Single Base Extension Research Articles

Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

Background:Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform.Methods:A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility.Results:Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform.Conclusions:We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.

Association of miR-146a, miR-149, miR-196a2, miR-499 gene polymorphisms with ischemic stroke in a Chinese people.

This study aimed to investigate genetic polymorphisms of miR-146a, miR-149, miR-196a2, and miR-499 and genetic susceptibility of ischemic stroke in the population of Guangxi in China. A case–control study was used to investigate miRNAs genetic polymorphisms in 298 patients with ischemic stroke and 303 healthy controls. Single-base extension polymerase chain reaction genotyping principle was used to detect genetic polymorphisms of miRNAs,and the relationship of genotype in each group and blood lipid was compared and analyzed. The genetic polymorphism of miR-499A>G (rs3746444) was associated with ischemic stroke (P < 0.05), and the risk of ischemic stroke was high in patients with G allele (OR = 1.455; 95% CI = 0.531–2.381; P = 0.039) and AG (OR = 1.339; 95% CI = 1.126–1.967; P = 0.037) genotype. The levels of low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, homocysteine, and lipoprotein in the ischemic stroke group were higher than those in the control group (P < 0.05). The genetic polymorphism of miR-499A>G (rs3746444) was related to ischemic stroke, and G allele and AG genotype may increase the risk of ischemic stroke in the population of Guangxi in China.

HRM and SNaPshot as alternative forensic SNP genotyping methods

Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.

Rs12603332 is associated with male asthma patients specifically in urban areas of Lahore, Pakistan

ABSTRACTObjective: rs12603332, an important regulatory site variant, is known to alter the regulatory motif E2A that is involved in the maturation of B-lymphocytes. The study was designed to check whether different environmental exposures alter its risk allele association with asthma or not. Methods: 200 Physician-diagnosed asthma patients and 108 healthy individuals were enrolled from hospitals of Lahore. After quantitation of DNA extracted from peripheral blood, amplification of genomic region with rs12603332, followed by single base extension (SBE), was performed. Allele and genotype frequencies were calculated by SHEsis and Haploview software packages. Statistical analyses on PLINK were also performed, taking different factors as covariates. HaploReg analysis was done to predict the effect of risk allele on different regulatory motifs. Results: Risk allele for rs12603332 i.e., “C” allele was found to be significantly associated with male patients residing in urban localities. Conclusion: The finding suggests that on exposure with urban environment, risk allele carriers tend to develop asthma symptoms via epigenetic regulation of motif associated with maturation of B-lymphocytes.

Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers

The SNPforID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single-base-extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP-7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP-4™ on ABI 310 PRISM® and polymers POP-4™, POP-6™ and POP-7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP-6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP-7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.

Somatic DNA Mutation Analysis.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

CD3Z hypermethylation is associated with severe clinical manifestations in systemic lupus erythematosus and reduces CD3ζ-chain expression in T cells.

The importance of hypomethylation in SLE is well recognized; however, the significance of hypermethylation has not been well characterized. We screened hypermethylated marks in SLE and investigated their possible implications. DNA methylation marks were screened in SLE whole-blood DNA by microarray, and two marks ( CD3Z and VHL hypermethylations) were confirmed by a methylation single-base extension method in two independent ethnic cohorts consisting of 207 SLE patients and 151 controls. The correlation with clinical manifestations and the genetic influence on those epigenetic marks were analysed. Two epigenetic marks, CD3Z and VHL hypermethylation, were significantly correlated with SLE: CD3Z hypermethylation (odds ratio = 7.76; P = 1.71 × 10 -13 ) and VHL hypermethylation (odds ratio = 3.77; P = 3.20 × 10 -8 ), and the increased CD3Z methylation was correlated with downregulation of the CD3ζ-chain in SLE T cells. In addition, less genetic influence on CD3Z methylation relative to VHL methylation was found in analyses of longitudinal and twin samples. Furthermore, a higher CD3Z methylation level was significantly correlated with a higher SLE disease activity index and more severe clinical manifestations, such as proteinuria, haemolytic anaemia and thrombocytopenia, whereas VHL hypermethylation was not. CD3Z hypermethylation is an SLE risk factor that can be modified by environmental factors and is associated with more severe SLE clinical manifestations, which are related to deranged T cell function by downregulating the CD3ζ-chain.

