Allele-specific Primers Research Articles

Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentication of Meat Products.

Single-nucleotide polymorphisms (SNPs) are powerful molecular markers for the identification and differentiation of closely related organisms. A variety of methods can be used to determine the allele that is present at a specific locus in the genome, including real-time PCR by using an allele-specific primer. In order to increase the selectivity for the target allele, deliberate mismatch bases at the 3' end of the allele-specific primer may be introduced. This strategy has already been used for the identification and differentiation of microorganisms and plants. We have recently developed real-time PCR assays involving mismatch primers for the identification and differentiation of closely related deer species (red deer, fallow deer, sika deer) or the discrimination of wild boar and domestic pig in game meat products. These methods are applicable to detect meat species adulteration in food products.In this chapter, we offer a protocol for the design of PCR primer/probe systems suitable for meat species authentication in food. We address the retrieval and alignment of sequences, primer design by using a commercial software and the introduction of deliberate mismatch bases. In addition, we describe how the suitability of primer/probe systems can be tested in silico and in practice. We use the design of PCR primer/probe systems for wild boar and domestic pig as example.

Association between Transcription Factor 7-like-2 Polymorphisms and Type 2 Diabetes Mellitus in a Ghanaian Population

Type 2 diabetes mellitus (T2DM) has been strongly associated with single nucleotide polymorphisms (SNPs) in the TCF7L2 gene. This study investigated the association between rs12255372, rs7903146 in the TCF7L2 gene and T2DM in a Ghanaian population. A case-control study design was used for this study. A total of 106 T2DM patients and 110 control participants were selected. Basic data collected included body mass index, blood pressure and socio-demographics. Fasting blood samples were collected and processed for: serum lipid analysis, plasma glucose estimation and plasma HbA1c estimation. Parts of the whole blood samples were used for DNA extraction using a modified salting-out method. Common and allele-specific primers were designed for genotyping using the Modified Tetra-Primer Amplification assay. Associations were evaluated using logistic regression models. The rs7903146 risk variant was significantly associated with 2.16 vs. 4.06 increased odds for T2DM in patients <60 years vs. ≥60 years. Both rs7903146 and rs12255372 were significantly associated with increased odds of T2DM in women, overweight/obese, T2DM negative family history (T2DM-NFH) and low-HDL-C. In a multivariate model, rs7903146 but not rs12255372 was significantly associated with 2.18, 5.01 and 2.25 increased odds of T2DM, under the codominant, recessive and additive model, respectively (p < 0.05). The association between rs7903146 and rs12255372 with T2DM is more highly associated in a subgroup—women and those with T2DM-NFH, yet who have cardiometabolic risk.

Multiplex Fragment analysis detects all COVID-19 variants of concern

Introduction/ObjectiveThe majority of tracking methods have employed whole genome sequencing, which can be very expensive and time consuming. An alternative method has been to use genotyping of specific mutations to identify variants. However, tracking SARS-CoV-2 variants by targeted methods has been a moving target. Most methods only multiplex four targets per reaction, but we have multiplexed 8 targets in a single tube using fragment analysis.Methods/Case ReportFluorescently labeled primers targeted a combination of insertion/ deletion mutations and single nucleotide mutations. The PCR amplified products, amplicons, were separated by capillary electrophoresis. Primers were designed to detect changes in size indicative of insertion or deletion mutations including: ORF1A:Del3675_3677, S:Del69_70, S:Del144, S:Del157_158, S:Del242_244, ORF8:Del119_120, and ORF8:ins28269-28273. Allele-specific primers were designed to detect both the wild-type and mutated versions of S:N501Y, S:E484K, and S:L452R.Residual nasopharyngeal and nasal specimens testing positive for SARS-CoV-2 by RT-PCR or isothermal amplification (IDnow) methods were selected from May 1- June 24, 2021. Variant analysis was performed by multiplex targeted PCR and whole genome sequencing in parallel on the same specimens to determine positive percent agreement.Results (if a Case Study enter NA)Variant analysis was performed on 250 specimens detecting each of the major variants of concern Alpha (B.1.1.7, U.K. origin, n= 108), Beta (B.1.351, South Africa origin, n=3), Gamma (P.1, Brazil origin, n=12), Delta (B.1.617.2, Indian origin, n=17), and Iota (B.1.526, New York, n=5). Some specimens with low viral load were detected by only PCR (n=18), only WGS (n=41), or neither (n=20). Overall positive percent agreement was 95% (163/171).ConclusionThis adjustable method robustly and accurately identifies COVID-19 VOCs utilizing a platform amenable to multiple targets (20-40 targets ranging from 100-500b.p. across four fluorescent channels) using equipment commonly found in routine molecular pathology laboratories. Future directions include adjusting targets to detect new variants.

