A Laboratory Exercise for Genotyping Two Human Single Nucleotide Polymorphisms

The dramatic decrease in the cost of sequencing a human genome is leading to an era in which a wide range of students will benefit from having an understanding of human genetic variation. Since over 90% of sequence variation between humans is in the form of single nucleotide polymorphisms (SNPs), a laboratory exercise has been devised in order to illustrate the importance of SNPs. Two separate SNPs are analysed, one of which has a significant effect on a person’s phenotype and one which does not. The genotyping protocol is relatively inexpensive and uses standard molecular biology reagents and equipment.     

hRFT2基因单核苷酸多态性的生物信息学分析

旨在筛选可能与人类疾病有关的hRFT2基因单核苷酸多态性(single nucleotide polymorphisms,nsSNPs)和突变位点,从SNP数据库中检索并筛选出395个有效的hRFT2基因SNPs,其中包括30个同义SNPs(synonymous SNPs,sSNPs)和31个非同义SNPs(non-synonymous single nucleotide polymorphisms,nsSNPs)。分别采用SIFT、SNPs3D和PolyPhen-2方法分析nsSNPs引起的氨基酸替换是否可能影响hRFT2的功能。结果表明,5个nsSNPs(rs11477762、rs146302587、rs146492942、rs76947760和rs145431028)可能严重影响hRFT2蛋白的功能,其中rs76947760和rs145431028的影响已得到临床证明,另外3个nsSNPs(rs148387972、rs140391358和rs3746802)也可能对hRFT2有较大的影响。     

Arg753Gln Polymorphism of the Human Toll-like Receptor-2 Gene in Children with Recurrent Febrile Infections

Polymorphisms in toll-like receptors (TLRs) have been reported to increase susceptibility for some diseases. TLR-2 gene polymorphisms in Turkish children with recurrent respiratory tract infections and without well-known humoral immunodeficiencies were examined. The study consisted of 52 children with recurrent infections (study group) and 91 healthy children with a maximum of two infections in a year (control group). Recurrent infection was defined as the presence of at least six febrile bacterial infection episodes in a year. Not only TLR-2 gene polymorphisms but also immunoglobulins (IgG, IgM, IgA), IgG subsets (G1, G2, G3), and specific antibody levels (anti-tetanus and anti-hemophilus influenza) were determined to exclude humoral immunodeficiencies. The Arg753Gln and Arg677Trp polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism. The Arg753Arg genotype was significantly decreased in the study group compared to the control (P < 0.05). Children with recurrent infections were also found to be more frequently Arg753Gln heterozygous (P < 0.05), and their Gln allele distribution was higher than that of the control subjects (23% vs. 4.9%; P < 0.001). In contrast to these results, we did not detect any case with Arg677Trp polymorphism in both groups. These results have indicated that there is a strong significant relationship between susceptibility to recurrent bacterial infections and Arg753Gln polymorphism of the TLR-2 gene.     

A Brief Review on the Human Encyclopedia of DNA Elements (ENCODE) Project

The ENCyclopedia Of DNA Elements (ENCODE) project is an international research consortium that aims to identify all functional elements in the human genome sequence. The second phase of the project comprised 1640 datasets from 147 different cell types, yielding a set of 30 publications across several journals. These data revealed that 80.4% of the human genome displays some functionality in at least one cell type. Many of these regulatory elements are physically associated with one another and further form a network or three-dimensional conformation to affect gene expression. These elements are also related to sequence variants associated with diseases or traits. All these findings provide us new insights into the organization and regulation of genes and genome, and serve as an expansive resource for understanding human health and disease.     

Epigenetic states and expression of imprinted genes in human embryonic stem cells

AIM: To investigate the epigenetic states and expression of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan.METHODS: The heterozygous alleles of single nucleotide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expression profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray.RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW, PEG10, NESP55, KCNQ1, ATP10A, TCEB3C and IGF2, were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10, PEG10, SGCE, MEST, SDHD, SNRPN, SNURF, NDN, IPW and NESP55, were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73, COPG2, OSBPL5, IGF2 and ATP10A) were found to be up-regulated in differentiated tissues.CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.     

Vitamin Pharmacogenomics: New Insight into Individual Differences in Diseases and Drug Responses

Vitamins are vital to sustain normal physiological function, metabolism, and growth for all living organisms. Being an integral component of coenzyme, vitamins can affect the catalytic activities of many enzymes and the expression of drug transporters. Genetic variations in metabolism and/or transporter genes of drugs can influence the exposure of the human body to drugs and/or their active metabolites, thus contributing to the variations in drug responses and toxicities. Nonetheless, pharmacogenomics studies on nutrients have been rarely summarized. In this article, we reviewed recent progress on vitamin pharmacogenomics, for a better understanding on the influence of vitamin-related gene polymorphisms on inter-individual differences in diseases and drug efficacy and safety.     

