Polymorphic Region Research Articles

The serotonin reuptake transporter modulates mitochondrial copy number and mitochondrial respiratory complex gene expression in the frontal cortex and cerebellum in a sexually dimorphic manner.

Neuropsychiatric and neurodevelopmental disorders such as major depressive disorder (MDD) and autism spectrum disorder (ASD) are complex conditions attributed to both genetic and environmental factors. There is a growing body of evidence showing that serotonergic signaling and mitochondrial dysfunction contribute to the pathophysiology of these disorders and are linked as signaling through specific serotonin (5-HT) receptors drives mitochondrial biogenesis. The serotonin transporter (SERT) is important in these disorders as it regulates synaptic serotonin and therapeutically is the target of selective serotonin reuptake inhibitors which are a major class of anti-depressant drug. Human allelic variants of the serotonin transporter-linked polymorphic region (5-HTTLPR) such as the S/S variant, are associated with reduced SERT expression and increased susceptibility for developing neuropsychiatric disorders. Using a rat model that is haploinsufficient for SERT and displays reduced SERT expression similar to the human S/S variant, we demonstrate that reduced SERT expression modulates mitochondrial copy number and expression of respiratory chain electron transfer components in the brain. In the frontal cortex, genotype-related trends were opposing for males and females, such that reduced SERT expression led to increased expression of the Complex I subunit mt-Nd1 in males but reduced expression in females. Our findings suggest that SERT expression and serotonergic signaling have a role in regulating mitochondrial biogenesis and adenosine triphosphate (ATP) production in the brain. We speculate that the sexual dimorphism in mitochondrial abundance and gene expression contributes to the sex bias found in the incidence of neuropsychiatric disorders such as MDD and ASD.

Using synthetic chromosome controls to evaluate the sequencing of difficult regions within the human genome

BackgroundNext-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS.ResultsTo evaluate the accuracy by which these difficult regions are analyzed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically important human genome regions, including repeats, microsatellites, HLA genes, and immune receptors. These controls provide a known ground-truth reference against which to measure the performance of diverse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools and identify the errors and systematic bias that confound our resolution of these remaining difficult regions.ConclusionsThis study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.

Role of serotonin transporter and receptor gene polymorphisms in treatment response to selective serotonin reuptake inhibitors in major depressive disorder.

Significant challenges in the management of major depressive disorder include the lag period from treatment initiation to an evident response, low response rates and unpredictable disparities in outcome between patients. As a large part of these has been linked to genetic mechanisms, we tried to establish a relationship between genes associated with serotonin neurotransmission and outcome to selective serotonin reuptake inhibitor (SSRI) treatment. One hundred and twenty-five patients with moderate to severe depression [at least 15 on the Hamilton Depression (HAM-D) Rating Scale] being started on SSRI were recruited. Those with a reduction of at least 50% from baseline or an absolute score of 7 or less after 8weeks of treatment were considered as responders. The serotonin transporter linked polymorphic region 5HTTLPR, serotonin transporter intron 2 (STin2) polymorphism and the 5-HT receptor 1A rs6295 polymorphisms were studied in association with outcome. The l/l genotype of the 5HTTLPR was associated with greater likelihood of response (OR: 4.65, CI: 1.74-12.38, p=0.003). Patients with the 12/12 repeat variant of the STin2 VNTR polymorphism showed a greater reduction in HAM-D score, compared to patients with the 10/10 genotype (OR: 0.12, CI: 0.03-0.44, p=0.001). We found no association of the 5HTR1Ars6295 polymorphism with response. The 5HTTLPR polymorphism and the SLC6A4 intron 2 polymorphism were associated with treatment response, with the l/l genotype and 12-copy allele showing a tendency towards better outcomes, respectively.

Long range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

BackgroundShort read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections.ResultsTotal substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations.ConclusionsQuantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Rs62139665 Polymorphism in the Promoter Region of EpCAM Is Associated With Hepatitis C Virus-Related Hepatocellular Carcinoma Risk in Egyptians.