English

The purpose of this study was to apply an intentional mismatch design technique for primer-based detection of gene targets. In this study, wild-type versus mutant cystic fibrosis transmembrane conductance regulator alleles as well as two related adenovirus B7 and E4 genomes were used to test discrimination of primers. Lambda viral DNA genome template as well as published primers versus purified human genome template was used as controls. The presence of the ΔF508 mutation and adenovirus B7 could be accurately detected using allele-specific primers, with the reverse ΔF508 primers containing an intentional mismatch, to increase the steric hindrance, therefore both diagnosing CFTR and associated viruses. Key words: cystic fibrosis, genetic diagnosis, assay, PCR, electrophoresis.

Abstract 399: Pan-cancer survey of HLA loss of heterozygosity using a robustly validated NGS-based machine learning algorithm

Abstract HLA loss of heterozygosity (LOH) is increasingly being recognized as an important immune escape mechanism in response to checkpoint inhibitor therapy. HLA LOH reduces the repertoire of neoantigens displayed on the cell surface of cancer cells, limiting the efficacy of the immune system to detect and eliminate them. Though highly accurate HLA LOH detection algorithms are needed to allow clinical utility, the field lacks robust, allele-specific validation approaches. Moreover, algorithms of unknown sensitivity and specificity have led to significant discrepancies in the estimated occurrence of HLA LOH as an immune escape mechanism across tumor types. To address these challenges, we have developed a machine learning algorithm to detect HLA LOH (DASH - Deletion of Allele-Specific HLAs), established the accuracy of the algorithm with an allele-specific PCR validation strategy, investigated the frequencies of HLA LOH across 14 tumor types in a cohort of over 800 patients and observed allele-specific neoantigen expansion in response to immunotherapy. To build DASH, we profiled 279 patients on the ImmunoID NeXT Platform to create a training dataset. Our novel features, which account for allele-specific differences in exome probe capture and capitalize on our whole exome platform by including information about copy number alterations in the regions flanking the HLA genes, were used to train an XGBoost model. Orthogonal, allele-specific validation was required to accurately assess sensitivity and specificity for clinical utility. Thus, we profiled over 30 paired tumor-normal cell lines on the ImmunoID NeXT Platform® and identified cell lines with HLA LOH. Using in silico mixtures, we found 100% sensitivity and specificity for tumors with at least 36% tumor purity. Next, we designed a digital PCR (dPCR) assay using patient-specific, allele-specific primers that target a single HLA allele while avoiding all other HLA alleles and tested the limit of detection of the assay in the same cell lines. Then, we performed dPCR with patient-specific primers on 20 tumor and normal sample pairs and found 94% sensitivity. After establishing the high sensitivity and specificity of DASH, we profiled over 800 patients spanning 14 tumor types on the ImmunoID NeXT Platform. We found that over 25% of patients in the majority of tumor types had at least one HLA LOH event. Further, we observed that novel neoantigens that arose during checkpoint treatment were significantly more likely to bind to deleted HLA alleles as compared to the remaining HLA alleles in a head and neck carcinoma cohort treated with anti-PD-1 therapy, shedding light on the mechanism of immune escape in response to checkpoint inhibitors. In summary, we introduced an HLA LOH detection method, performed allele-specific validation, exposed widespread HLA across tumor types and observed the mechanism of immune escape in response to immunotherapy. Citation Format: Rachel Marty Pyke, Datta Mellacheruvu, Charles Abbott, Steven Dea, Eric Levy, Simo V. Zhang, Nikita Bedi, A. Dimitrios Colevas, Devayani Bhave, Manju Chinnappa, Gabor Bartha, John Lyle, John West, Michael Snyder, John Sunwoo, Richard Chen, Sean Michael Boyle. Pan-cancer survey of HLA loss of heterozygosity using a robustly validated NGS-based machine learning algorithm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 399.

Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.

Gene Expression Modification by an Autosomal Inversion Associated With Three Male Mating Morphs.

