SNaPshot Assay Research Articles

DIP-microhaplotypes: new markers for detection of unbalanced DNA mixtures.

The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (I value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.

Polymorphisms in endoplasmic reticulum aminopeptidase genes are associated with cervical cancer risk in a Chinese Han population

BackgroundAntigen-processing machinery molecules play crucial roles in infectious diseases and cancers. Studies have shown that polymorphisms in endoplasmic reticulum aminopeptidase (ERAP) genes can influence the enzymatic activity of ERAP proteins and are associated with the risk of diseases. In the current study, we evaluated the influence of ERAP gene (ERAP1 and ERAP2) polymorphisms on susceptibility to cervical intraepithelial neoplasia (CIN) and cervical cancer.MethodsSix single nucleotide polymorphisms (SNPs) in ERAP1 and 5 SNPs in ERAP2 were selected and genotyped in 556 CIN patients, 1072 cervical cancer patients, and 1262 healthy control individuals. Candidate SNPs were genotyped using SNaPshot assay. And the association of these SNPs with CIN and cervical cancer was analysed.ResultsThe results showed that allelic and genotypic frequencies of rs26653 in ERAP1 were significantly different between cervical cancer and control groups (P = 0.001 and 0.004). The allelic frequencies of rs27044 in ERAP1 and rs2287988 in ERAP2 were significantly different between control and cervical cancer groups (P = 0.003 and 0.004). Inheritance model analysis showed that genotypes of rs27044, rs26618, rs26653 and rs2287988 SNPs may be associated with the risk of cervical cancer (P = 0.003, 0.004, 0.001 and 0.002). Additionally, haplotype analysis results showed that the ERAP1 haplotype, rs27044C-rs30187T-rs26618T-rs26653G-rs3734016C, was associated with a lower risk of cervical cancer (P = 0.001). The ERAP2 haplotypes rs2549782G- rs2548538A-rs2248374A-rs2287988G-rs1056893T (P = 0.009 and 0.006) and rs2549782T-rs2548538T-rs2248374G-rs2287988A-rs1056893T (P = 0.003 and 0.009) might be associated with cervical cancer and the development from CIN to cervical cancer.ConclusionOur results indicated that rs27044, rs26618 and rs26653 in ERAP1 and rs2287988 in ERAP2 influenced susceptibility to cervical cancer.

DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1–94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28–86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Evaluation of miR-34b/c polymorphisms to the risk of ischemic stroke.

Polymorphisms in microRNA genes are related to the risk of ischemic stroke, but the association between miR-34b/c polymorphisms and the risk of ischemic stroke has not been reported. MiR-34b/c rs2187473 and rs4938723 polymorphisms were genotyped by Snapshot assay among 495 controls and 492 ischemic stroke patients. Expression levels of miR-34b and miR-34c were quantified by real-time PCR. Transcriptional activity of miR-34b/c promoter was measured by luciferase reporter assay. Rs4938723 was associated with an increased risk of ischemic stroke in our study (CC versus TT: OR = 2.34, 95% CI = 1.47-3.72, P = 0.001; C versus T: OR = 1.37, 95% CI = 1.12-1.68, P = 0.002; CC versus TT + TC: OR = 2.12, 95% CI = 1.37-3.29, P = 0.001). The expression levels of miR-34b and miR-34c were significantly downregulated in cases by contrast with controls (P < 0.05). Further analysis demonstrated that the expression levels of miR-34b and miR-34c were also downregulated in the individuals carrying rs4938723 CC genotype by contrast with that carrying TT + TC genotypes (P < 0.05). The result of luciferase reporter assay showed that rs4938723C allele decreased the transcriptional activity of miR-34b/c promoter compared with rs4938723 T allele. Our study showed a positive relation between the miR-34b/c rs4938723 polymorphism and the risk of ischemic stroke, which indicated that rs4938723 may be used for ischemic stroke prediction or therapy in the future.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n= 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

Effect of Cytochrome P450 7A1 (CYP7A1) Polymorphism on Lipid Responses to Simvastatin Treatment.

