Construction and evaluation of in-house methylation-sensitive SNaPshot system and three classification prediction models for identifying the tissue origin of body fluid / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The identification of tissue origin of body fluid can provide clues and evidence for criminal case investigations. To establish an efficient method for identifying body fluid in forensic cases, eight novel body fluid-specific DNA methylation markers were selected in this study, and a multiplex singlebase extension reaction (SNaPshot) system for these markers was constructed for the identification of five common body fluids (venous blood, saliva, menstrual blood, vaginal fluid, and semen). The results indicated that the in-house system showed good species specificity, sensitivity, and ability to identify mixed biological samples. At the same time, an artificial body fluid prediction model and two machine learning prediction models based on the support vector machine (SVM) and random forest (RF) algorithms were constructed using previous research data, and these models were validated using the detection data obtained in this study (n=95). The accuracy of the prediction model based on experience was 95.79%; the prediction accuracy of the SVM prediction model was 100.00% for four kinds of body fluids except saliva (96.84%); and the prediction accuracy of the RF prediction model was 100.00% for all five kinds of body fluids. In conclusion, the in-house SNaPshot system and RF prediction model could achieve accurate tissue origin identification of body fluids.

Diseño y validación de un ensayo de minisecuenciación múltiple para detectar polimorfismos asociados con Síndrome Metabólico / Design and validation of a multiple minisequencing assay to detect polymorphisms associated with Metabolic Syndrome

Resumen Introducción: Es importante identificar los polimorfismos de interés clínico en patologías complejas como el Síndrome Metabólico. Por esto, las metodologías para su evaluación deben estar diseñadas y validadas correctamente, esto permite optimizar recursos y tiempo en la genotipificación y detección correcta de los alelos presentes en los individuos. Objetivo: Diseñar y validar una PCR múltiple, seguida de detección por minisecuenciación, para la genotipificación de ocho polimorfismos de nucleótido simple ubicados en el gen del Receptor Beta 3-Adrenérgico (rs4994 y rs4998), gen de la Apolipoproteina A5 (rs3135506 y rs2075291), gen de la Adiponectina (rs1501299 y rs2241766) y gen del Receptor Activador de la Proliferación de los Peroxisomas tipo gamma (rs1801282 y rs1800571), asociados con el síndrome metabólico. Materiales y métodos: Se diseñaron 24 cebadores para la amplificación y detección de ocho polimorfismos de nucleótido sencillo ubicados en cuatro genes candidatos a estar asociados con el síndrome metabólico, usando el software Primer3®. Dieciséis fueron diseñados para amplificar los polimorfismos y ocho para detectarlos por minisecuenciación. Las estructuras secundarias entre los cebadores se verificaron con el software Autodimer. Los polimorfismos se amplificaron simultáneamente y los fragmentos amplificados se acoplaron a las sondas diseñadas para detectar por minisecuenciación el alelo presente, por medio de bases marcadas con fluorocromos. Finalmente, los alelos fueron detectados por electroforesis capilar en un analizador genético ABI 310 y se interpretaron con el software GeneMapper®. La validación del multiplex se realizó genotipando 20 muestras de individuos, cada uno de ellos autorizó este procedimiento por medio del consentimiento informado. Resultados: Se obtuvieron los perfiles genéticos de los 20 controles genotipados, a partir de la amplificación múltiple, seguida de minisecuenciación, diseñada y validada para detectar los ocho polimorfismos. Conclusión: Se diseñó y validó un ensayo para la detección simultánea de los polimorfismos, ubicados en cuatro genes asociados con el Síndrome metabólico. Los cuales pueden ser empleados como referencia para futuros estudios poblacionales.

