Single Nucleotide Primer Extension Research Articles

Association of iron supplementation, HFE and AMPD1 polymorphisms and biochemical iron metabolism parameters in the performance of a men’s World Tour cycling team: A pilot study

BackgroundNutritional strategies with iron supplementation have been shown to be effective in preventing the decline of blood biochemical parameters and sports performance. The aim of the study was to describe biochemical iron metabolism parameters in association with iron supplementation and HFE and AMPD1 polymorphisms in a Union Cycliste Internationale (UCI) World Tour cycling team to evaluate performance during a whole season MethodsTwenty-eight professional men cyclists took part in this longitudinal observational pilot study. AMPD1 c.34 C>T (rs17602729) and HFE c.187 C>G (rs1799945) polymorphisms were genotyped using Single Nucleotide Primer Extension (SNPE). All the professional cyclists took oral iron supplementation throughout the season. Four complete blood analyses were carried out corresponding to UCI controls in January (1st), April (2nd), June (3rd) and October (4th). Data on participation in three-week Grand Tours, kms of competition and wins were analyzed. Results: In performance, especially in wins, there was a significant effect in HFE on biochemical hemoglobin (F = 4.255; p = 0.021) and biochemical hematocrit (F = 5.335; p = 0.009) and a hematocrit biochemical × genotype interaction (F = 3.418; p = 0.041), with higher values in professional cyclist with GC genotype. In AMPD1 there were significant effects in the biochemical iron x genotype interaction in three-week Grand Tours (F = 3.874; p = 0.029) and wins (F = 3.930; p = 0.028) ConclusionsBlood biochemical iron metabolism parameters could be related to performance in the season due to increasing hemoglobin and hematocrit concentration under iron supplementation, associated with winning in the professional cyclists with GC genotype of the HFE polymorphism.

Pilot Study on the Prevalence of Diamine Oxidase Gene Variants in Patients with Symptoms of Histamine Intolerance.

A retrospective pilot study was carried out to investigate the prevalence of four variants of the diamine oxidase (DAO) encoding gene (AOC1) in Caucasian adults with symptoms of histamine intolerance. In a cohort of 100 patients and 100 healthy individuals, DAO-encoding gene non-synonymous Single Nucleotide Variations (SNVs) were genotyped by multiplex single-nucleotide primer extension (SNPE) and capillary electrophoresis, and serum DAO activity was analyzed with a radio-extraction assay. The study found that 79% of individuals with symptoms of histamine intolerance harbored one or more of the four SNVs associated with reduced DAO activity. No significant differences were found in the prevalence of any variant between the group of patients and healthy controls. However, when considering the status of the alleles associated with DAO deficiency, more homozygous alleles were observed in histamine-intolerant patients. Moreover, a slightly but statistically higher percentage of patients had a high genetic risk score, reflecting the cumulative effect of carrying multiple DAO deficiency-associated gene variants and a high load of risk alleles (homozygous). A relationship between serum DAO activity and the genetic load of one specific SNV was observed, with DAO activity being significantly lower in patients homozygous for rs2052129. These results potentially support that carrying multiple DAO deficiency-associated gene variants and a high load of risk alleles (homozygous) is more relevant than the mere presence of one or more SNVs. Further studies are needed to determine the predictive value of these DAO-encoding gene variants.

Predictive Factors and ACE-2 Gene Polymorphisms in Susceptibility to Long COVID-19 Syndrome.