Hepatocellular carcinoma (HCC) is a universal health problem that is particularly alarming in Egypt. The major risk factor for HCC is hepatitis C virus (HCV) infection which is a main burden in Egypt. The epithelial cell adhesion molecule (EpCAM) is a stem cell marker involved in the tumorigenesis and progression of many malignancies, including HCC. We investigated the association of -935 C/G single nucleotide polymorphism in EpCAM promoter region (rs62139665) with HCC risk, EpCAM expression and overall survival in Egyptians. A total of 266 patients (128 HCV and 138 HCC cases) and 117 age- and sex-matched controls participated in this study. Genotyping, performed using allelic discrimination and confirmed by sequencing, revealed a significant association between EpCAM rs62139665 and HCC susceptibility, with higher GG genotype and G allele distribution in HCC patients than in non-HCC subjects. Such association was not detected in HCV patients compared to controls. EpCAM gene expression levels, determined in blood by RT-qPCR, and its serum protein expression levels, determined by ELISA, were significantly higher in GG relative to GC+CC genotype carriers in HCV and HCC patients in a recessive model. ROC analysis of EpCAM protein levels revealed significant discriminatory power between HCC patients and non-HCC subjects, with improved diagnostic accuracy when combining α-fetoprotein and EpCAM compared to that of α-fetoprotein alone. Altogether, EpCAM rs62139665 polymorphism is significantly associated with HCC and with EpCAM gene and protein expression levels in the Egyptian population. Moreover, serum EpCAM levels may hold promise for HCC diagnosis and for improving the diagnostic accuracy of α-fetoprotein.

Evaluation of basal hormone levels and androgen receptor gene mutations in individuals with recurrent abortion.

A recurrent miscarriage is at least two consecutive miscarriages in the first trimester of pregnancy. Due to the dependence of pregnancy on endocrine changes in the menstrual cycle, its disorders can also affect the outcome of pregnancy. In addition to hormonal disorders, genetic changes are essential factors in recurrent miscarriage. The development and maturation of ovulation depend on the molecular signaling pathways that respond to androgens. Hundreds of mutations leading to resistance to androgen receptor (AR) gene function have been recorded, including the 5'UTR polymorphic region. Therefore, considering the role of androgen receptors and hormonal changes in recurrent miscarriage, this study was performed to investigate the relationship between hormonal changes and AR gene mutations in patients with recurrent miscarriage. In this regard, a case-control study was performed on 150 patients with miscarriage referred to the infertility center. Hysterosalpingography, parental karyotype, vaginal ultrasound, antiphospholipid antibody measurement, anticardiolipin antibody, history and physical examination were performed to evaluate the possible causes of recurrent miscarriage. Hormone levels of LH, FSH, TSH, and Prolactin were measured and compared in two groups with known and unknown causes. Blood samples were also taken from patients, and after DNA extraction, the PCR method was used to determine AR gene mutations. The mean age was 30.2 ± 7.1 years, the mean number of abortions was 2.6 ± 1.2, and the mean duration of marriage was 6.1 ± 2.1 years. The mean of hormones in the two groups with known and unknown causes was compared, that TSH was significantly lower in the group with unknown cause (P = 0.031) and prolactin was higher in recurrent miscarriage patients with polycystic ovaries (P = 0.048). Regarding genetic evaluation, in the 5'UTR region of the androgen receptor gene, deletion of T nucleotide was observed in the +25 position, but no significant difference was found between the two groups. Generally, the findings of this study showed that thyroid dysfunction and hyperprolactinemia should be considered as an endocrine disorder in people with recurrent miscarriage, and genetic evaluation showed that the AR gene mutation was not associated with recurrent miscarriage.

First Report of Pyrus pyrifolia 'Cuiguan' Fruit Rot Caused by Monilinia fructicola in Southern China.