Abstract Introduction: It is important to identify the polymorphisms of clinical interest in complex pathologies such as Metabolic Syndrome. Therefore, the methodologies for its evaluation must be designed and validated correctly, this permits optimization of resources and time in genotyping and correct detection of the alleles present in individuals. Objective: To design and validate a multiplex PCR, followed by detection by minisequencing, for the genotyping of eight single nucleotide polymorphisms located in the Beta 3-Adrenergic Receptor gene (rs4994 and rs4998), Apolipoprotein A5 gene (rs3135506 and rs2075291), Adiponectin gene (rs1501299 and rs2241766) and gamma-type Peroxisome Proliferation Activating Receptor gene (rs1801282 and rs1800571), associated with metabolic syndrome. Materials and methods: Twenty-four primers were designed for the amplification and detection of eight single nucleotide polymorphisms located in four candidate genes to be associated with the metabolic syndrome, using the Primer3® software. Sixteen were designed to amplify the polymorphisms and eight to detect them by minisequencing. The secondary structures between the primers were verified with Autodimer software. The polymorphisms were simultaneously amplified, and the amplified fragments were coupled to probes designed to minisequence the present allele using fluorochrome-labeled bases. Finally, the alleles were detected by capillary electrophoresis using an ABI 310 genetic analyzer and analyzed with the GeneMapper® software. The validation of the multiplex was performed by genotyping 20 individual samples, each of them authorized this procedure through informed consent. Results: The genetic profiles of the 20 genotyped controls were obtained, from multiple amplification, followed by minisequencing, designed and validated to detect the eight polymorphisms. Conclusion: An essay was designed and validated for the simultaneous detection of polymorphisms, located in four genes associated with metabolic syndrome, and can used as a reference for future population studies.

SNP of AHSA2 gene in three cattle breeds using snapshot technology

Droughtmaster is a tropical breed of beef cattle that can survive in hot climates and easily adapt to torrid environments. These traits are important in livestock breeding. In this study, we genotyped five single-nucleotide polymorphisms (SNPs) of the AHSA2 gene from 190 cattle belonging to three different breeds (Droughtmaster, Angus and Simmental) by using snapshot technology. This work aimed to identify the valuable molecular marker of heat resistance in cattle. Results showed that Droughtmaster exhibited higher expected heterozygosity and polymorphic information content compared with the two other breeds. The AHSA2-1 locus deviated from the Hardy– Weinberg equilibrium in the Droughtmaster breed (P \ 0.05). Two SNPs in Droughtmaster diverged significantly from Angus and Simmental. The SNPs were identified as AHSA2-3 and AHSA2-4, which were closely linked to the three breeds based on pair-wise FST. AHSA2-4 involved a missense mutation. In summary, the GG genotypes in AHSA2-3 and AHSA2-4 may be candidate genotypes associated with heat resistance traits and may serve as valuable genetic markers for breeding of heat-tolerant beef cattle in the future.

Analysis of genetic susceptibility to proliferative diabetic retinopathy in Han patients with type 2 diabetes from Southern China by whole exome sequencing and SnaPshot / 中华实验眼科杂志

Objective To research the genetic susceptibility of proliferative diabetic retinopathy (PDR) in Han patients with type 2 diabetes from Southern China.Methods A cross-sectional study was performed under the informed consent of the patients.Patients with type 2 diabetes in the Dongguan Eye Study from September 2011 to February 2012 and relative patients treated in Guangdong General Hospital from July 2017 to March 2018 were included in this study,including 100 patients with diabetes mellitus(DM) and 120 patients with PDR.Whole exome sequencing was used to identify DNA mutation in peripheral blood samples from 22 type 2 diabetic patients without retinopathy (DM group) and 23 diabetic patients with PDR (PDR group).Genotype and allele of the nine selected single-nucleotide polymorphisms (SNPs) were tested and analyzed by SnaPshot technology in another 78 DM patients without retinopathy and 97 PDR patients.Results A total of 75 SNPs were associated with PDR (P＜0.01),involving 53 genes.Eleven gene loci were in the exon region and 7 were non-synonymous mutations.Nine exon loci of 8 genes with significant differences were screened out for the verification.SnaPshot SNP genotyping technique found that there were no significant differences in allele and genotype frequency in the nine selected SNPs between PDR group and DM group (all at P＞0.05).However,7 haplotypes distribution frequencies were significantly different between PDR group and DM group (all at P＜0.01).Hapl and Hap4 might reduce the risk of PDR (both at OR＜1,P＜0.05),and Hap2 might increase the risk of PDR (OR＞ 1,P＜0.05).Conclusions The occurrence of PDR probably has a genetic susceptibility in type 2 DM patients of Han nationality in Southern China.

