin signaling pathway, which might be a main cause of FEVR. In conclusion, our findings reveal that variants in EMC1 gene cause compromised β-catenin signaling activity, which may be associated with the pathogenesis of FEVR.

DNA Probe as Biosensor Candidate for Clavibacter michiganensis subsp. michiganensis on Tomato Plants

The study was conducted to evaluate gene detection technique using a specific DNA probe to detect tomA gene in Clavibacter michiganensis subsp. michiganensis, a bacteria causing cancer in tomatoes. The probe was designed using Primer3Plus program, labeled with the non-radioactive molecule digoxigenin (DIG) and used in the hybridization method with the dot blot technique. The test samples consisted of two types, i.e. genomic DNA samples from pure bacterial cultures and from artificially infected tomato seeds with C. michiganensis subsp. michiganensis. Samples derived from pure bacterial cultures showed positive hybridization results at all levels of DNA concentration; while samples from tomato seeds only showed positive reactions at concentrations of 10, 8, 6, and 4 g L-1. This study concludes that the designed probe has the potential to be used in the development of biosensor-based detection methods for C. michiganensis subsp. michiganensis in tomato seeds and is quite specific because there is no cross-reaction with non-target bacterial groups.

Isolation, screening and molecular identification of antagonistic bacteria against Colletotrichum gloeosporioides in mango

The study was conducted at the Postharvest and plant Biotechnology laboratory, Department of Horticulture, Patuakhali Science and Technology University, Patuakhali, Bangladesh during the period from January to July 2017 to isolate, screening and molecular identification of antagonistic bacteria against anthracnose of mango. All treatments were arranged in a completely randomized design (CRD) with replications and repeated twice. Epiphytic bacteria, isolated from leaf and fruit surfaces of mango, were tested as biocontrol agent against anthracnose disease caused by Colletotrichum gloeosporioides wherein 20 strains were confirmed as antagonistic. Molecular characterization of the three potential strains of bacteria were done by the amplification of 16S rDNA gene following the extraction of genomic DNA, polymerase chain reaction (PCR) amplification, gel electrophoresis and gel documentation. The PCR amplified products and the genomic DNA samples were sent to the Macrogen Company through Sunchon National University, Seoul, South Korea for molecular identification by sequence analysis. Among the 20 antagonistic bacteria screened in vitro by dual and concomitant tests, two isolates, namely GB6 (PSTU-Hort-8), and GB19 (PSTU-Hort-14) were recognized as antagonistics to the test fungus. Using the molecular identification systems, isolated bacterial strains PSTU-Hort-8 was identified as B. subtilis with National Center for Biotechnology Information (NCBI) accession numbers MW659188; on the other hand, strain PSTU-Hort-14 was identified as Stenotrophomonas rhizophila with NCBI accession number MW659190.

Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing.

Genotyping of folate metabolism genes is of great importance in disease diagnosis and prevention. However, most current detection methods used for folate metabolism gene genotyping are based on sequencing and chips, which suffer from a high cost and laborious and time-consuming procedures. Herein, we reported a multiplex asymmetric PCR-HRM strategy for identifying genotypes of folate metabolism genes in a single tube. The proposed multiplex PCR-HRM assay has been successfully applied to identify the genotypes of folate metabolism genes, methylene tetrahydrofolate reductase (C677T, A1298C) and methionine synthase reductase A66G, on 1 μL of genomic DNA (gDNA) samples directly released from blood specimens, and the genotyping results were 100% consistent with the results of sequencing. The assay allows us to accurately detect the genotypes of gDNA with the detection limit down to 1 ng, which meets the clinical requirement. What is more, the capacity of resistance to aerosol pollution of the multiplex asymmetric PCR-HRM biosensing was first addressed and has been evaluated as it can withstand contamination of roughly 12.5-25% interfering nucleic acids. Because of the advantages of multiplex detection, high accuracy, and resistance to aerosol pollution and having no open tube procedure, this approach would pave the way for establishing a fast and cost-effective platform for folate metabolism gene genotyping and other SNP genotyping in clinical diagnostics.

Association of the ACTN3 rs1815739 Polymorphism with Physical Performance and Injury Incidence in Professional Women Football Players.