Identifying patients with high risk of low response to statin therapy is important for optimization of lipid-lowering therapy. Cholesterol 7α-hydroxylase, a rate-limiting enzyme encoded by cytochrome P450 7A1 (CYP7A1) gene, is considered to be associated with statin efficacy. This study aimed to investigate the association between a novel CYP7A1 single nucleotide polymorphism rs3824260 and statin treatment response for hypercholesteremic patients in Chinese Han population. A total of 336 subjects were prescribed with simvastatin for 12 weeks after enrollment. Plasma lipid parameters were measured at enrollment and after 12-week simvastatin treatment separately. Subjects were classified into high- and low-response groups depending on their total cholesterol, low-density lipoprotein cholesterol (LDL-C) and TG changes and increase or reduction groups according to their high-density lipoprotein cholesterol (HDL-C) levels changing after simvastatin treatment. The CYP7A1 rs3824260 was genotyped from blood samples with a SNaPshot assay. At baseline, the LDL-C level and TG level were significantly higher in the AA genotype, while the HDL-C level was significantly higher in the GG genotype of CYP7A1 rs3824260. Patients carrying AA genotype are at an increased risk of low response for LDL-C reduction (odds ratio = 2.295, 95% confidence interval = 1.164-4.524, P = 0.016). Furthermore, the GG genotype of rs3824260 was significantly associated with a high risk of HDL-C reduction response after simvastatin therapy (odds ratio = 2.240, 95% confidence interval = 1.137-4.413, P = 0.025). The CYP7A1 gene polymorphism rs3824260 is related to inappropriate response of simvastatin treatment for hypercholesterolemia patients in Chinese Han population.

The evaluation of seven age-related CpGs for forensic purpose in blood from Chinese Han population

Age prediction of biological samples is one of the important tasks in forensic DNA phenotyping, and DNA methylation is regarded as the most promising biomarker for forensic age prediction. To date, numerous CpG sites have been reported to be age-related based on the changes in methylation. In this study, seven age-related CpG (AR-CpG) sites, cg02228185 (ASPA), cg09809672 (EDARADD), cg19283806 (CCDC102B), cg04208403 (ZNF423), chr17: 44,390,358 of GRCh38/hg38 (ITGA2B), cg14361627 (KLF14), and cg06639320 (FHL2), were selected and analyzed in 310 blood samples using a multiplex methylation SNaPshot assay to evaluate the value of selected AR-CpGs in age prediction in blood from Chinese Han population. The study confirmed the correlation of all the investigated markers with human age, and the correlation of cg19283806 with age is the highest while cg04208403 is the lowest in the Chinese Han population. Two different age prediction models, stepwise regression and support vector regression (SVR), were established based on the methylation SNaPshot data using 230 blood samples from donors aged 2–86 years old. The stepwise regression model included six AR-CpGs (except cg09809672) and enabled age prediction with R2 = 0.85, mean absolute deviation (MAD) = 4.22, while the SVR model enabled age prediction with R2 = 0.86, MAD = 4.01. An independent set of 80 samples was used to test the two models′ performance and the prediction MAD for the validation set was 4.71 and 4.56 for the stepwise regression and SVR models, respectively. The number of correct predictions for ±5 years achieved a high level of 67.50 % and 73.75 %, respectively for the stepwise regression and SVR models. In general, the SVR model was superior to the linear regression model in age prediction. These results suggest that these seven CpG sites would be useful for age prediction in blood samples from the Chinese Han population.

A Case-Control Study of the Association Between the SPP1 Gene SNPs and the Susceptibility to Breast Cancer in Guangxi, China.