Long COVID-19 syndrome is present in 5-10% of patients infected with SARS-CoV-2, and there is still little information on the predisposing factors that lead to its development. The purpose of the study was to evaluate the predictive factors in early symptoms, clinical features and the role of Angiotensin-Converting Enzyme-2 (ACE-2) c.513-1451G>A (rs2106806) and c.15643279T>C (rs6629110) polymorphisms in the susceptibility to developing Long COVID-19 syndrome subsequent to COVID-19 infectionA total of 29 patients who suffered COVID-19 were recruited in a descriptive longitudinal study of two groups: Long COVID-19 (n = 16) and non-Long COVID-19 (n = 13). Early symptoms and clinical features during COVID-19 were classified by a medical service. ACE-2 polymorphisms were genotyped by using a Single Nucleotide Primer Extension (SNPE). Of the early symptoms, fatigue, myalgia and headache showed a high risk of increasing Long COVID-19 susceptibility. Clinical features such as emergency care, SARS-CoV-2 reinfection, previous diseases, respiratory disease and brain fog also had a high risk of increasing Long COVID-19 susceptibility. The A allele in the rs2106806 variant was associated with an odds ratio (OR) of 4.214 (95% CI 2.521-8.853; p < 0.001), and the T allele in the rs6629110 variant was associated with an OR of 3.754 (95% CI 1.785-6.105; p = 0.002) of increasing Long COVID-19 susceptibility. This study shows the risk of ACE-2 polymorphisms, different early symptoms and clinical features during SARS-CoV-2 infection in susceptibility to Long COVID-19.

Cumulative effect of AOC1 gene variants on symptoms and pathological conditions in adult women with fibromyalgia: a pilot study.

Introduction: The amine oxidase copper-containing 1 (AOC1) gene encodes for the diamine oxidase (DAO) enzyme. DAO is an enzyme that catabolizes some molecules, including histamine, and is the degradative enzyme in the polyamine catabolic pathway that is active in intestinal mucosal cells. Variants of AOC1 are associated with reduced DAO activity, resulting in accumulation of high levels of histamine and causing a wide range of neurological, gastrointestinal, and epidermal disorders, which are present in people with fibromyalgia. This study aimed to evaluate the impact of four AOC1 gene variants, namely, rs10156191, rs1049742, rs1049793, and rs2052129, on fibromyalgia symptoms measured by the Fibromyalgia Impact Questionnaire (FIQ), such as sleep disorders, atopic dermatitis, migraine, gastrointestinal (GI) disorders, allergies, and intolerances, in adult women with fibromyalgia. Methods: The sample consisted of 100 unrelated women with fibromyalgia between 33 and 60years of age (48.48years ±7.35), whose were diagnosed by a rheumatologist based on symptoms such as pain, stiffness, and fatigue. Single-nucleotide polymorphisms (SNPs) of AOC1 were identified using oral mucosa samples collected following a standard hygiene protocol. DNA was extracted, and gene variants of interest were analyzed using multiplex single-nucleotide primer extension (SNPE). Clinical data were collected using the FIQ and a series of variables that quantified the intensity and frequency of the symptoms. Results: The minor allele frequencies of rs10156191, rs1049742, rs1049793, and rs2052129 were 31.5, 10, 32.5, and 27%, respectively. Each variant was found to be in Hardy-Weinberg equilibrium, but partial linkage disequilibrium between AOC1 SNPs is suspected. The results show that fibromyalgia symptoms measured using the FIQ tend to increase with the number of risk alleles and that the intensity of dry skin and low stool consistency may be associated with an increase in the number of these alleles. Conclusion: This study constitutes the first step in investigating associations between fibromyalgia symptoms and candidate variants of the AOC1 gene in DAO enzyme activity. Identification of reduced DAO activity may improve the quality of life and treatment of symptoms in fibromyalgia patients.