Chromosomal inversions are structural rearrangements that frequently provide genomic substrate for phenotypic diversity. In the ruff Philomachus pugnax, three distinct male reproductive morphs (Independents, Satellites and Faeders) are genetically determined by a 4.5 Mb autosomal inversion. Here we test how this stable inversion polymorphism affects gene expression in males during the lekking season. Gene expression may be altered through disruptions at the breakpoints and the accumulation of mutations due to suppressed recombination. We used quantitative PCR to measure expression of 11 candidate inversion genes across three different tissues (liver, adrenal glands and gonads) and tested for allelic imbalance in four inversion genes across 12 males of all three morphs (8 Independents, 2 Satellites, 2 Faeders). We quantified transcripts of CENPN, an essential gene disrupted by the inversion at the proximal breakpoint, at different exons distributed near and across the breakpoint region. Consistent with dosage dependent gene expression for the breakpoint gene CENPN, we found that expression in Independents was broadly similar for transcripts segments from inside and outside the inversion regions, whereas for Satellites and Faeders, transcript segments outside of the inversion showed at least twofold higher expression than those spanning over the breakpoint. Within the inversion, observed expression differences for inversion males across all four genes with allele-specific primers were consistent with allelic imbalance. We further analyzed gonadal expression of two inversion genes, HSD17B2 and SDR42E1, along with 12 non-inversion genes related to steroid metabolism and signaling in 25 males (13 Independents, 7 Satellites, 5 Faeders). Although we did not find clear morph differentiation for many individual genes, all three morphs could be separated based on gene expression differences when using linear discriminant analysis (LDA), regardless of genomic location (i.e., inside or outside of the inversion). This was robust to the removal of genes with the highest loadings. Pairwise correlations in the expression of genes showed significant correlations for 9–18 pairs of genes within morphs. However, between morphs, we only found a single association between genes SDR42E1 and AROM for Independents and Satellites. Our results suggest complex and wide-ranging changes in gene expression caused by structural variants.

Polymorphism of pro-inflammatory cytokine genes in acute intestinal infections

Acute intestinal infections are widespread and hold the second place among infectious diseases, giving way solely to respiratory diseases. In this regard, much attention has been paid to examining acute intestinal infections, including immunopathogenetic mechanisms. And since proinflammatory and antiinflammatory cytokines play an important role in development of inflammatory reactions affecting disease severity and outcome, it becomes reasonable to study polymorphism of genes governing production of related molecules. Thus, the aim of our study was to examine the polymorphism in the IL-1β Т31С and IL-2 T330G genes; such mutations were characterized by nucleotide replacement affecting the gene promoters, which influenced production rate and level of the relevant cytokines. There were enrolled 108 patients with acute intestinal infections comprising main group as well as 94 apparently healthy subjects in the control group. Genomic DNA was isolated from the whole blood leukocytes by using a DNA-express-blood reagent, followed by conducting amplification reaction with two pairs of allele-specific primers. The polymorphism in the IL-1β, IL-2 genes was determined by PCR with primers purchased from Litech LLC (St. Petersburg). Data processing was carried out by using the statistical Statistica 6 suite software. While assessing the carriage rate of IL-1β T31C gene polymorphic markers by using the multiplicative inheritance model in both groups, the prevalence of the normal T allele and, respectively of the —31TT and —31TC genotypes with OR = 1.83 and an interval of 1.04—3.22 (χ2 = 6.35, p = 0.04, df = 2) was found, which allowed us to identify a relationship between the carriage of IL-1β в gene heterozygous variant and potentially elevated risk of AII. Regarding the IL-2 T330G gene, it was found that pathological G alleles was more markedly abundant in patients with acute intestinal infections compared to control group. Analyzing diverse IL-2 T330G carriage rate in patients with acute intestinal infections revealed that carriers of the TG heterozygous variant predominated — 56.48% (χ2 = 17.75, F = 0.000031), whereas pathological genotype GG was found in 13.89% (χ2 = 12.31, F = 0.000663, p 0.05), with high probability of the relationship between carriage of these genotypes and a risk of disease development (OR — 3.63 [1.97—6.68] and OR — 6.91 [2.12—22.59]). Hence, the carriage of polymorphic variants of the IL-1β T31C and IL-2 T330G genes was associated with elevated risk of developing AII in case of infection with pathogenic microorganisms.