Association of RTN4 gene rs2864052 and rs6545468 with the susceptibility of nasopharyngeal carcinoma in Guangxi Zhuang population

Objective:To study the relationship of the polymorphism of RTN4 gene rs2864052 and rs6545468 and haplotype with the susceptibility of nasopharyngeal carcinoma in Guangxi Zhuang population. Method:The polymorphism of Nogo gene (rs2864052,rs6545468) and haplotype were analyzed using the method of single-base extension PCR and DNA sequencing in 282 cases of nasopharyngeal carcinoma (NPC) and 199 healthy persons (control group) in Guangxi Zhuang Autonomous Region. Result:There were no differences between the NPC's patients and controls in the genotype and allele frequencies of RTN4 gene rs2864052 site,or rs6545468 site. The frequency of AG haplotype in the NPC's patients was significantly lower than in the controls(P=0.004, OR=0.14,95%CI=0.31-0.68). Conclusion:The haplotype AG of RTN4 gene rs2864052 and rs6545468 sites may reduce the risk of nasopharyngeal carcinoma in Guangxi Zhuang population.

Forensically relevant SNaPshot® assays for human DNA SNP analysis: a review.

Short tandem repeats are the gold standard for human identification but are not informative for forensic DNA phenotyping (FDP). Single-nucleotide polymorphisms (SNPs) as genetic markers can be applied to both identification and FDP. The concept of DNA intelligence emerged with the potential for SNPs to infer biogeographical ancestry (BGA) and externally visible characteristics (EVCs), which together enable the FDP process. For more than a decade, the SNaPshot® technique has been utilised to analyse identity and FDP-associated SNPs in forensic DNA analysis. SNaPshot is a single-base extension (SBE) assay with capillary electrophoresis as its detection system. This multiplexing technique offers the advantage of easy integration into operational forensic laboratories without the requirement for any additional equipment. Further, the SNP panels from SNaPshot® assays can be incorporated into customised panels for massively parallel sequencing (MPS). Many SNaPshot® assays are available for identity, BGA and EVC profiling with examples including the well-known SNPforID 52-plex identity assay, the SNPforID 34-plex BGA assay and the HIrisPlex EVC assay. This review lists the major forensically relevant SNaPshot® assays for human DNA SNP analysis and can be used as a guide for selecting the appropriate assay for specific identity and FDP applications.

A collaborative exercise on DNA methylation based body fluid typing.

A collaborative exercise on DNA methylation based body fluid identification was conducted by seven laboratories. For this project, a multiplex methylation SNaPshot reaction composed of seven CpG markers was used for the identification of four body fluids, including blood, saliva, semen, and vaginal fluid. A total of 30 specimens were prepared and distributed to participating laboratories after thorough testing. The required experiments included four increasingly complex tasks: (1) CE of a purified single-base extension reaction product, (2) multiplex PCR and multiplex single-base extension reaction of bisulfite-modified DNA, (3) bisulfite conversion of genomic DNA, and (4) extraction of genomic DNA from body fluid samples. In tasks 2, 3 and 4, one or more mixtures were analyzed, and specimens containing both known and unknown body fluid sources were used. Six of the laboratories generated consistent body fluid typing results for specimens of bisulfite-converted DNA and genomic DNA. One laboratory failed to set up appropriate conditions for capillary analysis of reference single-base extension products. In general, variation in the values obtained for DNA methylation analysis between laboratories increased with the complexity of the required experiments. However, all laboratories concurred on the interpretation of the DNA methylation profiles produced. Although the establishment of interpretational guidelines on DNA methylation based body fluid identification has yet to be performed, this study supports the addition of DNA methylation profiling to forensic body fluid typing.