Secreted phosphoprotein-1 (SPP1) has been reported to be involved in the pathogenesis of breast cancer (BRC), but the influence of SPP1 single nucleotide polymorphisms on the BRC susceptibility has been rarely reported. In this study, we explored the association between rs11730582, rs2853750, and rs35893069 in the SPP1 gene and the BRC susceptibility. We used Snapshot assay to detect SPP1 single nucleotide polymorphisms in 471 BRC patients and 471 controls. The plasma SPP1 level was measured by ELISA. We found that the CC genotype and C allele of rs11730582 were associated with a significantly decreased BRC risk compared with the TT genotype and T allele, respectively [CC vs. TT: odds ratio (OR) = 0.59, 95% CI = 0.37–0.94, P = 0.026; C vs. T: OR = 0.79, 95% CI = 0.65–0.96, P = 0.022]. In addition, BRC patients and controls with the rs11730582 CC genotype had a lower plasma SPP1 level than did BRC patients and controls with TT genotype (P = 0.007 and P = 0.011, respectively). Moreover, the proportions of rs11730582 CC genotype and C allele were decreased in BRC patients with clinical stages I–III compared with those with clinical stage IV (P = 0.012 and P = 0.003, respectively). Besides, the C-G-T haplotype was associated with a significantly decreased BRC risk compared with the T-A-T haplotype (OR = 0.69, 95% CI = 0.52–0.93, P = 0.015). However, there was no significant association between rs2853750 or rs35893069 and the BRC risk. In summary, our study found the association between rs11730582 and the risk of BRC and suggested that rs11730582 may promote the occurrence and development of BRC by regulating SPP1 expression.

GAD1 but not GAD2 polymorphisms are associated with heroin addiction phenotypes

IntroductionHeroin addiction is a chronic complex brain disease that contains multiple phenotypes, which vary widely among addicts and may be affected by genetic factors. A total of 801 unrelated heroin addicts were recruited and divided into different subgroups according to eight phenotypes of heroin addiction. Polymorphisms in GAD1 (rs3762555, rs3762556, rs3791878, rs3749034, rs11532313 and rs769395) and GAD2 (rs2839669, rs2839670 and rs2236418) were genotyped using the SNaPshot assay. Associations between genetic variants and the eight phenotypes were mainly assessed by binary logistic regression. ResultsWe found that the frequencies of G allele of GAD1 rs3749034 and rs3762555 were associated with daily dose of methadone use and memory change after heroin addiction. The C allele frequency of GAD1 rs3762556 was associated with lower daily dose of methadone use. In GAD1, SNPs rs3762556, rs3762555, rs3791878 and rs3749034 had strong linkage, and the frequency of the C-G-C-A haplotype was higher in the lower dose of methadone group. Patients with the TT genotype of rs11542313 were maintained on lower dose of methadone than patients with the CC genotype. The G alleles of rs3762555 and rs3749034 were lower, while the T allele of rs11542313 was higher, in the memory decreased group. The results of association analyses of GAD2 with phenotypes of heroin addiction showed no significant differences. ConclusionGAD1 polymorphisms were associated with phenotypes of heroin addiction, especially the daily dose of methadone use and memory change in the Han Chinese population. These results may provide individualized guidance for the treatment of heroin addiction.

Multiple methods used for type detection of uniparental disomy in paternity testing.

Uniparental disomy (UPD) has attracted more attention recently in paternity testing, though it is an infrequent genetic event. Although short tandem repeat (STR) profiling has been widely used in paternity testing, it is not sufficient to use STR only to judge the genetic relationship, because the existence of UPD will inevitably affect the results of genotyping. Compared with complete UPD, segmental UPD is more difficult to detect because it does not affect all genotypes on the same chromosome. It is necessary to determine the type of UPD with multiple methods because a single method is not sufficient. Therefore, it is advisable to detect UPD in paternity testing with multiple methods. In this study, after autosomal STR profiling was used, we found that there were several gene loci on the same chromosome that did not conform to Mendelian genetic law, thus we highly suspected the existence of UPD and performed X-STR profiling immediately. Then whole-genome single nucleotide polymorphism (SNP) array analysis was performed to identify the type, and the results provided straightforward evidence for distinguishing complete from segmental UPD. Lastly, we used deletion insertion polymorphism (DIP)-SNP SNaPshot assay and Miseq FGx sequencing (for SNP and STR) to determine whether the mutation source is maternal uniparental disomy (mUPD) or paternal uniparental disomy (pUPD). To avoid false exclusion of kinship, it is vital to determine the type of UPD in paternity testing and effective strategies based on multiple methods to detect the type of UPD are provided in this study.