The early-ripening pear (i.e., Asian pear; Pyrus pyrifolia (Burm. f.) Nak.) cultivar 'Cuiguan' is an important fruit crop in southern China. In July 2020, an unknown fruit rot was observed on pear, which were harvested from an orchard in Xiajiang County of Jiangxi Province (27.62° N, 115.33° E), during storage in postharvest lab of Jiangxi Agricultural University. The incidence of this disease was 15% of 1000 post-harvest pear fruit (P. pyrifolia cv 'Cuiguan') after 10 d in storage room (20°C, 90% relative humidity). Initial symptoms were small brown nearly circular (diameter 10-20 mm) lesions with water-soaked edges on the fruit surface. The lesions expanded as concentric circles, and a gray-white mold developed in the center of the lesions. Ten fruits with typical symptoms were collected and surface-sterilized with 75% ethanol for 30 s. Small fragments (5×5 mm) at the junction of diseased and healthy tissues were disinfected with 1% sodium hypochlorite for 2 min, washed with sterile water 3 times, transferred to potato dextrose agar (PDA), and incubated at 28 ± 1℃ for 3 days. Five fungal colonies that looked similar were single-spored. The resultant fungal colonies produced irregular concentric rings with abundant gray-white aerial mycelia and gradually turned gray-brown. Conidia were unicellular, hyaline, lemon-shaped, 11.1-24.8 × 10.3-17.4 μm (n=50) in size and were produced in branched monilioid chains. These morphological characteristics were consistent with Monilinia fructicola (G. Winter) Honey (Hilber-Bodmer et al. 2010; Peter et al. 2015). To further confirm the species identification, genomic DNA of a representative isolate was obtained using an extraction kit (Biocolor, Shanghai, China), and primers ITS1/ITS4 (Munda et al. 2010), IMf F/IMf R (Ma et al. 2003), MO368-5 F/MO368-10 R (Côté et al. 2004), Mon-TubF1/Mon-TubR1, and Mon-G3pdhF/Mon-G3pdhR (Hu et al. 2011) were used to amplify the internal transcribed spacer (ITS), microsatellite, and polymorphic regions, as well as the glyceraldehyde-3-phosphate dehydrogenase (g3pdh) and beta-tubulin (tub2) genes, respectively. A BLAST search with obtained DNA sequences (GenBank Accession No. MW740236, MW788382, MW788383, MZ243141, and MZ243142, respectively) indicated 100% identity with M. fructicola (GenBank Accession No. LC312668.1 (513/513 bp), AY237426.1 (438/438 bp), FM994904.1 (490/500 bp), MN709392.1 (744/744 bp), and HQ908768.1 (1534/1534 bp), respectively). To confirm pathogenicity, 20 μl of a spore suspension (1.0 × 106 spores/ml) prepared from 7-day old PDA colonies of each of the five isolates was applied to the surface of 10 needle-wounded and 10 nonwounded, surface-disinfected 'Cuiguan' pear fruit. Ten wounded and nonwounded pears were inoculated with sterile water as controls. The experiment was repeated three times. All fruit were incubated at 25℃, 90% relative humidity. After 5 days, all wounded and nonwounded pears inoculated with M. fructicola showed symptoms similar to those observed in the storage room. Symptoms of nonwounded pears were milder than the wounded inoculated pears, while the control fruit remained healthy. A fungus with similar morphology to M. fructicola was re-isolated from the inoculated fruits, and thus, Koch's postulates were fulfilled. To our knowledge, M. fructicola has been reported to cause brown rot of pear fruit in northern China (Zhu et al. 2016), but this is the first report of M. fructicola causing rot on P. pyrifolia in southern China. As an emerging rot disease in this region, and based on its economic importance in other pear growing regions, its presence is of concern the 'Cuiguan' pear fruit industry.

Association of rs780094 and rs1260326 glucokinase regulatory protein gene polymorphisms with dyslipidemia in a group of Serbian acute ischemic stroke patients

Although genetic variations rs780094 and rs1260326 of the glucokinase regulatory protein gene (GCKR) could be associated with lipid profile imbalance, their influence on acute ischemic stroke (AIS) risk has not yet been established. The aim of this study was to investigate the influence of GCKR single nucleotide polymorphisms (SNPs) rs780094 and rs1260326 on lipid profile parameters in patients with AIS, and to evaluate the association of these SNPs with the risk of AIS. In a case-control study, a total of 148 subjects were screened for GCKR rs780094 and rs1260326 SNPs using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The lipid profile was determined based on serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triacylglycerol (TG) concentrations. The frequencies of the minor rs780094T allele and the minor rs1260326T allele were significantly lower in AIS patients compared to controls. The rs780094TT genotype and the rs1260326TT genotype were associated with decreased risk of AIS compared to wildtype carriers. In conclusion, this is the first study implying that decreased risk of AIS in rs780094 and rs1260326 homozygous minor allele carriers is not caused by dyslipidemia, but possibly by the lack of coagulation factor glycosylation.