Prevalence of Genetic Diamine Oxidase (DAO) Deficiency in Female Patients with Fibromyalgia in Spain

Diamine oxidase (DAO) is an enzyme that metabolizes intestinal histamine. Single nucleotide polymorphisms (SNPs) of the Amine Oxidase Copper Containing 1 (AOC1) gene can lead to low enzymatic activity or functionality in histamine metabolism. This study aimed to determine the prevalence of DAO deficiency for four variants of the AOC1 gene, p.Thr16Met (rs10156191), p.Ser332Phe (rs1049742), p.His664Asp (rs1049793), and c.691G > T (rs2052129), in 98 Spanish women with fibromyalgia between the ages of 33 and 60 years, and compare the distribution of allelic and genotypic frequencies with those of European population samples in Hardy-Weinberg equilibrium extracted from the Allele Frequency Aggregator (ALFA) database. The patients' DNA was extracted, and analyzed using SNPE Multiplex (Single Nucleotide Primer Extension). The prevalence of genetic DAO deficiency was 74.5% based on the four variants of the AOC1 gene. SNP deficits were found at frequencies of 53.1% for p.Thr16Met, 49% for c.691G > T, 48% for p.His664Asp, and 19.4% for p.Ser332Phe. The allele and genotypic frequencies of the women with fibromyalgia did not differ from the European population. Variants of the AOC1 gene that are associated with genetic DAO deficiency could serve as a disruptive biomarker in patients with fibromyalgia. This study was registered in ClinicalTrials.gov Identifier: NCT05389761.

Epigenetic aging in patients diagnosed with coronary artery disease: results of the LipidCardio study

IntroductionPeople age biologically at different rates. Epigenetic clock-derived DNA methylation age acceleration (DNAmAA) is among the most promising markers proposed to assess the interindividual differences in biological age. Further research is needed to evaluate the characteristics of the different epigenetic clock biomarkers available with respect to the health domains they reflect best.MethodsIn this study, we have analyzed 779 participants of the LipidCardio study (mean chronological age 69.9 ± 11.0 years, 30.6% women) who underwent diagnostic angiography at the Charité University Hospital in Berlin, Germany. DNA methylation age (DNAm age) was measured by methylation-sensitive single nucleotide primer extension (MS-SNuPE) and calculated with the 7-CpG clock. We compared the biological age as assessed as DNAmAA of participants with an angiographically confirmed coronary artery disease (CAD, n = 554) with participants with lumen reduction of 50% or less (n = 90) and patients with a normal angiogram (n = 135).ResultsParticipants with a confirmed CAD had on average a 2.5-year higher DNAmAA than patients with a normal angiogram. This association did not persist after adjustment for sex in a logistic regression analysis. High-density lipoprotein, low-density lipoprotein, triglycerides, lipoprotein (a), estimated glomerular filtration rate, physical activity, BMI, alcohol consumption, and smoking were not associated with DNAmAA.ConclusionThe association between higher DNAmAA and angiographically confirmed CAD seems to be mainly driven by sex.

Genetic profile in genes associated with muscle injuries and injury etiology in professional soccer players.

Many causes define injuries in professional soccer players. In recent years, the study of genetics in association with injuries has been of great interest. The purpose of this study was to examine the relationship between muscle injury-related genes, injury risk and injury etiology in professional soccer players. In a cross-sectional cohort study, one hundred and twenty-two male professional football players were recruited. AMPD1 (rs17602729), ACE (rs4646994), ACTN3 (rs1815739), CKM (rs8111989) and MLCK (rs2849757 and rs2700352) polymorphisms were genotyped by using Single Nucleotide Primer Extension (SNPE). The combined influence of the six polymorphisms studied was calculated using a total genotype score (TGS). A genotype score (GS) of 2 was assigned to the "protective" genotype for injuries, a GS of 1 was assigned to the heterozygous genotype while a GS of 0 was assigned to the "worst" genotype. Injury characteristics and etiology during the 2021/2022 season were classified following a Consensus Statement for injuries recording. The distribution of allelic frequencies in the AMPD1 and MLCK c.37885C>A polymorphisms were different between non-injured and injured soccer players (p < 0.001 and p = 0.003, respectively). The mean total genotype score (TGS) in non-injured soccer players (57.18 ± 14.43 arbitrary units [a.u.]) was different from that of injured soccer players (51.71 ± 12.82 a.u., p = 0.034). There was a TGS cut-off point (45.83 a.u.) to discriminate non-injured from injured soccer players. Players with a TGS beyond this cut-off had an odds ratio of 1.91 (95%CI: 1.14-2.91; p = 0.022) to suffer an injury when compared with players with lower TGS. In conclusion, TGS analysis in muscle injury-related genes presented a relationship with professional soccer players at increased risk of injury. Future studies will help to develop this TGS as a potential tool to predict injury risk and perform prevention methodology in this cohort of football players.