A Study on β-Casein Gene Polymorphism in Crossbred Cattle and Murrah / Graded Murrah Buffalo in Tamil Nadu

Background: Majority of the people believed that, only the native breeds have A2 milk than exotic or crossbred animals. There is no enough research on milk protein variants have been carried out in Indian zebu cattle. Present study was conducted to screen more number of crossbred cattles and buffaloes in Tamil Nadu to identify the frequency of A1 and A2 alleles in the population.Methods: The study was conducted on 68 cattle and 172 murrah / graded murrah buffaloes to explore the polymorphic variants of β-casein gene. Genomic DNA was extracted by phenol-chloroform method. Polymerase Chain Reaction (PCR) was performed with allele specific primers to amplify a 244 bp long fragment of beta-casein gene and visualized in 2% agarose gel electrophoresis. The population genetic indices were calculated based on the formulas.Result: In the present study revealed higher level of A2A2 genotype frequency (1.00) and fixation of A2 allele in kangayam cattle and murrah / graded murrah buffalo. The observed frequency of A1A1, A1A2 and A2A2 genotypes were 0.38,0.62 and 0.00 for jersey crossbred and 0.29, 0.71 and 0.00 for HF crossbred cattle. The range of Expected homozygosity (0.50 to 1.00), polymorphism information content (0.30 to 0.38), effective number of alleles (1.00 to 2.00) and level of possible variability realization value (44.54% to 100%) reflected existence of medium genetic variability in studied population.

Detection of cell-free fetal DNA in maternal plasma using two types of compound markers.

With the discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing became possible. However, analysis of low-level cffDNA against high background maternal DNA remains complicated and challenging. To circumvent this limitation, selective amplification of cffDNA was used in this study. Two kinds of compound markers (namely DIP-STR and SNP-STR), both based on selective amplification, were used here for targeting fetal DNA. By designing two allele-specific forward primers for DIP-STR and SNP-STR, DNA fragments with different DIP/SNP alleles can be selectively amplified. When analyzing maternal plasma DNA, these markers can selectively target paternally inherited fetal alleles whose DIP/SNP allele was not shared with the mother. In this study, 21 families were studied with six DIP-STRs and 11 SNP-STRs. Fetal DNA was successfully detected across plasma samples for at least one marker. Detection rate varied between DIP-STR and SNP-STR markers, and DIP-STR outperforms SNP-STR. Fetal alleles obtained from maternal plasma were double confirmed by genotyping paternal genomic DNA and fetal genomic DNA from amniocentesis. This study demonstrated that selective amplification strategy can be used to target cffDNA in maternal plasma, which will be a promising method for noninvasive prenatal paternity testing.

Evaluation of β-Thalassaemia Cases for Common Mutations in Western Rajasthan.

β-Thalassaemia, the most common monogenic disorder, is characterized by genetic heterogeneity at the molecular level. More than 300 mutations of the β globin gene have been characterized all over the world, however, few common mutations account for majority of the cases in various populations. The present study aimed to screen known cases of β-thalassaemia in the Western part of Rajasthan state for five common mutations. The study included 144 known cases of β-thalassaemia of all clinical phenotypes. Cases were diagnosed based on clinical features, haematology investigations including haemogram and Hb-HPLC. Blood samples from cases were taken for mutation analysis. After DNA extraction, mutations were characterized by the polymerase chain reaction method employing allele specific priming technique (AMRS) to study the five mutations including IVS-I-5 (G → C), IVS-I-1 (G → T), CD41/42 (-TCTT), CD 8/9 (+G) and 619bp deletion from the 3' end of the β-globin gene using a total of seven different primers. Of all 144 cases, 74 (51.38% of all) cases were of β-thalassaemia major, five (3.4% of all) cases were of β-thalassaemia intermedia and 65 (45.14% of all) cases were of β-thalassaemia minor. Mutation analysis revealed that five common mutations were present in 130 (90.27% of all) cases. Among identified mutations, highest frequency of mutation was of IVS-I-5 (G → C) identified in 73 cases (50.7% of all cases). In 11 (7.63% of all) cases, more than one mutation was identified. β-Thalassaemias are common in Western Rajasthan; however, there is dearth of literature from this part of the country. We observed that five common mutations are common in this part of the country also. These observations are helping us in forming the basis for comprehensive diagnostic database that would not only be useful for genetic counselling but also for prenatal diagnosis.

A Novel SNP-STR System Based on a Capillary Electrophoresis Platform.