Силикаты магния при высоких динамических нагрузках

Research on the dynamic compression of geological materials is important for understanding composition and physical condition of the deep interior of the Earth and other planets. It also provides some data on the interaction processes related to the formation and evolution of planets. Magnesium silicates dominate in Earth's mantle and, thus, are expected to become the major phases in rocky exoplanets. In particular, enstatite Mg2[Si2O6] and forsterite Mg2SiO4 are essential constituents of Earth's mantles. Strong emphasis is put on the phase transition possibility for magnesium silicates under study. A remarkable fact is the dissociation of Mg2SiO4 into the following oxides: MgO and SiO2 (stishovite). The experiments have been carried out at a pressure value of 33 GPa, which corresponds to that in Earth's mantle at a depth of 1000 km. In this paper, the results of modeling the shock-wave loading of enstatite and forsterite as the mixtures of quartz SiO2 and periclase MgO are presented. The proposed model assumes that the components of the mixture under shock-wave loading are in thermodynamic equilibrium. The components of the material under study are considered in a phase transition region as a mixture of low- and high-pressure phases. The model is also valid for a polymorphic phase transition region. The calculations of magnesium silicates are performed with account for the polymorphic phase transition of quartz and periclase. The results are validated using the data obtained in dynamic experiments.

Structure Transition and Electric Properties in Lead-Free (Na 0.5 K 0.5 ) 1- X Ag X Nb 1- X Ta X O 3 Ceramics With the Polymorphic Phase Boundary Region

Structure Transition and Electric Properties in Lead-Free (Na 0.5 K 0.5 ) 1- X Ag X Nb 1- X Ta X O 3 Ceramics With the Polymorphic Phase Boundary Region

HLA-G 5'URR regulatory polymorphisms and pre-eclampsia: correspondence.

LETTER TO THE EDITOR Keywords.

Association of Butyrophilin gene polymorphism (A465G) with milk production traits in Holstein Friesian crossbred cattle of Kerala

The butyrophilin (BTN1A1) gene is found at a quantitative trait locus (QTL) in cattle for milk production traits. The effect of a single nucleotide polymorphism (SNP) A465G in exon 8, which causes a lysine to arginine transition, on milk production attributes in Holstein Friesian crossbred cattle from Kerala was investigated in this study (151 numbers). Using custom synthesised primers, a 90-bp fragment encompassing the polymorphic region was amplified from genomic DNA isolated.Genotyping was carried out by high resolution melt curve analysis (HRM) and two genotypes KK (0.25) and KL (0.75) were detected based on melting temperature and melt curve patterns.Sanger’s sequencing and sequence analysis of representative samples confirmed the genotypes.Chi-square test showed that the population was not distributed as per Hardy-Weinberg equilibrium(p≤0.05). The relationship between the A465G transition and milk production traits like 305 daymilk, fat, SNF yields, fat and SNF per cent was determined by general linear model-analysis of variance (GLM-ANOVA). In the model herd, season of calving, parity of animal (non-geneticfactors), and genotype were considered fixed variables and milk production traits as dependent variable. The study revealed significantly higher (p≤0.01) milk fat and SNF per cent for KK genotype(4.14±0.08 %; 7.86±0.06 %) than KL genotype (3.91±0.07 %; 7.73±0.05 %). The butyrophilin genepolymorphism (A465G) can be recommended as a marker for higher milk fat and SNF per cent infuture breeding programmes in crossbred cattle of Kerala.

Interleukin-1 gene (IL-1) polymorphism in patients with Behçet’s Disease, and its relationship with disease manifestations

Objective The objective of this study was to investigate whether interleukin-1 (IL-1) gene polymorphisms are associated with susceptibility to Behçet’s disease (BD) and clinical manifestations of the disease. Methods In this cross-sectional study, we enrolled 110 patients with BD and age-and gender-matched 120 healthy controls. Five polymorphic regions of the IL-1 gene including rs 1800587 (IL1A-889 C/T), rs 2234650 (IL1R1), rs 16944 (IL1B-511 C/T), rs 315952 (IL1RN), and rs1143634 (IL1B-3954 C/T) were analyzed by using the real-time polymerase chain reaction system. Allele frequencies and genotypes were compared between groups. p ≤ 0.05 was accepted as statistically significant. Results The mean age of the patients with BD was 41 ± 12.4 years. The two groups were similar in terms of the age and gender distribution. The vast majority of BD patients had mucocutaneous involvement. The mean disease duration was 83.7 ± 67.8 months among the patients. The frequencies of each polymorphism in the IL-1 gene were similar between patients with BD and healthy controls. Conclusion The frequencies of variable IL-1 gene polymorphisms were similar between patients with BD and healthy controls.