Selective Unnatural Base Pairing and Recognition of 2-Hydroxy-2'-deoxyadenosine in DNA Using Pseudo-dC Derivatives.

The formation of unnatural base pairs within duplex DNA would facilitate DNA nanotechnology and biotechnology. Iso-2'-deoxyguanosine (iso-dG) forms base pairs with iso-2'-deoxycytidine, and its use as an unnatural base pair was investigated. Iso-dG is one of the tautomers of 2-hydroxy-2'-deoxyadenosine (2-OH-dA), known as an oxidatively damaged nucleobase, and its selective recognition in DNA plays an important role in the diagnosis and pathogenesis of disease. Therefore, we focused on pseudo-dC (ψdC) as a suitable molecule that recognizes 2-OH-dA in DNA. Since 2-OH-dA shows tautomeric structures in DNA, we designed and used ψdC, which also has a tautomeric structure. We successfully synthesized a ψdC phosphoramidite compound for the synthesis of oligonucleotides (ODNs) as well as its triphosphate derivative (ψdCTP). Tm measurements revealed that ODNs including ψdC showed stable base pair formation with ODNs having 2-OH-dA. In contrast, low Tm values were observed for other bases (dG, dA, dC, and T). The results obtained for the single-nucleotide primer extension reaction revealed that ψdCTP was incorporated into the complementary position of 2-OH-dA in template DNA with high selectivity. In addition, the primer elongation reaction was confirmed to proceed in the presence of dNTPs. The present study reports an artificial nucleic acid that selectively and stably forms unnatural base pairs with 2-OH-dA in DNA.

DNA methylation analysis for smoking status prediction in the Chinese population based on the methylation-sensitive single-nucleotide primer extension method

In some criminal cases, the identity of suspect is unknown and there is no matching DNA profile in the DNA database. Forensic DNA Phenotyping can provide useful investigative information for these cases. Most forensic studies focus on visible characteristics rather than behavioral characteristics. However, smoking is prevalent in the Chinese population, and DNA methylation is the most promising biomarker for smoking. We collected 204 whole blood samples from the Chinese population and measured methylation levels of 9 smoking-related CpG loci using the methylation-sensitive single-nucleotide primer extension method (Ms-SnuPE). But the single-base extension primers of loci cg12803068 and cg21566642 contained other CpG sites, which may introduce bias, and only the other 7 CpG loci were included in subsequent statistical analysis. The methylation level of locus cg05575921 near the aromatic hydrocarbon receptor repressor (AHRR) gene was much lower in the current smoker group than in the never smoker group. To evaluate the ability of each of 7 CpG loci to predict smoking status, the logistic regression (LR) models were established separately, and locus cg05575921 had the best ability to predict smoking status compared with the other 6 loci. Then, combined (including loci cg19572487, cg05575921, cg23480021, cg23576855, cg21161138, cg01940273, and cg09935388) and stepwise (including loci cg05575921 and cg01940273) multinomial logistic regression (MLR) models were also established. Both combined and stepwise MLR models had good efficiencies in predicting smoking status, and outperformed the above 7 LR models. However, the accuracy, specificity and area under the curve (AUC) of stepwise MLR model in the testing dataset were slightly higher than those of combined MLR model, and the stepwise MLR model required less loci information. Therefore, the stepwise MLR model based on 2 significant CpG loci was more recommended model for predicting smoking status in the Chinese population, and the formula was as follow: P = 1/(1 +e-(10.621–10.005*cg05575921–8.770*cg01940273)). Mainly 2 CpG loci (cg05575921 and cg01940273) played a major role in the prediction of smoking status, and the other 5 CpG loci contributed less. Moreover, for evaluating the ability of each of 7 CpG loci to predict cigarette consumption, the polynomial regression formulas were established separately. As the adjusted R2 was between 0.00 and 0.20, the methylation levels of these 7 loci were not closely associated with the cigarette consumption. Our methylation assay is simple, economical, and available in conventional forensic laboratories, and may be useful in assessing the smoking status of unknown suspects.