Evaluation of the iPLEX® ADME PGx Pro Panel and allele frequencies of pharmacogenetic markers in Danes

ObjectivesThe iPlex® ADME PGx Pro Panel was developed to investigate 191 polymorphisms including single nucleotide polymorphisms (SNPs), insertion-deletions (INDELS), and copy number variations (CNV) relevant for absorption, distribution, metabolism, and excretion (ADME) of drugs. The purpose of this study was to perform a technical evaluation of the iPlex® ADME PGx Pro Panel by genotyping 50 unrelated Danes and estimate preliminary genotype frequencies among Danes. Design and methodsThe investigations were performed by the use of PCR, single base extension (SBE) and Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). ResultsThe typing quality of 161 SNP assays was categorized as well performing or acceptable, whereas 22 SNP assays were categorized as either questionable or unacceptable. The frequencies of the genotypes observed in the Danish population were compared to those of the European reference population from the 1000 Genome Project. Three SNPs (rs737865, rs35167514, and rs34305973) showed statistically significantly differences between the frequencies of the 1000 Genomes Europeans and the Danes. The CNV assays could only be used as a guideline. ConclusionIn conclusion, the iPlex® ADME PGx Pro Panel is a cost-effective way of genotyping genes relevant for ADME.

ERBB3-rs2292239 as primary type 1 diabetes association locus among non-HLA genes in Chinese

Type 1 diabetes (T1D) is an autoimmune disease that has strong contribution of genetic factors to its etiology. We aimed to assess the genetic association between non-HLA genes and T1D in a Chinese case-control cohort recruited from multiple centers consisting of 364 patients with T1D and 719 unrelated healthy children. We genotyped 55 single nucleotide polymorphisms (SNP) markers located in 16 non-HLA genes (VTCN1, PTPN22, CTLA4, SUMO4, CD274, IL2RA, INS, DHCR7, ERBB3, VDR, CYP27B1, CD69, CD276, PTPN2, UBASH3A, and IL2RB) using SNaPshot multiple single-base extension methods. After multivariate analysis and correction for multiple comparisons, we identified the SNP rs2292239 in ERBB3 gene were significantly associated with T1D. The frequency of the major G allele was significantly decreased in patients with T1D (68.8% in T1D vs 77.3% in controls, OR 0.65, 95% CI 0.53–0.79, P=0.02), and the minor allele T was associated with an increased risk of T1D (OR 1.55, 95% CI 1.26–1.90, P=0.02). Our haplotype analysis confirmed that rs2292239 was the primary T1D association locus in our current investigation. These results indicated that the ERBB3-rs2292239 was the primary T1D association locus among the investigated 55 SNPs in 16 non-HLA genes in Chinese Han population.

Association between cytokine gene polymorphisms and outcome of hepatitis B virus infection in southern Brazil.

A number of studies have demonstrated associations between cytokine gene polymorphisms and outcome of hepatitis B virus (HBV) infection. However, no general consensus has been reached, possibly due to differences between ethnic groups. In this study, 345 individuals living in southern Brazil, including 196 chronic HBV carriers and 149 subjects who had spontaneously recovered from acute infection, were enrolled to evaluate the influence of cytokine gene polymorphisms on the outcome of HBV infection. Most participants were of European descent. Genotyping of IL2-330 G/T, IL4-589C/T, IL6-174 G/C, IL10-592C/A, IL10-1082 A/G, IL17A-197 G/A, IL17A-692 T/C, TNF-α-238 G/A, and TNF-α-308 G/A single nucleotide polymorphisms was performed by using the minisequencing (single base extension) method. By multivariable analysis, a statistically significant association was found between genotypic profile AA + GA in TNF-α-308 and chronic HBV infection (OR, 1.82; 95%CI, 1.01-3.27; P = 0.046). In southern Brazil, the carriers of the -308A allele in the TNF-α gene promoter have a moderately higher risk of becoming chronic carriers in case of HBV infection. In addition, patients with chronic active hepatitis B (n = 60) exhibited a decreased frequency (3.3%) of the TNF-238A allele when compared to that (14.8%) found among asymptomatic HBV carriers (n = 136), suggesting that this could be a protective factor against liver injury (OR, 0.17; 95%CI, 0.04-0.076; P = 0.023). J. Med. Virol. 88:1759-1766, 2016. © 2016 Wiley Periodicals, Inc.

Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system.