A new approach to detect a set of SNP-SNP markers: Combining ARMS-PCR with SNaPshot technology

Microhaplotypes have become a new promising forensic genetic marker in recent years. The microhaplotype composed of two SNPs, SNP-SNP, indicates strong application potential because of the shortest fragment and good polymorphism and without the interference of stutter and high mutation rate as short tandem repeats (STR) and low polymorphism as a single SNP. Currently, the most common method to detect microhaplotypes is massively parallel sequencing (MPS), however its high cost and the need for special instruments limit its use in general forensic laboratories. In this study, we screened out 8 new SNP-SNP loci and established a new detection method by associating multiplex ARMS-PCR and SNaPshot technology. Firstly, we introduced ARMS-based PCR for SNP1. Then, SBE primers for SNaPshot assay were designed as 20–25 bp upstream complementary sequence next to the position of SNP2. Finally, 8 loci were built into one panel based on different SBE primer lengths and fluorescence colors. In brief, by combing ARMS-PCR and SNaPshot technology, it is easy and fast to profile the SNP1 and SNP2 orderly of the SNP-SNP microhaplotype based on CE platform. Our results suggested that the 8 loci have relatively high polymorphism as well as robust performance.

A Useful SNP Panel to Distinguish Two Cockle Species, Cerastoderma edule and C. glaucum, Co-Occurring in Some European Beds, and Their Putative Hybrids.

Cockles are highly appreciated mollusks and provide important services in coastal areas. The two European species, edible (Cerastoderma edule) and lagoon (Cerastoderma glaucum) cockles, are not easily distinguishable, especially when young. Interestingly, the species show different resistance to Marteilia cochillia, the parasite responsible for marteiliosis outbreaks, which is devastating cockle production in some areas. C. edule is severely affected by the parasite, while C. glaucum seems to be resistant, although underlying reasons are still unknown. Hybrids between both species might be interesting to introgress allelic variants responsible for tolerance, either naturally or through artificial selection, from lagoon into edible cockle. Here, we used 2b restriction site-associated DNA sequencing (2b–RAD) to identify single nucleotide polymorphisms (SNP) diagnostic for cockle discrimination (fixed for alternative allelic variants). Among the nine diagnostic SNPs selected, seven were validated using a SNaPshot assay in samples covering most of the distribution range of both species. The validated SNPs were used to check cockles that were suggested to be hybrids by a claimed diagnostic tool based on the internal transcribed spacers of the ribosomal RNA. Although these were shown to be false positives, we cannot rule out the fact that hybrids can occur and be viable. The SNP tool here developed will be valuable for their identification and management.

Genetic variants of ADAMTS7 confer risk for ischaemic stroke in the Chinese population.

Large-scale genome-wide association analyses show an association between ADAMTS7 variations and coronary risk. However, the link between ADAMTS7 variability and ischaemic stroke (IS) has yet to be determined. This study evaluated ADAMTS7 variants with respect to the risk of IS. Genetic association analyses were performed in two independent case-control cohorts with 1279 patients with IS and 1268 age-matched healthy controls. Four variant genotypes of the ADAMTS7 gene were identified using the Multiplex SNaPshot assay. The rs3825807, rs11634042, and rs7173743 variants of ADAMTS7 were related to lower IS risk in both initial and replication cohort. The G-T-T-C and G-T-C-C haplotypes are significantly less prevalent in the IS group than in the control group. Further stratification according to IS subtypes indicated that carriers with the variant alleles of the rs3825807, rs11634042 and rs7173743 variants of ADAMTS7conferred a lower risk of developing large-artery atherosclerosis stroke subtype. Also, the mutated rs3825807 G allele, as well as the mutated rs11634042 T allele of ADAMTS7, are linked to a significant reduction of ADAMTS7 in patients with IS. Our findings confirm the role of ADAMTS7 in the pathophysiology of IS, with potentially significant implications for the prevention, treatment, and development of novel therapies for IS.