Vitamin D supplementation is associated with slower epigenetic aging

Adverse effects of low vitamin D level on mortality and morbidity are controversially discussed. Especially older people are at risk for vitamin D deficiency and therefore exposed to its potentially harmful consequences. A way of measuring differences in the biological age is through DNA methylation age (DNAm age) and its deviation from chronological age, DNAm age acceleration (DNAmAA). We previously reported on an association between vitamin D deficiency and higher 7-CpG DNAmAA in participants of the Berlin Aging Study II (BASE-II). In this study, we employ a quasi-interventional study design to assess the relationship between DNAmAA of five epigenetic clocks and vitamin D supplementation. Longitudinal data were available for 1,036 participants of BASE-II that were reexamined on average 7.4 years later in the GendAge study (mean age at follow-up: 75.6 years, SD = 3.8 years, age range: 64.9–94.1 years, 51.9% female). DNAmAA was estimated with the 7-CpG clock, Horvath’s clock, Hannum’s clock, PhenoAge, and GrimAge. Methylation data were obtained through methylation-sensitive single nucleotide primer extension (MS-SNuPE) or Illumina’s Infinium “MethylationEPIC” array. Vitamin D–deficient participants who chose to start vitamin D supplementation after baseline examination showed a 2.6-year lower 7-CpG DNAmAA (p = 0.011) and 1.3-year lower Horvath DNAmAA (p = 0.042) compared to untreated and vitamin D–deficient participants. DNAmAA did not statistically differ between participants with successfully treated vitamin D deficiency and healthy controls (p > 0.16). Therefore, we conclude that intake of vitamin D supplement is associated with lower DNAmAA in participants with vitamin D deficiency.

Seven-CpG DNA Methylation Age Determined by Single Nucleotide Primer Extension and Illumina's Infinium MethylationEPIC Array Provide Highly Comparable Results.

DNA methylation age (DNAm age, epigenetic clock) is a novel and promising biomarker of aging. It is calculated from the methylation fraction of specific cytosine phosphate guanine sites (CpG sites) of genomic DNA. Several groups have proposed epigenetic clock algorithms and these differ mostly regarding the number and location of the CpG sites considered and the method used to assess the methylation status. Most epigenetic clocks are based on a large number of CpGs, e.g. as measured by DNAm microarrays. We have recently evaluated an epigenetic clock based on the methylation fraction of seven CpGs that were determined by methylation-sensitive single nucleotide primer extension (MS-SNuPE). This method is more cost-effective when compared to array-based technologies as only a few CpGs need to be examined. However, there is only little data on the correspondence in epigenetic age estimation using the 7-CpG clock and other algorithms. To bridge this gap, in this study we measured the 7-CpG DNAm age using two methods, via MS-SNuPE and via the MethylationEPIC array, in a sample of 1,058 participants of the Berlin Aging Study II (BASE-II), assessed as part of the GendAge study. On average, participants were 75.6 years old (SD: 3.7, age range: 64.9–90.0, 52.6% female). Agreement between methods was assessed by Bland-Altman plots. DNAm age was highly correlated between methods (Pearson’s r = 0.9) and Bland-Altman plots showed a difference of 3.1 years. DNAm age by the 7-CpG formula was 71.2 years (SD: 6.9 years, SNuPE) and 68.1 years (SD: 6.4 years, EPIC array). The mean of difference in methylation fraction between methods for the seven individual CpG sites was between 0.7 and 13 percent. To allow direct conversion of DNAm age obtained from both methods we developed an adjustment formula with a randomly selected training set of 529 participants using linear regression. After conversion of the Illumina data in a second and independent validation set, the adjusted DNAm age was 71.44 years (SD: 6.1 years, n = 529). In summary, we found the results of DNAm clocks to be highly comparable. Furthermore, we developed an adjustment formula that allows for direct conversion of DNAm age estimates between methods and enables one singular clock to be used in studies that employ either the Illumina or the SNuPE method.