Knowledge of the composition of complex body fluid mixtures may aid forensic investigations greatly. However, many of the traditional tests are presumptive in nature and can lead to ambiguous results. The aim of this study is to establish a reliable method to identify various biofluids via analysis of their DNA methylation profiles. A total of eight biofluid-specific methylated markers for saliva, venous blood, vaginal fluids, and semen were isolated from the open database of Infinium HumanMethylation450 BeadChip. These biofluid-specific markers, a control marker to confirm bisulfite conversion, and a gender marker, were combined into a 10-plex methylation-specific PCR single-base-extension (MSP-SBE) system. Analysis of 65 DNA samples isolated from venous blood, semen, vaginal fluid, saliva, and menstrual blood that had been treated with bisulfite, resulted in all eight markers detecting the body fluid to which they were designed. Unambiguous body fluid identification occurred from both single sources of body fluids and complex mixtures. A threshold was devised for each marker to minimize the chance of a false inclusion. The efficacy of the assay and application to forensic practice was demonstrated using five non-probative samples from real alleged sexual assault cases. The system unambiguously determined the biofluid types for the non-probative forensic samples that previously resulted in inconclusive or conflicting results using traditional tests. The results demonstrated the 10-plex MSP-SBE system established in this study is both sensitive and specific when applied to body fluid identification and can be readily adopted into forensic practice.

Expansion of a SNaPshot assay to a 55-SNP multiplex: Assay enhancements, validation, and power in forensic science.

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty-four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single-base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father-child-mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10(-22) and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.

IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS ON CATTLE BREEDS IN INDONESIA USING BOVINE 50K

&lt;p&gt;Single nucleotide polymorphisms (SNPs) abundant in bovine genome influence genetic variation in biological mechanism. The study aimed to identify SNPs on Indonesian cattle breeds and analyze their genetic diversity using Bovine 50K SNP chip. Twenty eight "Ongole Grade" (OG) beef cattle and 20 "Holstein Friesian" (HF) dairy cattle were used for the Infinium II assay test. This assay included amplification of genomic DNA, fragmenta-tion, precipitation, resuspension, hybridization, processing bead chip for single-base extension, and imaging at iScan. Data and clusters were analyzed using GenomeStudio software. The Bovine 50K SNP chip containing 54,609 SNPs was observed spanning all chromosomes of bovine genome. Genotyping for the total SNPs was successfull based on Call Rate, GeneCall and GeneTrain scores. Most SNP markers had alleles that shared among the individuals or breeds, or had specific alleles at distinctive frequencies. Minor allele frequency (MAF) spreads equally with intervals of 0-0.5. The breeds of OG and HF tended to be separated in different clusters without considering their genetic history and twin or normal. This result suggests that most individuals are closely related to one another, regardless of the same breed. Some genes identified on chromosomes 3, 4, 5, 7, 13, 17 and 18 were located in the loci/regions that contained SNPs with specific alleles of either HF or OG breed. These SNPs were more powerful for differentiation of beef cattle and dairy cattle than among individuals in the same breed. These SNP variations and genetic relatedness among individuals and breeds serve basic information for cattle breeding in Indonesia.&lt;/p&gt;

IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS ON CATTLE BREEDS IN INDONESIA USING BOVINE 50K

&lt;p&gt;Single nucleotide polymorphisms (SNPs) abundant in bovine genome influence genetic variation in biological mechanism. The study aimed to identify SNPs on Indonesian cattle breeds and analyze their genetic diversity using Bovine 50K SNP chip. Twenty eight "Ongole Grade" (OG) beef cattle and 20 "Holstein Friesian" (HF) dairy cattle were used for the Infinium II assay test. This assay included amplification of genomic DNA, fragmenta-tion, precipitation, resuspension, hybridization, processing bead chip for single-base extension, and imaging at iScan. Data and clusters were analyzed using GenomeStudio software. The Bovine 50K SNP chip containing 54,609 SNPs was observed spanning all chromosomes of bovine genome. Genotyping for the total SNPs was successfull based on Call Rate, GeneCall and GeneTrain scores. Most SNP markers had alleles that shared among the individuals or breeds, or had specific alleles at distinctive frequencies. Minor allele frequency (MAF) spreads equally with intervals of 0-0.5. The breeds of OG and HF tended to be separated in different clusters without considering their genetic history and twin or normal. This result suggests that most individuals are closely related to one another, regardless of the same breed. Some genes identified on chromosomes 3, 4, 5, 7, 13, 17 and 18 were located in the loci/regions that contained SNPs with specific alleles of either HF or OG breed. These SNPs were more powerful for differentiation of beef cattle and dairy cattle than among individuals in the same breed. These SNP variations and genetic relatedness among individuals and breeds serve basic information for cattle breeding in Indonesia.&lt;/p&gt;