Various compound markers encompassing two or more variants within a small region can be regarded as generalized microhaplotypes. Many of these markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. SNP-STR is a compound biomarker composed of a single nucleotide polymorphism (SNP) and a closely linked short tandem repeat polymorphism (STR), and possess the advantages of both SNPs and STRs. In addition, in conjunction with a polymerase chain reaction (PCR) technique based on the amplification refractory mutation system (ARMS), SNP-STRs can be used for forensic unbalanced DNA mixture analysis based on capillary electrophoresis (CE), which is the most commonly used platform in worldwide forensic laboratories. Our previous research reported 11 SNP-STRs, but few of them are derived from the commonly used STR loci, for which existing STR databases can be used as a reference. For maximum compatibility with existing DNA databases, in this study, we screened 18 SNP-STR loci, of which 14 were derived from the expanded CODIS core loci set. Stable and sensitive SNP-STR multiplex PCR panels based on the CE platform were established. Assays on simulated two-person DNA mixtures showed that all allele-specific primers could detect minor DNA components in 1:500 mixtures. Population data based on 113 unrelated Chengdu Han individuals were investigated. A Bayesian framework was developed for the likelihood ratio (LR) evaluation of SNP-STR profiling results obtained from two-person mixtures. Furthermore, we report on the first use of SNP-STRs in casework to show the advantages and limitations for use in practice. Compared to 2.86 × 103 for autosomal STR kits, the combined LR reached 7.14 × 107 using the SNP-STR method in this casework example.

Genotyping of RHD c.1227G>A allele by melting curve analysis

BackgroundDEL is the weakest known D-positive phenotype and is detectable only by adsorption and elution tests. RHD c.1227G>A is an important marker for DEL phenotype in East Asians. The aim of this study was to develop a method for RHD c.1227G>A genotyping by single-tube PCR with melting curve analysis. MethodsTwo GC-rich tails of different lengths were attached to the 5′-end of allele-specific primers for RHD 1227G and 1227A alleles, such that RHD c.1227G>A could be distinguished by the melting temperature. A total of 145 D-negative Chinese Han blood donors were genotyped for RHD c.1227G>A by melting curve analysis, conventional polymerase chain reaction with sequence-specific primers (PCR-SSP), and sequencing. ResultsIn 143 subjects (143/145, 98.6%), PCR-SSP and melting curve analysis produced consistent results with RHD exon 9 sequencings. Two samples were genotyped as RHD 1227G/A by PCR-SSP, but as RHD 1227A/A or A/- by melting curve analysis. These two samples were confirmed to be RHD 1227A/A or A/-. Based on RHD exon 9 sequencing, the accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the melting curve analysis for detecting both RHD 1227A and 1227G were all 100%. In contrast, the accuracy, specificity and positive predictive value of PCR-SSP for RHD 1227G detection were 98.62%, 98.21% and 94.29%, respectively, which were lower than those observed with the melting curve analysis. ConclusionMelting curve analysis for RHD c.1227G>A genotyping is a simple, rapid, and reliable method, superior to conventional PCR-SSP.

ТЕХНОЛОГИЯ REAL-TIME ПЦР-ГЕНОТИПИРОВАНИЯ BOS TAURUS ПО ВАРИАНТАМ АЛЛЕЛЕЙ А И B ГЕНА КАППА-КАЗЕИНА

The main goal of the study was to develop and test an effective technology for cattle genotyping by the CSN3 gene based on real-time PCR with hybridization-fluorescence detection. There was developed a method for real-time PCR for cattle genotyping by A and B alleles of the CSN3 gene in the format of hybridization-fluorescence detection, involving the use of two 5/-fluorescence-labeled forward allele-specific primers, one reverse common primer, and one anti-primer labeled with a fluorescence quencher at the 3/-end of the oligonucleotide. As a result of practical studies aimed at testing the developed method, we obtained the technical result provided by the proposed technology, expressed in the effective identification of the desired genotypes due to correct interpretation of these curves of increasing fluorescence intensity, the results reliability of which was also confirmed by the well-known PCR-RFLP analysis technique for Bos taurus genotyping for similar allelic variants of the kappa-casein gene.