Abstract 4704: Identification of melanoma mutational tumor heterogeneity using BRAF, NRAS and TERT-promoter mutation-detection assays

Abstract Purpose: The mutational spectra of melanoma has been well characterized; however, the presence of distinct subclones among multiple tumors from a given patient has been less well described. As mutational heterogeneity has been associated with decreased responses to treatments in other cancers, we sought to estimate the occurrence of distinct subclones within individual melanoma patients by analyzing commonly mutated melanoma genes using a multi-platform mutation-detection approach. Methods: We analyzed 271 formalin-fixed, paraffin embedded tumors from 99 patients with stage III or IV melanoma enrolled in the NYU Melanoma Biorepository. All patients had two or more available tumor specimens, and complete clinical data. All samples were reviewed for adequate tumor content, and extracted DNA was assessed for mutations at BRAFV600, NRASQ61, and TERT-124C&amp;gt;T and TERT-146C&amp;gt;T using a combination of multiplex SNaPshot assays, Sanger Sequencing, Allele-specific real-time PCR, or droplet digital PCR (ddPCR). Samples undergoing ddPCR analysis for TERT mutations were treated with uracil-DNA glycolase prior to amplification to remove C&amp;gt;T artifacts from formalin-fixation. Results: Sixty patients had a primary plus one or more metastatic tumors available; 39 patients had multiple metastatic tumors, but no primary tumor available. Overall, 88% of patients had tumors with at least one BRAF, NRAS and/or TERT mutation. We identified inter-tumor mutational heterogeneity in 20/99 (20%) patients, with TERT mutational heterogeneity present in 15 of these patients. Among 14 patients with heterogeneity between their primary and metastatic tumors, 6/14 (43%) had additional mutations in their metastases compared to their primaries. Most interestingly, 8/14 (57%) patients had mutations in their primary that were undetectable in at least one of their metastases; 5 of these patients had TERT mutational heterogeneity. Three patients had a BRAF mutation in their primary that was undetectable in at least 1 of their metastases. One patient had a BRAFV600E/NRASWILD-TYPE/TERTWILD-TYPE primary on the leg and 3 regional metastases lacking BRAF mutations; but carrying NRASQ61K and TERT-124C&amp;gt;T mutations. Another patient had 3 different metastatic tumors, with 3 different mutational spectra. We did not detect any tumors with simultaneous BRAF and NRAS mutations; however, we did detect both TERT-124C&amp;gt;T and TERT-146C&amp;gt;T mutations in 7 tumors from 7 individual patients. Four of these were primary tumors, and metastases from these patients lacked 1 of the 2 TERT mutations identified in the primary. Conclusion: Clonal heterogeneity in melanoma is fairly common as evidenced by divergent detection of TERT, BRAF and NRAS mutations using high sensitivity multi-platform mutation detection analyses of multiple melanoma tumors from individual patients. Heterogeneity appears to occur in primary tumors. Citation Format: Gregory Chang, Broderick Corless, Nathaniel Fleming, Cindy Spittle, Farbod Darvishian, Anna Pavlick, Russell Berman, Richard Shapiro, George Karlin-Neumann, Iman Osman, David Polsky. Identification of melanoma mutational tumor heterogeneity using BRAF, NRAS and TERT-promoter mutation-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4704.

A new set of DIP-SNP markers for detection of unbalanced and degraded DNA mixtures.

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP-SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP-SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP-SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP-SNPs in a Chinese Han population. The DIP-SNPs were capable of detecting the minor contributor's allele in home-made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1-10ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP-SNPs may serve as a valuable tool in detection of UDM in forensic medicine.