The p.R577X polymorphism (rs1815739) in the ACTN3 gene causes individuals with the XX genotype to be deficient in functional α-actinin-3. Previous investigations have found that XX athletes are more prone to suffer non-contact muscle injuries, in comparison with RR and RX athletes who produce a functional α-actinin-3 in their fast-twitch fibers. This investigation aimed to determine the influence of the ACTN3 R577X polymorphism on physical performance and injury incidence of players competing in the women’s Spanish first division of football (soccer). Using a cross-sectional experiment, football-specific performance and epidemiology of non-contact football-related injuries were recorded in a group of 191 professional football players. ACTN3 R577X genotype was obtained for each player using genomic DNA samples obtained through buccal swabs. A battery of physical tests, including a countermovement jump, a 20 m sprint test, the sit-and-reach test and ankle dorsiflexion, were performed during the preseason. Injury incidence and characteristics of non-contact injuries were obtained according to the International Olympic Committee (IOC) statement for one season. From the study sample, 28.3% of players had the RR genotype, 52.9% had the RX genotype, and 18.8% had the XX genotype. Differences among genotypes were identified with one-way analysis of variance (numerical variables) or chi-square tests (categorical variables). Jump height (p = 0.087), sprint time (p = 0.210), sit-and-reach distance (p = 0.361), and dorsiflexion in the right (p = 0.550) and left ankle (p = 0.992) were similar in RR, RX, and XX football players. A total of 356 non-contact injuries were recorded in 144 football players while the remaining 47 did not sustain any non-contact injuries during the season. Injury incidence was 10.4 ± 8.6, 8.2 ± 5.7, and 8.9 ± 5.3 injuries per/1000 h of football exposure, without differences among genotypes (p = 0.222). Injury rates during training (from 3.6 ± 3.7 to 4.8 ± 2.1 injuries per/1000 h of training exposure, p = 0.100) and match (from 47.8 ± 9.5 to 54.1 ± 6.3 injuries per/1000 h of match exposure, p = 0.209) were also similar in RR, RX, and XX football players. The ACTN3 genotype did not affect the mode of onset, the time needed to return to play, the type of injury, or the distribution of body locations of the injuries. In summary, women football players with different genotypes of the p.R577X ACTN3 polymorphism had similar values of football-specific performance and injury incidence. From a practical perspective, the ACTN3 genotyping may not be useful to predict performance or injury incidence in professional women football players.

Role of CYP1A1, CYP2D6, and NOS3 gene polymorphisms in idiopathic recurrent pregnancy loss in the Iranian Azeri population: A case-control study.

Background It is estimated that 1-5% of couples suffer from recurrent pregnancy loss (RPL). Recent studies have shown the effects of gene polymorphisms in RPL.ObjectiveThe aim of this study was to evaluate 3 gene polymorphisms including rs1048943 of CYP1A1, rs28371725 of CYP2D6, and rs7830 of NOS3 in idiopathic RPL to identify their association with RPL.Materials and MethodsBlood samples were collected from 136 women with at least 2 consecutive idiopathic miscarriages (case group) and 136 women with no history of miscarriage and at least one successful pregnancy (control group) from the Iranian Azeri population. This study was carried out between April 2018-April 2020. Amplification-refractory mutation system polymerase chain reaction was used for the rs7830, rs1048943 and rs28371725 polymorphisms in order to genotype each extracted genomic DNA sample. After that, Chi-square, Fisher's exact test and logistic regression were used to investigate whether each of these polymorphisms is associated with RPL.ResultsAmong these polymorphisms, only rs1048943 of CYP1A1 showed a statistically significant association with RPL in the Iranian Azeri women studied.ConclusionOur results suggest that CYP1A1 gene polymorphisms might be associated with a reduced risk of RPL. Further studies in other populations and in the same population with a larger sample size, as well as functional genomics analyses such as gene expression analyses or epigenetic studies are required to validate our results.

The mediating role of transmembrane protein 132D methylation in predicting the occurrence of panic disorder in physical abuse.

ObjectiveGenome Wide Association study (GWAS) has revealed that the transmembrane protein 132D (TMEM132D) is a gene of sensitive for panic disorder (PD). As the main type of childhood trauma experience, childhood abuse has become a public health issue attracting much attention at home and abroad, and has been proved to be a risk factor for the onset of PD. However, how it affects the occurrence and development of panic disorder has not yet been revealed. We examined the relationship between TMEM132D methylation, childhood abuse and symptoms based on this finding.Materials and methodsThirty-two patients with PD and 22 healthy controls (HCs) were recruited after age, gender, and the education level were matched. The DNA methylation levels of CpG sites across the genome were examined with genomic DNA samples (PD, N = 32, controls, N = 22) extracted from subjects’ elbow venous blood. A mediation model was used to explore the relationship between the methylation degree of different CpG sites and childhood maltreatment and clinical symptoms.ResultsWe found that the PD group had significantly lower methylation at CpG1, CpG2, CpG3, CpG4, CpG5, CpG6, CpG7, CpG8, CpG11, CpG14, and CpG18 than did the HCs (p < 0.05). The CpG2 (r = 0.5953, p = 0.0117) site in the priming region of TEME132D gene were positively associated with PDSS score. The CpG2 (r = 0.4889, p = 0.046) site in the priming region of TEME132D gene were positively associated with physical abuse. Furthermore, path analyses showed that the methylation of CpG2 of TMEM132D played a fully mediating role in the relationship between physical abuse and PD symptom severity (95ConclusionChildhood abuse experiences, especially physical abuse, are significantly related to PD. The methylation of CpG2 of TMEM132D was shown to have a fully mediating effect between panic disorder and physical abuse. The interaction between TMEM132D methylation and physical abuse can predict panic disorder.