Multiple-turnover single nucleotide primer extension reactions to detect 8-oxo-2'-deoxyguanosine in DNA.

The identification of the position of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is important to clarify the pathogenesis of many diseases. We herein developed a purine-1,3-diazaphenoxazine triphosphate (dPdapTP) and described the first example of detecting the presence of 8-oxo-dG by amplifying it several hundred times after the multiple-turnover single nucleotide primer extension reactions.

Development of a novel SNP assay to detect lactase persistence associated genetic variants

BackgroundIn adulthood the activity of the lactase enzyme is inherited as autosomal dominant form associated to Single nucleotide polymorphisms (SNPs). The present research was aimed to develop a novel genetic method to test lactase non persistence more powerfully.Methods and resultsIn our study, we selected eight different SNPs that are associated with lactase persistence from Caucasian, Arabian Bedouins, sub-Saharian Africans and Asian populations to set up an approach to detect all the eight different SNPs at the same time in the same sample. This technique is centred on the identification of SNPs with a single nucleotide primer extension method using Sanger sequencing and capillary electrophoresis.ConclusionsOur method allowed us to check the genotype asset of eight SNPs related to lactase persistence simultaneously and in a very efficient manner. It could be applied to a higher number of SNPs in a single reaction.

Evaluation of CD109, PCP4 and SEMA3D genes for their association with Ovine Johne’s disease in Turkish sheep

Johne’s disease is a chronic, contagious, zoonotic disease that affects numerous species including livestock and sometimes humans. The disease is globally distributed in sheep populations and caused by Mycobacterium avium Subsp. paratuberculosis (MAP). A previous genome-wide association study identified single nucleotide polymorphism (SNP) markers associated with OJD serostatus in CD109, PCP4, and SEMA3D genes. Our aim was to evaluate the same markers for association with OJD seroprevalence in Turkish sheep in a retrospective matched case-control study. The serological status for OJD in 1801 sheep was determined for four native and four composite breeds from three research flocks. One hundred eleven matched case-control pairs were constructed according to breed type and age from 1750 comingled ewes reared in the same environment. A Single Nucleotide Primer Extension (SNuPE) assay was designed to genotype PCP4-Intron 1, PCP4-3’UTR, SEMA3D, CD109-intron 2 and CD109-intron 8 markers and a McNemar’s test was performed on the matched pairs. An association with these five markers was not detected with the OJD serostatus in Turkish sheep (power of detection, 0.95; odds ratio >3; McNemar’s p < .05). Thus, a wider search may be needed to identify any major underlying genetic risk factors for OJD in Turkish sheep.