A Genetic Variant of miR-34a Contributes to Susceptibility of Ischemic Stroke Among Chinese Population.

miRNAs are small non-coding RNAs modulating gene expression, and variants in miRNA genes are involved in the pathogenesis of ischemic stroke (IS). However, the effect of miR-34a polymorphisms on IS susceptibility has rarely been reported. In the present study, we investigated the association between rs12128240, rs2666433, and rs6577555 of the miR-34a gene and IS susceptibility. Snapshot assay was used to detect miR-34a polymorphisms in 548 IS patients and 560 controls. Relative expression of miR-34a was measured by quantitative real-time PCR. We found that rs2666433 was associated with a significantly increased risk of IS (AA vs. GG: OR = 1.61, 95% CI = 1.05–2.52, P = 0.031; AA vs. GG+GA: OR = 1.58, 95% CI = 1.05–2.45, P = 0.026). For the IS subtypes, rs2666433 was associated with large artery atherosclerosis (AA vs. GG: OR = 2.09, 95% CI = 1.16–3.51, P = 0.007; AA vs. GG+GA: OR = 2.02, 95% CI = 1.15–3.33, P = 0.007; A vs. G: OR = 1.36, 95% CI = 1.07–1.81, P = 0.021). Additionally, the level of miR-34a was significantly up-regulated in IS patients compared to the controls (P < 0.001), and patients with rs2666433 AA genotype had a higher level of miR-34a than those with GG+GA genotypes (P < 0.001). Furthermore, increased level of homocysteine was observed in IS patients compared to the controls (P < 0.001), especially in patients carrying the rs2666433AA genotype compared to those carrying the rs2666433 GG+GA genotypes (P < 0.001). However, no significant association between rs12128240 or rs6577555 and IS was found. Collectively, our study found the association between miR-34a polymorphisms and the risk of IS among the Chinese population. The results may provide an explanation for etiology of IS and a potential biomarker or therapeutic target for IS. HIGHLIGHTS-MiR-34a rs2666433 polymorphism was associated with an increased risk of ischemic stroke.-The level of miR-34a was significantly up-regulated in ischemic stroke patients compared with controls, and patients with rs2666433 AA genotype had a higher level miR-34a than those with GG+GA genotypes.-Furthermore, increased level of homocysteine was showed in IS patients compared to controls, and in patients carrying the rs2666433AA compared to those carrying the rs2666433 GG+GA.

Applying a multiplexed primer extension method on dried blood spots increased the detection of carriers at risk of glucose-6-phosphate dehydrogenase deficiency in newborn screening program

BackgroundPatients with glucose-6-phosphate dehydrogenase deficiency might develop acute hemolytic anemia, chronic hemolytic anemia, and neonatal hyperbilirubinemia when exposed to high levels of oxidative stress. Severe hemolysis may occur in not only patients but also female carriers under certain conditions. However, 80%–85% of female carriers were undetected in an existing newborn screening program because of their wide-ranging levels of enzyme activity. MethodsWe developed a cost- and time-efficient multiplex SNaPshot assay using dried blood spots. ResultsBy detecting 21 common mutations in Taiwan and Southeast Asia, the assay could determine 98.2% of the mutant alleles in our cohort of Taiwanese newborns. The 9 undetermined mutant alleles were consequently detected by Sanger sequencing, of which 5 unpublished variations—c.187G > A (Pingtung), c.585G > C (Tainan), c.586A > T (Changhua), c.743G > A (Chiayi), and c.1330G > A (Tainan-2)—were detected. Furthermore, 13% of mild mutations were missed in male infants whose enzyme levels at 6.1–7.0 U/gHb in the newborn screening program when set the cutoff value at 6.0 U/gHb. We therefore suggest increasing the cutoff value and applying the multiplex SNaPshot assay as the second tier for neonatal screening. ConclusionsOur approach could significantly increase the detection rate of male patients and female carriers with a reasonable cost and a reasonable number of clinic referrals.