Prevalence of the A2058G mutation in 23S rRNA gene, which determines Treponema pallidum macrolide resistance in Russian population

Objective. To investigate prevalence of the A2058G mutation in the Russian population of T. pallidum and its association with molecular subtypes. Materials and Methods. We analyzed DNA isolated from 325 samples of clinical material obtained from patients of dermatovenereological treatment and prophylactic institutions in 6 federal districts of the Russia in the period from 2014 to 2021. Patients were diagnosed with primary syphilis of the genital organs, primary syphilis of other sites, or secondary syphilis of the skin and mucous membranes. DNA was isolated using the Proba-NK reagent kit (DNA-technology, Russia) according to the manufacturer’s instructions. The presence of T. pallidum genetic material was confirmed by PCR with primers for the species-specific polA gene. Molecular typing was performed based on the analysis of polymorphic regions of species-specific T. pallidum genes. The primary decoding of the nucleotide sequences of the 23S rRNA gene fragment was carried out using the Sequencing Analysis 5.3.1 program. The analyzed fragments were aligned using the Mega 5 program. Results. Eight molecular subtypes of T. pallidum – 14d/f, 14d/g, 14b/f, 14c/f, 14i/f, 9d/f, 14b/g and 14e/f with stable dominance of subtype 14d/f – were identified in the Russian Federation in the interval 2014–2021. Three subtypes, 14d/g, 14b/g and 14b/f, carrying the A2058G mutation associated with azithromycin resistance, were identified. Conclusions. Studies on molecular typing of T. pallidum strains in the Russia have shown significant population heterogeneity. Three sublines containing the A2058G mutation were shown to exist, one of which – 14b/f – is described as rare. The obtained data confirm the relevance of continuous monitoring of the emergence of resistant strains and the development of new mutations.

Genetic variability of genetic chicken breeds estimated based on SNPS analysis in the PPARG gene

Obesity of chickens significantly reduces the efficiency of feeding, egg production, palatability of meat carcasses, slaughter yield, consumer value, and there is also such an ecological aspect as the problem of fat utilization. The search for SNPs in genes involved in lipid metabolism is an important task today for modern research in the field of poultry farming. Differences in carcass fat between breeds and lines within a breed illustrate the importance of genetic factors in lipid metabolism in chickens. The aim of the study was to find and analyze SNPs in the PPARG gene by sequencing in various chicken breeds (n = 83). The objects of the study were chickens from the VNIIGZh bioresource collection "Genetic collection of rare and endangered chicken breeds" (Pushkin, St. Petersburg) of various productivity trends (n = 83). Blood for DNA isolation was taken from hens at the age of 330 days from a wing vein into a microtube containing 50 μl of 0.5 mM EDTA as an anticoagulant. Prior to use, blood samples were stored at -200C. Genomic DNA was isolated using a standard phenolic-detergent method. The concentration and purity of the samples were determined using a NanoDrop 2000 instrument (Thermo Fisher, USA).Analysis of the polymorphism of the regulatory region of the PPARG gene was carried out by sequencing. The primer design was carried out on the basis of information from the Internet database.The studies were carried out on the basis of the laboratory of molecular genetics of the RRIFAGB. Genetic variability for rs314476701 of the PPARG gene was determined in the analyzed breeds. SNP rs316237745 revealed a strong shift of the A.

VEGF and eNOS genes polymorphism features in patients with diabetes mellitus with and without initial non-proliferative diabetic retinopathy