Prevalence and distribution of major β-thalassemia mutations and HbE/β-thalassemia variant in Nepalese ethnic groups

BackgroundBeta-thalassemia is a genetic disorder that is inherited in an autosomal recessive pattern. This genetic disease leads to a defective beta-globin hemoglobin chain causing partial or complete beta-globin chain synthesis loss. Beta-thalassemia major patients need a continuous blood transfusion and iron chelation to maintain the normal homeostasis of red blood cells (RBCs) and other systems in the body. Patients also require treatment procedures that are costly and tedious, resulting in a serious health burden for developing nations such as Nepal. MethodsA total of 61 individuals clinically diagnosed to have thalassemia were genotyped with multiplex amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Twenty-one major mutations were investigated using allele-specific primers grouped into six different panels. ResultsThe most common mutations found (23%) were IVS 1–5 (G-C) and Cd 26 (G-A) (HbE), followed by 619 deletion, Cd 8/9 (+G), Cd 16 (–C), Cd 41/42 (–TTCT), IVS 1–1 (G–T), Cd 19 (A-G), and Cd 17 (A-T) at 20%, 12%, 8%, 6%, 4%, 3%, and 1%, respectively. ConclusionThe results of this study revealed that Nepal's mutational profile is comparable to that of its neighboring countries, such as India and Myanmar. This study also showed that thalassemia could be detected across 17 Nepal's ethnic groups, especially those whose ancestors originated from India and Central Asia.

АССОЦИАЦИЯ ПОЛИМОРФИЗМОВ Ser447Ter ГЕНА LPL И rs9939609 ГЕНА FTO С ОЖИРЕНИЕМ У ДЕТЕЙ И ПОДРОСТКОВ В ПОПУЛЯЦИИ РОСТОВА-НА-ДОНУ

The aim of this study was to study the correlation between the Ser447ter (C-G) polymorphisms of the LPL gene and rs9939609 of the FTO gene and obesity in children and adolescents of the Rostov region (Russia). A case-control study examined the relationship between the rs9939609 polymorphisms of the FTO gene and Ser447Ter (S447X) of the LPL gene with obesity in 520 children and adolescents of both sexes aged 3 to 18 years: the main group consisted of 370 obese children and adolescents, and the control group - 150 children and adolescents without obesity. Genotyping of polymorphisms T / A rs9939609 of the FTO gene and C / G Ser447Ter of the LPL gene was performed using PCR- allele-specific primers. Polymorphisms of the FTO rs9939609 and LPL Ser447Ter genes in donor DNA samples were typed by the electrophoretic method using commercial test systems from the Litekh research and production com-pany (Russia). The relationship between the rs9939609 polymorphism of the FTO gene and obesity was revealed, differences (P<0.05) were established between the main and control groups in the frequency of occurrence of the AA genotype (P = 0.0079) and allele A (P = 0.005; OR 0.67; 95% CI 0.51–0.88) of the rs9939609 polymorphism of the FTO gene. Combinations of genotypes with increased and decreased risk of obesity in the child and adolescent population of Rostov-on-Don were determined. Antagonistic rela-tionships between the rs9939609 polymorphisms of the FTO gene and Ser447Ter of the LPL gene are shown. Using the MDR method, combinations of genotypes with an increased and decreased risk of obe-sity in the child and adolescent population of Rostov-on-Don were established.

Association between Fertility HCD Haplotype, Milk Production, and Reproductive Performance in Holstein Cows

The survey results of testing the Holstein cows for carriage of the fertility haplotype for cholesterol deficiency (HCD) associated with the fetal death detected at various stages of prenatal and early postnatal ontogenesis has been reported. Economically important traits of the animals tested as carrying the defect caused by HCD in their genotypes are examined. The survey sample consisted of the animals (n = 583) born in 2007–2017 on the breeding farm in Leningrad oblast. A comparative assessment of the HCD+-affected animals (tested carriers of a genetic defect caused by cholesterol deficiency) and the HCD–-unaffected animals (tested noncarriers of a genetic defect caused by cholesterol deficiency) was performed based on their milk production and reproductive performance parameters. DNA extracted from animal blood served as the testing material. The genotypes were determined based on the polymerase chain reaction with the use of allele-specific primers. It was found that 35 animals (6.0%) had a mutant allele at the ApoB gene locus present in their genotypes. These cows were not inferior to their genetic defect-free peers in breeding value for milk yields and milk protein and fat percentage. The HCD+-affected cows born in 2014 were characterized by higher values for the milk protein content (3.29 ± 0.05%) when compared to the HCD–-unaffected cows (3.17 ± 0.01%). A smaller number of sperm doses were used to reach effective insemination in the HCD carrier cows born in 2013 (n = 11) and 2014 (n = 11), which significantly affected the terms of the first effective-insemination. The cows born in 2014 carrying a mutant allele at the ApoB gene locus present in their genotypes had less service interval length when compared to their peers. The HCD+-affected cows used in production have no impact on overall herd performance and fertility. However, the animals with the HCD+ status should be identified and registered in a timely manner. The record-keeping system can allow us to reduce the number of carriers of this genetic defect in subsequent generations.