Understanding epigenetic modifications in response to abiotic stresses in plants

Abstract Plants have to cope up with environmental stresses during different developmental stages of their life cycle. To overcome the stresses, epigenetic regulations play a significant role in their adaptation. Epigenetic modification has already been well understood. In fact, there are numerous reports related to an epigenetic modification in plants under abiotic stresses. However, there is still a gap in understanding the correlation among different epigenetic phenomena occurring under various abiotic stresses in plants. Therefore, in this review, an attempt has been made to correlate the various epigenetic modifications in the plant under abiotic stresses. Further, recent high-throughput technologies that are being used in the epigenetic analysis have also been highlighted. Various aspects of epigenetic modification in plants have been studied giving emphasis towards abiotic stresses. DNA methylation and histone modification have been focused including the RdDM pathway, ATP-dependent chromatin remodeling, and methylation-sensitive amplified polymorphism (MSAP). With the advancement of technology, various techniques have been applied to detect DNA methylation patterns in plants. These may include bisulfite sequencing, combined bisulfite restriction analysis (COBRA), chromatin immunoprecipitation (ChIP), methylation-sensitive single nucleotide primer extension (Ms-SNuPE), cytosine assay and many more, of which bisulfite genomic sequencing, is the most commonly used. In this report, bisulfite sequencing and chromatin immunoprecipitation were described in detail. Moreover, with the advent of various bioinformatics tools and techniques, epigenetic research has been facilitated, few of which were discussed in this review.

Epigenetic age signatures in bones

Age prediction can help identify skeletal remains by limiting the search range for a missing person. Although age prediction methods based on odontology and anthropology are frequently used in the forensic field, DNA methylation is particularly promising age-predictive biomarker. In this study, we generated genome-wide DNA methylation profiles of bone samples from 32 identified skeletal remains with an age at death ranging from 31 to 96 years. Only 12 provided more than 800 K quality-filtered CpG methylation values using Illumina’s Infinium MethylationEPIC BeadChip array. Methylation ages of the bone samples calculated using a recently developed skin & blood clock composed of 391 CpG sites were found to be very similar to their actual ages (MAD = 6.4 years). However, the low success rate in methylation profiling of bone DNA samples may prevent researchers from applying the array to this type of samples. Therefore, we selected a set of CpG sites that would enable age prediction based on only a few CpG sites in bone DNA samples. Nineteen age-associated CpG marker candidates were selected from 720 K quality-filtered CpG values of 21 male skeletal remain samples. Because age signatures for blood, such as markers on the ELOVL2, FHL2, KLF14 and TRIM59 genes, had showed strong age associations in 12 bone samples, we further tested the age association of the 5 well-known markers in a blood-based model and the 13 out of 19 CpG markers from the array of 21 bone samples with an independent set of 30 skeletal remain samples using SNaPshot multiplex based on single nucleotide primer extension. Four CpG sites on TMEM51, TRIM59, ELOVL2, and EPHA6 genes showed moderate or weak correlations between methylation and age, which suggests further investigation of these markers to predict the age of bones.

Practical genotyping by single-nucleotide primer extension.

Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given approach. We have adapted a single-nucleotide primer extension (SNuPE) method that may be tailored to genotype a required variant, and implemented it as a useful general laboratory protocol. We demonstrate reliable application for production-scale genotyping, successfully converting 87% of SNPs and indels for assay with an estimated error rate of 0.003. Our implementation of the SNuPE genotyping assay is a viable addition to existing alternative methods; it is readily customizable, scalable, and uses standard reagents and a laboratory plate reader.

Effects of the 2-Substituted Adenosine-1,3-diazaphenoxazine 5'-Triphosphate Derivatives on the Single Nucleotide Primer Extension Reaction by DNA Polymerase.

The adenosine triphosphate derivatives of 2-oxo-1,3-diazaphenoxazine (dAdapTP) showed a significant discrimination ability for the template strand including that between 8-oxo-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) by the single nucleotide primer extension reaction using the Klenow Fragment. In this study, we synthesized new dAdapTP derivatives, i.e., 2-amino-dAdapTP, 2-chloro-dAdapTP and 2-iodo-dAdapTP, to investigate the effect on the selectivity and efficiency of incorporation for the primer extension reaction using a variety of DNA polymerases. In contrast to the previously tested dAdapTP, the selectivity and efficiency of the 2-halo-dAdapTP incorporation were dramatically decreased using the Klenow Fragment. Moreover, the efficiency of the 2-amino-dAdapTP incorporation into the T-containing template was almost the same with that of dAdapTP. In the case of the Bsu DNA polymerase, the efficiency of all the dAdapTP derivatives decreased compared to that using the Klenow Fragment. However, the incorporation selectivity of dAdapTP had improved against the oxodG-containing template for all the template sequences including the T-containing template. Moreover, 2-amino-dAdapTP showed a better efficiency than dAdapTP using the Bsu DNA polymerase. The 2-amino group of the adenosine unit may interact with syn-oxodG at the active site of the Bsu DNA polymerase during the single primer extension reaction.