The endothelial NO synthase (eNOS) and vascular endothelial growth factor (VEGF) imbalance and the polymorphism of these genes may be the predisposition for diabetic retinopathy (DR) development and progression.The aim: to analyze VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes polymorphism and their combinations in patients with type 2 diabetes mellitus (DM2) with and without initial non-proliferative DR.Materials and methods. The study included 200 patients with type 2 diabetes (155 women and 45 men, age – 43–70 years): 111 people without and 89 people with DR. The polymorphism of the regulatory regions of VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes was studied using restriction fragment length polymorphism analysis and TaqMan Real-Time PCR by. Statistical processing was carried out using the software packages Statistica 10.0, SPSS Statistics 23 and the package of original programs for volumetric processing of bioinformation.Results. The VEGF-2578 heterozygosity and two complex genotypes – VEGF-2578CA:VEGF+936CC and NOS3-786CT:VEGF-2578CA:VEGF+936CC – signifi cantly decreased in patients with DR. The predisposition to early DR development to minor genotype of eNOS gene in the NOS3-786CC:VEGF+936CT complex and signifi cantly decreased the homozygous wild-type eNOS genotype in DM2 patients with ophthalmopathology were shown. NOS3-86TT:VEGF2578AA genotype signifi cantly decreased in group with retinopathy developing and the glycated hemoglobin high level.Conclusion. Along with the clinical risk factors for the development of DR in DM2, the genetic polymorphism of the regulatory regions of the genes analyzed by us has a signifi cant weight. When analyzing potential genetic markers, it is important to consider possible joint epistatic/hypostatic effects. The complex analysis of polymorphic gene can help early prognosis of the DR development.

Allele and genotype frequency for milk beta-casein in dairy cattle in the northern region of Tocantins State, Brazil

At present, there is a concern about the quality of milk and diseases related to its consumption, as it can generate discomfort and allergic reactions in some individuals due to its protein components. Thus, the present study was developed to identify the allele and genotype frequencies of genes for β casein, A1 and A2, in dairy herds in the region of Araguaína-TO, Brazil. Genetic material from 421 animals (crossbred dairy cattle in lactation) was used. All animals were numbered for identification, and DNA samples were extracted from hair bulbs. Samples for two markers from the polymorphic regions were characterized and confirmed by real time PCR using the ABI Prism® 7500 Sequence Detection System (Applied Biosystems). Allele and genotype frequencies were determined using the TaqMan™ detection system, where the primer and probe release different fluorescence signals for each allele of the polymorphism. The sampled herd showed frequencies of 28.27% for the A1 allele and 71.73% for the A2 allele. Genotype frequencies were 52.96% (223/421) for A2A2; 37.53% (158/421) for the A1A2 genotype; and 9.50% (40/421) for the A1A1 genotype. The frequency of the A1 allele for β-casein in dairy herds from the northern region of Tocantins was low and is per the results of previous studies. Although the A2A2 genotype of β-casein had a high relative frequency, the A1A2 genotype is still rather frequent, warranting greater selection pressure.

A summary-statistics-based approach to examine the role of serotonin transporter promoter tandem repeat polymorphism in psychiatric phenotypes

In genetic studies of psychiatric disorders in the pre-genome-wide association study (GWAS) era, one of the most commonly studied loci is the serotonin transporter (SLC6A4) promoter polymorphism, a 43-base-pair insertion/deletion polymorphism in the promoter region (5-HTTLPR). The genetic association signals between 5-HTTLPR and psychiatric phenotypes, however, have been inconsistent across many studies. Since the polymorphism cannot be tested via available SNP arrays, we had previously proposed an efficient machine learning algorithm to predict the genotypes of 5-HTTLPR based on the genotypes of eight nearby SNPs, which requires access to individual-level genotype and phenotype data. To utilize the advantage of publicly available GWAS summary statistics obtained from studies with very large sample sizes, we develop a GWAS summary-statistics-based approach for testing the variable number of tandem repeat (VNTR) associations with various phenotypes. We first cross-verify the accuracy of the summary-statistics-based approach for 61 phenotypes in the UK Biobank. Since we observed a strong similarity between the predicted individual-level 5-HTTLPR genotype-based approach and the summary-statistics-based approach, we applied our method to the available neurobehavioral GWAS summary statistics data obtained from large-scale GWAS. We found no genome-wide significant evidence for association between 5-HTTLPR and any of the neurobehavioral traits. We did observe, however, genome-wide significant evidence for association between this locus and human adult height, BMI, and total cholesterol. Our summary-statistics-based approach provides a systematic way to examine the role of VNTRs and related types of genetic polymorphisms in disease risk and trait susceptibility of phenotypes for which large-scale GWAS summary statistics data are available.

