Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay

A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost.     

Identification of the gene variations in human CD22

 CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases. Received: 14 July 1998 / Revised: 7 September 1998     

Comparative Evaluation of Quantitative Polymerase Chain Reaction Methods for Routine Enumeration of Specific Bacterial DNA in Aquatic Samples

Summary Three quantitative polymerase chain reaction (PCR) methods, the internal standard method (IS-PCR), competitive PCR (cPCR) and most probable number-PCR (MPN-PCR), were compared in terms of their ability to quantify specific bacterial DNA in environmental samples. Serially diluted Pseudomonas putida BH, the target bacterium, was inoculated into sterilized potassium phosphate buffer (PPB), river water and activated sludge, total DNA was extracted, and the number of pheB genes carried by P. putida BH in each sample was enumerated. IS-PCR and cPCR could not quantify the pheB gene at low concentrations (1.0 × 10³ copies ml^-1 in all samples and 1.0 × 10⁴ copies ml^--1 in some samples) and tended to give overestimations because of differences in amplification efficiencies between pheB gene and the internal standard/competitor in a reaction tube. Although reproducibility of MPN-PCR was slightly lower than that of the other two methods, MPN-PCR was the most sensitive, enabling us to quantify the pheB gene at 1.0 × 10³ copies ml^--1, and it had a good correlation with the inoculum size of P. putida BH. These results suggest that MPN-PCR is the best suited for routine microbial monitoring in natural environmental samples because of the simple handling, the ease of modification as occasion demands and the wide detection range, especially at low cell densities of the target microbe.     

Lack of Association of the HbE Variant with Protection from Cerebral Malaria in Thailand

Hemoglobin E (HbE; beta26Glu --> Lys) is the most common variant of the beta-globin gene in Southeast Asia; it has been suggested that it confers resistance against Plasmodium falciparum malaria. In this study 306 adult patients with P. falciparum malaria (198 mild and 108 cerebral malaria patients) living in northwest Thailand were investigated to examine whether the HbE variant is associated with protection from cerebral malaria. Our results revealed that the sample allele frequency of HbE was not significantly different between mild (7.3%) and cerebral malaria (7.4%) patients. Thus, the HbA/HbE polymorphism would not be a major genetic factor influencing the onset of cerebral malaria in Thailand.     

A replication study confirmed the <Emphasis Type="Italic">EDAR</Emphasis> gene to be a major contributor to population differentiation regarding head hair thickness in Asia

Hair morphology is a highly divergent phenotype among human populations. We recently reported that a nonsynonymous SNP in the ectodysplasin A receptor (EDAR 1540T/C) is associated with head hair fiber thickness in an ethnic group in Thailand (Thai-Mai) and an Indonesian population. However, these Southeast Asian populations are genetically and geographically close, and thus the genetic contribution of EDAR to hair morphological variation in the other Asian populations has remained unclear. In this study, we examined the association of 1540T/C with hair morphology in a Japanese population (Northeast Asian). As observed in our previous study, 1540T/C showed a significant association with hair cross-sectional area (P = 2.7 × 10⁻⁶) in Japanese. When all populations (Thai-Mai, Indonesian, and Japanese) were combined, the association of 1540T/C was stronger (P = 3.8 × 10⁻¹⁰) than those of age, sex, and population. These results indicate that EDAR is the genetic determinant of hair thickness as well as a strong contributor to hair fiber thickness variation among Asian populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene flow and natural selection in oceanic human populations inferred from genome-wide SNP typing

It is suggested that the major prehistoric human colonizations of Oceania occurred twice, namely, about 50,000 and 4,000 years ago. The first settlers are considered as ancestors of indigenous people in New Guinea and Australia. The second settlers are Austronesian-speaking people who dispersed by voyaging in the Pacific Ocean. In this study, we performed genome-wide single-nucleotide polymorphism (SNP) typing on an indigenous Melanesian (Papuan) population, Gidra, and a Polynesian population, Tongans, by using the Affymetrix 500K assay. The SNP data were analyzed together with the data of the HapMap samples provided by Affymetrix. In agreement with previous studies, our phylogenetic analysis indicated that indigenous Melanesians are genetically closer to Asians than to Africans and European Americans. Population structure analyses revealed that the Tongan population is genetically originated from Asians at 70% and indigenous Melanesians at 30%, which thus supports the so-called Slow train model. We also applied the SNP data to genome-wide scans for positive selection by examining haplotypic variation and identified many candidates of locally selected genes. Providing a clue to understand human adaptation to environments, our approach based on evolutionary genetics must contribute to revealing unknown gene functions as well as functional differences between alleles. Conversely, this approach can also shed some light onto the invisible phenotypic differences between populations.     

Thymidine phosphorylase inhibits apoptosis induced by cisplatin

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.     

alpha 1-Adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1

Three distinct subtypes of alpha(1)-adrenergic receptors (alpha(1)A-, alpha(1)B-, and alpha(1)D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of alpha(1)A- or alpha(1)B-AR inhibits serum-promoted cell proliferation, whereas alpha(1)D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G(1)/S transition after activation of alpha(1)A/alpha(1)B-AR but not of alpha(1)D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR, whereas all alpha(1)-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27(Kip1), was blocked after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR. These results show that alpha(1)-AR subtypes differentially activate the cAMP/p27(Kip1) pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where alpha(1)-AR subtypes are generally co-expressed.     

Activation and suppression of renin-angiotensin system in human dendritic cells

We previously identified the gene expression of renin-angiotensin system in human monocyte-derived dendritic cells (DCs). This study was conducted to examine the mechanisms by which angiotensin II and captopril, the inhibitor of the angiotensin-converting enzyme (ACE), affect human DCs. In DCs, lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-(IL)-1alpha, IL-10, IL-12, and IL-18 was significantly inhibited by captopril. In contrast, angiotensin II treatment resulted in a significant increase in TNF-alpha and IL-6 protein biosynthesis by DCs. In addition, we have studied the global expression of 2400 genes in DCs from two donors. Here, we demonstrated the specific down-regulation of the ACE gene expression in captopril-treated DCs. Our finding indicates the possible activation of NF-kappaB through the up-regulation of expressions of MEFV gene (encoding PYRIN protein) and heterogeneous nuclear ribonucleoprotein R in DCs. This is the first study on the modulation of cytokine and gene expression by angiotensin II and captopril in DCs.