Exploring the Relationship of Relative Telomere Length and the Epigenetic Clock in the LipidCardio Cohort.

Telomere length has been accepted widely as a biomarker of aging. Recently, a novel candidate biomarker has been suggested to predict an individual’s chronological age with high accuracy: The epigenetic clock is based on the weighted DNA methylation (DNAm) fraction of a number of cytosine-phosphate-guanine sites (CpGs) selected by penalized regression analysis. Here, an established methylation-sensitive single nucleotide primer extension method was adapted, to estimate the epigenetic age of the 1005 participants of the LipidCardio Study, a patient cohort characterised by high prevalence of cardiovascular disease, based on a seven CpGs epigenetic clock. Furthermore, we measured relative leukocyte telomere length (rLTL) to assess the relationship between the established and the promising new measure of biological age. Both rLTL (0.79 ± 0.14) and DNAm age (69.67 ± 7.27 years) were available for 773 subjects (31.6% female; mean chronological age= 69.68 ± 11.01 years; mean DNAm age acceleration = −0.01 ± 7.83 years). While we detected a significant correlation between chronological age and DNAm age (n = 779, R = 0.69), we found neither evidence of an association between rLTL and the DNAm age (β = 3.00, p = 0.18) nor rLTL and the DNAm age acceleration (β = 2.76, p = 0.22) in the studied cohort, suggesting that DNAm age and rLTL measure different aspects of biological age.

Association of interleukin-27 gene polymorphisms with susceptibility to HIV infection and disease progression.

Interleukin‐27 (IL‐27) gene polymorphisms are linked to infectious disease susceptibility and IL‐27 plasma level is associated with HIV infection. Therefore, we aimed to investigate the association between IL‐27 polymorphisms and susceptibility to HIV infection and disease progression. A total of 300 patients with HIV infection (48 long‐term nonprogressors and 252 typical progressors) and 300 healthy controls were genotyped for three IL‐27 polymorphisms, rs17855750, rs181206, rs40837 which were performed by using multiple single nucleotide primer extension technique. Significant association was found between IL‐27 rs40837 polymorphisms with susceptibility to HIV infection (AG vs AA: adjusted OR = 1.60, 95% CI, 1.11‐2.30, P = 0.012; AG+GG vs AA: adjusted OR = 1.44, 95% CI, 1.02‐2.03, P = 0.038) and disease progression (LTNP: AG vs AA: adjusted OR = 2.33, 95% CI, 1.13‐4.80, P = 0.021; TP: AG vs AA: adjusted OR = 1.50, 95% CI, 1.04‐2.24, P = 0.030). Serum IL‐27 levels were significantly lower in cases compared to controls (P < 0.001). There were lower serum IL‐27 levels in TPs than in LTNPs (P < 0.001). We further found that LTNPs with rs40837 AG or GG genotype had lower serum IL‐27 levels than with AA genotype (P < 0.05). The CD4+T counts in cases were significantly lower than controls (P < 0.001). In contrast, individuals with rs40837 AG genotype had lower CD4+T counts than with AA genotype in cases (P < 0.05). In addition, CD4+T counts in TPs were significantly lower than LTNPs (P < 0.001). IL‐27 rs40837 polymorphism might influence the susceptibility to HIV infection and disease progression probably by regulating the level of serum IL‐27 or the quantity of CD4+T.