Susceptibility to Plasmodium falciparum Malaria: Influence of Combined Polymorphisms of IgG3 Gm Allotypes and Fc Gamma Receptors IIA, IIIA, and IIIB.

The binding of immunoglobulin (Ig) to Fc gamma receptors (FcgR) at the immune cell surface is an important step to initiate immunological defense against malaria. However, polymorphisms in receptors and/or constant regions of the IgG heavy chains may modulate this binding. Here, we investigated whether polymorphisms located in FcgR and constant regions of the heavy chain of IgG are associated with susceptibility to P. falciparum malaria. For this purpose, a clinical and parasitological follow-up on malaria was conducted among 656 infants in southern Benin. G3m allotypes (from total IgG3) were determined by a serological method of hemagglutination inhibition. FcgRIIA 131R/H and FcgRIIIA 176F/V genotypes were determined using the TaqMan method and FcgRIIIB NA1/NA2 genotypes were assessed by polymerase chain reaction using allele-specific primers. Association analyses between the number of malaria infections during the follow-up and polymorphisms in IgG G3m allotypes and FcgR were studied independently by zero inflated binomial negative regression. The influence of combinations of G3m allotypes and FcgRIIA/FcgRIIIA/FcgRIIIB polymorphisms on the number of P. falciparum infections, and their potential interaction with environmental exposure to malaria was assessed by using the generalized multifactor dimensionality reduction (GMDR) method. Results showed that individual carriage of G3m24 single allotype and of G3m5,6,10,11,13,14,24 phenotype was independently associated with a high risk of malaria infection. A risk effect for G3m6 was observed only under high environmental exposure. FcgRIIIA 176VV single genotype and combined carriage of FcgRIIA 131RH/FcgRIIIA 176VV/FcgRIIIB NA1NA2, FcgRIIA 131HH/FcgRIIIA 176FF/FcgRIIIB NA1NA1, FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA2NA2 and FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA1NA2 genotypes were related to a high number of malaria infections. The risk was accentuated for FcgRIIIA 176VV when considering the influence of environmental exposure to malaria. Finally, the GMDR analysis including environmental exposure showed strengthened associations with a malaria risk when FcgRIIA/FcgRIIIA/FcgRIIIB genotypes were combined to G3m5,6,11,24 and G3m5,6,10,11,13,15,24 phenotypes or G3m10 and G3m13 single allotypes. Our results highlight the relevance of studying IgG heavy chain and FcgR polymorphisms, independently as well as in combination, in relation to the individual susceptibility to P. falciparum infection. The intensity of individual exposure to mosquito bites was demonstrated to impact the relationships found.

Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations.

The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.

Efficiency of S-genotyping for diversity screening and self-incompatible group identification of almond cultivars within the Mediterranean basin

ABSTRACT Molecular tools were largely used to characterise native almond (Prunus dulcis Mill.) cultivars, to elucidate their diversification and to establish their membership to Mediterranean germplasm pools. Hereby, the efficiency of the application of PCR techniques to study the genetic diversity of S alleles in almond was tested to identify self-incompatible cultivar combinations and groups. Hence, an analysis with consensus AS1II and AmyC5R and with allele-specific primers was performed for 25 almond accessions including 17 local Tunisian cultivars, 7 advanced selections, and 1 extra group from Italy in order to identify their S-genotype. Interestingly, all genotypes were assigned to their specific self-incompatible groups and a new group (LI) has been established. The two cultivars ‘Dillou’ and ‘Porto’ both considered as self-compatible have been demonstrated to be self-incompatible. Results reveal also a discrepancy for the S-genotype assigned to ‘Zahaaf’ preserved in the International almond collection. Thus, S genes genotyping of commercially used or newly released cultivars is very useful as we demonstrated that it may show that common knowledge may be partly inaccurate. For important varieties, this immediately has a consequence for cultivation practices, also for smallholder orchards as changing an incorrect practice may in time increase yield and also guarantee yield stability.