Association of mitochondrial haplogroup F with physical performance in Korean population.

Athletic performance is a complex multifactorial trait involving genetic and environmental factors. The heritability of an athlete status was reported to be about 70% in a twin study, and at least 155 genetic markers are known to be related with athlete status. Mitochondrial DNA (mtDNA) encodes essential proteins for oxidative phosphorylation, which is related to aerobic capacity. Thus, mtDNA is a candidate marker for determining physical performance. Recent studies have suggested that polymorphisms of mtDNA are associated with athlete status and/or physical performance in various populations. Therefore, we analyzed mtDNA haplogroups to assess their association with the physical performance of Korean population. The 20 mtDNA haplogroups were determined using the SNaPshot assay. Our result showed a significant association of the haplogroup F with athlete status (odds ratio, 3.04; 95% confidence interval, 1.094 to 8.464; p = 0.012). Athletes with haplogroup F (60.64 ± 3.04) also demonstrated a higher Sargent jump than athletes with other haplogroups (54.28 ± 1.23) (p = 0.041). Thus, our data imply that haplogroup F may play a crucial role in the physical performance of Korean athletes. Functional studies with larger sample sizes are necessary to further substantiate these findings.

Genotyping of 49-plex autosomal SNP panel in Iranian Turkmens ethnic group.

A total of 94 unrelated individuals from Turkmens ethnic group in Iran were typed for forty-nine of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex using the SNaPshot assay. Allele frequencies are presents for the 49 SNPs. No deviation from Hardy-Weinberg equilibrium (HWE) was observed in all but one of the 49 SNP systems and no significant linkage disequilibrium was detected for any SNP pairs. FIS and FST were estimated. A statistically significant global FST value was obtained when Turkmens ethnic group were compared with other 20 populations in Turkey, Israel, Pakistan, India, China, Taiwan, Japan, Thailand, Siberia, Algeria, Somali, Uganda, Mozambique, Angola, Nigeria, Russia, Slovenia, Sweden, France and Spain. All but 11 pairwise FST values were statistically significant. Multidimensional scaling plot drawn based on the pairwise FST values showed that the Turkmens ethnic grouped with populations geographically close to Iran and other Middle-Eastern populations. The cumulative values for the match probability using the 49 SNPs was 5.65 × 10-19 consistent to a combined power of discrimination of >99.99999% and the mean exclusion probability was 99.95%.

Association of sirtuin 1 gene polymorphisms with nephrolithiasis in Eastern chinese population

Sirtuin 1 (SIRT1), an NAD+-dependent deacylase, has been identified to be associated with renal tubular inflammatory conditions and metabolic disorders, which are risk factors of nephrolithiasis. To further confirm the role of the SIRT1 in kidney stone formation, the expression of SIRT1 was analyzed based on a mouse model and the genetic polymorphisms of SIRT1 gene was compared between patients with kidney stones and controls. The calcium oxalate (CaOx) crystal-induced renal injury model was established to analyzed the expression of SIRT1 in the kidney tissue of both wild-type and ApoE(−/−) mice. And a total of 430 Eastern Chinese subjects (215 patients with nephrolithiasis and 215 age- and gender-matched controls) were recruited for the present study to investigate the associations between 6 common single nucleotide polymorphisms (SNPs) (i.e., rs10509291, rs3740051, rs932658, rs33957861, rs3818292 and rs1467568) in the SIRT1 gene and the incidence of kidney stones. Pairwise linkage disequilibrium and the haplotypes of the 6 SNPs were also analyzed. The genotypes of SIRT1 gene polymorphisms were analyzed by a Snapshot assay. Reduced expression of SIRT1 was observed in the kidney of the mice in the crystal group, revealing the potential role of SIRT1 in the nephrolithiasis. However, we did not find a significant association between the 6 SNPs of the SIRT1 gene and kidney stone formation in the Eastern Chinese population.