Substitutions of Three Amino Acids in Human Heart/Muscle Type Carnitine Palmitoyltransferase I Caused by Single Nucleotide Polymorphisms

Heart/muscle type carnitine palmitoyltransferase I (M-CPTI) catalyzes the rate-limiting step of mitochondrial long-chain fatty acid (LCFA) oxidation in muscle and adipose tissue. Three replacements of nucleotides resulting in missense mutations of I66V, S427C, and E531K were observed in the M-CPTI gene of patients showing abnormal fatty acid metabolism. These nucleotide replacements were found to be common single nucleotide polymorphisms (SNPs) of this gene and not specific to patients. The question of whether these missense mutations caused by SNPs alter the functional properties of M-CPTI remains unanswered. Thus, we examined whether these missense mutations are associated with any changes in the enzymatic properties of M-CPTI. None of these mutations was found to cause remarkable alteration of its enzymatic properties. Based on the comparison of amino acid sequences of M-CPTI among different animal species, the roles of these amino acids in the enzyme are discussed.     

Single-nucleotide polymorphic variants of human glutathione transferase T1-1 differ in stability and functional properties

We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.     

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

焦磷酸测序测定SNP的常见问题与解决方法

目的:探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法:以VKORC1基因1639 GA位点、CYP2C19基因636 GA位点及UGT1A1基因TA重复序列(TA)6(TA)7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果:通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 GA位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列(TA)6(TA)7多态性的分型准确性。结论:本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

焦磷酸测序测定SNP的常见问题与解决方法

目的：探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法：以VKORC1基因1639 G〉A位点、CYP2C19基因636 G〉A位点及UGT1A1基因TA重复序列（TA）6〉（TA）7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果：通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 G〉A位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列（TA）6〉（TA）7多态性的分型准确性。结论：本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。     

单核苷酸多态性检测技术的研究进展

单核苷酸多态性是指在基因组水平上单个核苷酸变异引起的一种DNA序列多态性。因其具有密度高,遗传稳定,易于进行自动化、规模化分析等优势它已成为第三代分子标记,因此其检测技术也在近几年得到了快速的发展。将对未知单核苷酸突变位点的检测方法和已知单核苷酸突变位点的检测方法这两部分的研究进展作一综述。     

Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.     

Single nucleotide polymorphism rs1800872 in the promoter region of the IL10 gene is associated with predisposition to chronic hepatitis C in Russian population

Previously, we studied an association of two IL28B gene single nucleotide polymorphisms (SNPs) and three IL10 gene SNPs with predisposition to tick-borne encephalitis in a Russian population. In this study, a possible involvement of these SNPs in the development of predisposition to chronic hepatitis C (caused by structurally similar, related virus from the Flaviviridae family) was investigated in the same population. Only the IL10 promoter rs1800872 SNP was associated with predisposition to chronic hepatitis C. This SNP seems to be a common genetic marker of predisposition to two diseases caused by hepatitis C and tick-borne encephalitis viruses in Russian population.     

Development of Genotyping Methods for Single Nucleotide Polymorphism in the Human Pancreatic Ribonuclease Gene (RNASE1) and Their Application to Population Studies

Two potential single nucleotide polymorphisms [SNPs; rs1804215 (G979T) and rs11545379 (G1169T)] have been identified in the human pancreatic ribonuclease, RNase 1, gene (RNASE1) that could give rise to an amino acid substitution in the protein, but relevant population data are not available. We have developed genotyping methods for each SNP using the mismatched PCR-restriction fragment length polymorphism technique. These methods are advantageous in comparison with other SNP genotyping methods because they are technically simpler and do not require specialized instruments. We applied these genotyping methods to examine the genotype distribution of each SNP in four populations, including Japanese populations living in two prefectures, an Ovambo population, and a Turkish population. In all the populations studied, however, only a single genotype for each SNP was found. Therefore, irrespective of differences in ethnic groups, RNASE1 might show markedly low heterogeneity in its genetic structure with regard to these SNPs.     

Eotaxin-3基因多态性与类风湿性关节炎相关性研究

目的:研究湖北地区汉族成人eotaxin-3 77C/T和 2497T/G单核苷酸多态性与类风湿性关节炎易感性之间的关系。方法:用聚合酶链反应-单链构象多态性-四引物聚合酶链反应-限制性酶切的方法进行分析。结果:类风湿关节炎组与对照组eotax- in-3 2497位基因型频率及等位基因的频率差异有统计学意义(P<0.05); 77位基因型频率及等位基因的频率差异无统计学意义(P>0.05)。结论:eotaxin-3 2497T/G多态性跟类风湿关节炎易感性相关。