Study on phenotypic screening and molecular genetics of rare blood type Jk(a-b-) in donors of Fujian province / 临床检验杂志

Objective To investigate the frequency of Jk(a-b-) phenotype of Kidd blood type system in blood donors of Fujian province and its genetic characteristics.Methods The Jk (a-b-) phenotype in the blood samples obtained from 180 626 donors were screened by using urea lysis assay and the suspected Jk(a-b-) pbenotype individuals were confirmed using conventional serological method.The genomic DNA covering the sequence from exon 1 to exon 11 of JK gene and respective flanking area(50-150 bp),as well as the promoter were amplified by polymerase chain reaction,and the products of PCR were directly sequenced.The genotypes of 7 SNPs of JK gene in the blood samples from 200 blood donors of Fujian province were detected by SNaPshot assay.Results Of 180 626 blood donors,15 cases with Jk(a-b-) phenotype were identified.The genomic analysis for the 15 cases revealed the four recessive JK-null alleles,i.e.,JK*B(IVS5-1G＞A),JK*B(896G＞A),JK*A(130G＞A,220A ＞G) and JK* B(130G ＞A,956C ＞ T) were observed with frequency of 66.67％,23.33％,6.67％ and 3.33％,respectively.SNaPshot results showed the frequency of JK * B (IVS5-1 G ＞ A) was 0.75 ％ and G130A was the common polymorphism.No A220G,C222T,C956T and G896A mutation was found in the 200 blood donors.Conclusion The frequency of Jk (a-b-) blood type in the donors of Fujian population was estimated about 0.008％.JK * B(IVS5-1G ＞ A) and JK * B(896G ＞ A) alleles may be the predominate circulating genes in Fujian population with Jk (a-b-) phenotype.Direct DNA sequencing revealed a novel allele leading to JK-null,SLC14A1 130A,220G.

Genotyping of ABO Blood Group in Partial Population of Yunnan Province by SNaPshot Technology / 法医学杂志

OBJECTIVES@#To detect the genotype of ABO blood group by SNaPshot technology.@*METHODS@#DNA were extracted from the peripheral blood samples with known blood groups （obtained by serology） of 107 unrelated individuals in Yunnan. Six SNP loci of the 261th, 297th, 681th, 703th, 802th, and 803th nucleotide positions were detected by SNaPshot Multiplex kit, and relevant genetics parameters were calculated.@*RESULTS@#In 107 blood samples, the allele frequencies of types A, B, OA, and OG were 0.355 1, 0.168 2, 0.230 0 and 0.247 6, respectively, while that of types AG and cis AB were not detected. The genotyping results of ABO blood group were consistent with that of serologic testing.@*CONCLUSIONS@#SNaPshot technology can be adapted for genotyping of ABO blood group.

Promoter variants in interleukin-6 and tumor necrosis factor alpha and risk of coronary artery disease in a population from Western India.