Genetic variants of ADAM33 are associated with asthma susceptibility in the Punjabi population of Pakistan

Objective: A disintegrin and metalloproteinase 33 (ADAM33) gene has been considered as an asthma susceptibility gene due to its possible role in airway remodeling, abnormal cell proliferation, and differentiation. Association of this gene with asthma has been reported in several genetic studies on various populations. The current study aims to evaluate the association of ADAM33 gene polymorphisms with the risk of asthma in the Punjabi population of Pakistan. Method: A total of 101 asthma patients and 102 age-matched healthy controls from Lahore, a city in Punjab, were recruited. ADAM33 single nucleotide polymorphisms (SNPs) T + 1[rs2280089], T2[rs2280090], T1[rs2280091], ST + 5[rs597980], ST + 4[rs44707], S2[rs528557], Q − 1[rs612709], and F + 1[rs511898] were genotyped in both patients and controls using single base extension and capillary electrophoresis-based genetic analyzer. The basic allelic and genotypic model was analyzed for association of the SNPs with asthma using SHEsis software. Haploview software was used to calculate pairwise linkage disequilibrium (LD) among six of the genotyped SNPs. Results: Of the 8 SNPs genotyped, only S2[rs528557] showed significant association with asthma (Allele p = 0.0189, Genotype p = 0.021). SNPs T + 1[rs2280089], T2[rs2280090], T1[rs2280091], ST + 4[rs44707], S2[rs528557], and Q − 1[rs612709] were found to be in moderate to strong LD. The significantly higher frequency of haplotype “AAGTCG” in healthy controls suggests a protective effect against asthma risk in the studied population (p = 0.0059). Conclusion: These findings suggest that genetic variants of ADAM33 gene may play important roles in asthma susceptibility in the Punjabi population of Pakistan.

Association of CD40 gene polymorphisms and serum CD40 levels with the pathogenesis of systemic lupus erythematosus.

Objective To explore the association of CD40 gene single nucleotide polymorphisms (SNPs) and haplotypes with the susceptibility to systemic lupus erythematosus (SLE), as well as the association of serum levels and genotypes of CD40 with the occurrence of SLE. Methods A multiplex PCR single-base extension assay (PCR-SBE) and DNA sequencing were performed to analyze 4 SNPs of the CD40 gene, including rs1883832 C/T, rs13040307 C/T, rs752118 C/T and rs3765459 G/A, in 205 patients with SLE (SLE group) and 220 healthy human controls (control group). Enzyme-linked immunosorbent assay (ELISA) was conducted to measure serum levels of CD40 in these subjects. Results Compared with the control group, the SLE group showed significantly increased serum levels of CD40(P < 0.05). There were significant differences in genotype and allele frequencies of the SNP rs1883832 C/T in the CD40 gene between the SLE group and control group (all P < 0.01). Relative risk analysis showed that the risk of developing SLE in rs1883832 T allele carriers was 1.517 times that in rs1883832 C allele carriers (OR = 1.517, 95% CI: 1.157-1.990, P = 0.003). Moreover, serum levels of CD40 were significantly higher in rs1883832 T allele carriers than in rs1883832 C allele carriers (P < 0.01). The risk of developing SLE was significantly increased in TCCA haplotype carriers compared with the healthy controls (OR = 2.322, 95% CI: 1.181 -4.564, P = 0.012). Conclusion The CD40 gene rs1883832 C/T polymorphism and its TCCA haplotype were both associated with the occurrence of SLE, and the rs1883832 T allele may be a gene predisposing to SLE. Key words: Lupus erythematosus, systemic; Antigens, CD40; Polymorphism, single nucleotide; Haplotypes; Genotype; Alleles

SNP Miniplexes for Individual Identification of Random-Bred Domestic Cats.

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random‐bred domestic cats, focusing on individual and phenotypic identification. Seventy‐eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot®). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10−19 across all Western cat populations and the likelihood ratio was 1.52 × 1018. These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.