The role of maternal HLA-DR and HLA-G loci in determining the risk of sporadic congenital heart defects in the next generation

The paper considers the role of maternal HLA-DR (Human Leukocyte Antigens-DR) and HLA-G (Human Leukocyte Antigen-G) loci in determining the risk of the formation of sporadic congenital heart defects without chromosomal diseases in the next generation. The HLA-G molecule expressed on trophoblast performs a protective function by blocking killer receptors on natural killer cells (NK cells). At the same time, the maternal alleles of HLA-DRB1 restrict the immune response to allogeneic antigens of the paternal embryo, which may affect the severity of inflammation in the mother-embryo system and through this mechanism induce the formation of heart disease.Objective: to study the frequency distribution of the combinations of alleles and genotypes of HLA-G 3’UTR and HLA-DRB1 in women with children with sporadic congenital heart defects without chromosomal diseases. Children characteristics and research methods. There were formed 2 groups: Main Group (103 women with children with sporadic congenital heart defects without chromosomal diseases) and Control Group (103 women with conditionally healthy children). Genomic DNA was isolated by phenol-chloroform extraction. Typing of HLA-G 3’UTR 14-bp insertion/deletion was performed by amplification of polymorphic regions of genes by polymerase chain reaction with further electrophoretic detection in polyacrylamide gel 6.0. The frequency analysis of 14 alleles of the HLA-DRB1 gene was performed by real-time polymerase chain reaction. In the course of this work the authors identified predictor and protective combined genotypes.Conclusion. HLA-DRB1 and HLA-G 3’UTR 14-bp ins/del (rs 1704) make a significant contribution to determining the risk of the formation of sporadic congenital heart defects without chromosomal diseases in the next generation.

199. Polymorphisms in Key Regulatory Regions of the bla operon Correlate with the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus (MSSA)

BackgroundThe cefazolin inoculum effect (CzIE), defined as Cz minimum inhibitory concentration ≥ 16 µg/ml at high inoculum (HI-MIC), has been associated with poor clinical outcomes in patients with MSSA bacteremia or osteomyelitis. The CzIE is correlated with the presence of the blaZ gene, one of the components of the bla operon encoding the BlaZ β-lactamase (type A, B, C or D). Other portions of the bla operon include blaR and blaI (encoding the antibiotic sensor and transcriptional repressor, respectively) and the intergenic region with operator and promoter sequences (Figure 1). In BlaR, residue 293 mediates signal transduction, and the Z and R dyads in the intergenic region are the DNA-binding sites for BlaI (Figure 2). Previous experiments have shown that the regulatory portions of the bla operon play a key role in the CzIE. Here, we investigated the association between the CzIE and specific variations in the regulatory sequences of the bla operon. Figure 1. Functioning of the bla operon and the production of the staphylococcal β-lactamase BlaZ. Figure 2. Structure and key regions of the intergenic region of the bla operon, incluiding the promoter and the BlaI DNA-binding regions (Z and R dyads). MethodsA total of 437 MSSA containing blaZ were evaluated for the CzIE using broth microdilution at high inoculum. Using whole genome sequencing, the sequences of the bla operons were classified into cassettes based on unique changes in predicted amino acid sequences of BlaZ, BlaR and BlaI paired with specific nucleotide alterations in the intergenic region. The bla operon sequence of S. aureus ATCC29213 was used as reference (cassette 0).ResultsAmong 437 MSSA isolates, 46% exhibited the CzIE. We identified 55 unique bla cassettes. The bla cassettes were phylogenetically grouped in 7 clusters (Figure 3) which grouped cassettes with different BlaZ types and variations in the Z dyad, the -35 box, residue 293 of BlaR, and the blaI ribosomal binding site. Each cluster had an association to the CzIE and distinct Cz HI-MICs. The combination of: a BlaZ type A, C or D, an adenine in the position -66 of blaZ (-35 box of blaZ), a cytosine in the position -22 of blaZ(Z dyad), and either an arginine or a serine in position 293 of BlaR was a very strong predictor of the CzIE (Figure 4). Figure 3. Phylogenetical organization of bla operon cassettes into clusters, their association with polymorphisms in key regulatory regions and the CzIE. GM Cz-MIC: Geometric mean of the Cefazolin MIC at high Inoculum. Figure 4. Variations of the bla operon, their association with the CzIE, their GM (Geometric Mean) of the Cefazolin MIC and the MIC distribution of the strains with each specific combination of polymorphisms. ConclusionSpecific variations in regulatory portions of the bla operon, which are likely to influence BlaZ expression, are highly associated with the CzIE, supporting the notion that regulation of blaZ is the key factor responsible for the CzIE in MSSA. Disclosures Lorena Diaz, PhD , Nothing to disclose William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)