INTRODUCTION: A central component of the atherosclerotic process is inflammation. Single nucleotide polymorphisms (SNPs) present in the promoter region of various cytokines can lead to altered levels of the transcript and a state of low‑grade inflammation exacerbating the risk of coronary artery disease (CAD). The present work tries to understand the role of permissive promoter variants in the interleukin‑6 gene (IL‑6‑174G/C) and the tumor necrosis factor alpha (TNFα‑308G/A) in the causation of CAD and also dyslipidemia. MATERIALS AND METHODS: Genotyping was conducted on 100 cases of CAD and 150 controls by the allele termination assay SNaPshot. Biochemical parameters were determined by routine enzymatic endpoint methods. The results were analyzed by appropriate statistical methods. RESULTS: No differences in the minor allele frequency IL‑6‑174G/C SNP were seen between cases and controls (0.13 vs. 0.12). The differences in the allele frequency of TNFα‑308A between cases (6%) and controls (2%) have led to an odds ratio, 3.370; 95% confidence interval, 1.039‑11.543; P=0.033 in the univariate analysis. In the final logistic regression analysis, however none of the variants were associated with an increased risk of CAD. CONCLUSIONS: In summary, no association of the permissive promoter variants in the IL‑6 gene and the TNFa gene were seen with an increased CAD risk. These and other studies highlight the importance of doing population specific studies.

Typing of 24 mtDNA SNPs in a Chinese Population Using SNaPshot Minisequencing / 华中科技大学学报（医学）（英德文版）

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleo-tide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR) Ⅰ and Ⅱ, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both hu-man population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 3 i haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.

Application of SNaPshot for analysis of thiopurine methyltransferase gene polymorphism.

Background & objectives: Mercaptopurine, azathioprine, and thioguanine, used as antineoplastic agents and immunosuppressants are catabolized by thiopurine methyltransferase (TPMT) enzyme, which exhibits genetic polymorphism. Genotyping patients and the population to which the patients belong, is important for effective treatment and reducing toxicity. There is a need for faster methods for genotyping. Hence the present study was planned to test the application of SNaPshot technique for analysis of the three common TPMT alleles: TPMT*2, TPMT*3A, and TPMT*3C in DNA from healthy Indian volunteers as well as to apply the method on cDNA samples obtained from children with acute lymphoblastic leukaemia (ALL). Method: A total of 120 healthy volunteers and 25 patients were analysed by multiplexed SNaPshot reaction. Genomic DNA was the template for most of the analyses, but additionally the cDNA synthesized for translocation detection was used as the template in case of patients with ALL. The results of SNaPshot reaction were confi rmed by direct sequencing. Results: The TPMT genotype could be reliably identifi ed by SNaPshot analysis in multiplex reactions both in genomic DNA samples and cDNA. The overall frequency of the three common polymorphisms was observed to be 4.9 per cent, arising from heterozygosity for TPMT*3C (4.1%) and TPMT*3A (0.8%). Interpretation & conclusion: SNaPshot method for TPMT polymorphism analysis works faster with the potential for high throughput. By simultaneously interrogating the genotype at multiple sites, the method can provide future opportunity to multiplex, though multiplexing has not been done in the present analysis. Heterozygosity for TPMT*3C (719 A>G) was detected in 4.1 per cent of the study population and no homozygosity was observed. Our results indicated that TPMT*3C was the most common polymorphism in Indian population, while TPMT3*A, associated with the absence of catalytic activity of TPMT, was very rare.

Intraocular Injection of Muscimol Induces Illusory Motion Reversal in Goldfish

Induced activation of the gamma-aminobutyric acidA (GABA(A)) receptor in the retina of goldfish caused the fish to rotate in the opposite direction to that of the spinning pattern during an optomotor response (OMR) measurement. Muscimol, a GABA(A) receptor agonist, modified OMR in a concentration-dependent manner. The GABA(B) receptor agonist baclofen and GABA(C) receptor agonist CACA did not affect OMR. The observed modifications in OMR included decreased anterograde rotation (0.01~0.03 micrometer), coexistence of retrograde rotation and decreased anterograde rotation (0.1~30 micrometer) and only retrograde rotation (100 micrometer~1 mM). In contrast, the GABA(A) receptor antagonist bicuculline blocked muscimolinduced retrograde rotation. Based on these results, we inferred that the coding inducing retrograde movement of the goldfish retina is essentially associated with the GABA(A) receptor-related visual pathway. Furthermore, from our novel approach using observations of goldfish behavior the induced discrete snapshot duration was approximately 573 ms when the fish were under the influence of muscimol.