Dynamic Allele-specific Hybridization Research Articles

Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR.

Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS). Sixteen venous blood samples collected in K3-EDTA tubes (400μl of whole blood) were used for the spotting (4 circles each 100μl) on Ahlstrom 226 grad filter papers, for extraction and comparison. To ensure effectiveness, the extracted DNA was checked for quantity using the Quant-iT™ dsDNA Broad-Range Assay Kit and for quality by polymerase chain reaction (PCR) amplification of 344 bp segment of the HBB gene. Hybridization assays based on the dynamic allele specific hybridization (DASH) technique for two hemoglobin beta (HBB) mutations in genomic DNA extracted from DBS of ß-thalassemia patients were also performed to ensure the quality of extraction. The results revealed a compatible effectiveness of the superparamagnetic-bead based method in extracting DNA from DBS particularly when incubating the DBS with lysis buffers BL+BLM overnight. A mean concentration of 21ng/ μl was obtained with lysis buffers BL+BLM overnight incubation compared to 5.2 ng/μl for 2 h incubation with lysis buffers BL+BLM and 4.7 ng/μl when extraction performed using the lysis buffer BLM alone. Moreover, PCR amplification of 344 bp segment of the HBB showed a good quality of the extracted DNA. It was concluded that the superparamagnetic-bead based method is a reliable and effective method for DNA extraction from DBS and can be adopted for genetic diagnostic purposes.

Lack of association between ESR1 gene polymorphisms and premature ovarian failure in Serbian women

Objective It has previously been reported that estrogen receptor-alpha (ERα) gene (ESR1: estrogen receptor 1) polymorphisms are associated with premature ovarian failure (POF). The aim of this study was to investigate whether these genetic polymorphisms of ESR1 are associated with POF in Serbian women.Methods A series of 197 POF cases matched with 547 fertile controls was recruited by the Institute for Endocrinology, Diabetes and Metabolic Disorders of Serbia between 2007 and 2010. Genomic DNA was extracted from saliva using Oragene® DNA sample collection kits. Two single-nucleotide polymorphisms (SNPs), PvuII and XbaI, in ESR1 were genotyped by dynamic allele-specific hybridization. Haplotype analyses were performed with the restriction fragment length polymorphism method. SNP and haplotype effects were analyzed by logistic regression models.Results No significant difference was found in the distribution of ESR1 PvuII and XbaI polymorphisms or haplotypes between the POF and control groups.Conclusion The two ESR1 SNPs, PvuII and XbaI, are not commonly associated with POF in Serbian women and may not contribute to the genetic basis of the condition.

Single Nucleotide Polymorphism (SNPs) of DNA repair genes (hMLH1 and hMSH2) in Rheumatoid arthritis Patients and its association with rheumatoid arthritis by Smoking status

The purpose of this study is to examine the risk factors and the single nucleotide polymorphisms(SNPs) of DNA repair gene (hMLH1 and hMSH2) associated with rheumatoid arthritis by smoking status and environmental tobacco smoke(ETS). This study was planned to detect the susceptibility of the patients diagnosed by rheumatoid arthritis to the single nucleotide polymorphisms (SNPs) of DNA repair gene(hMLH1 and hMSH2) by smoking status and environmental tobacco smoke(ETS). The single nucleotide polymorphisms(SNPs) of DNA repair gene(hMLH1 and hMSH2) were analyzed by Dynamic allele specific hybridization(DASH) technique in rheumatoid arthritis patients and controls. The single nucleotide polymorphisms(SNPs) of DNA repair gene(hMLH1 and hMSH2) of patients were compared to those of age and sex-matched controls, which are statistically analyzed and adjusted by age, sex, smoking status, and ETS. This study showed that; The SNP of DNA repair gene hMLH1 was significantly associated with the susceptibility of rheumatoid arthritis, but that of hMSH2 was not. Differential effect of passive smoking on the association between the SNP of DNA repair gene hMSH2(CC type or CT type) and rheumatoid arthritis risk was found. It is suggested that the SNPs of DNA repair gene correlated with susceptibility to rheumatoid arthritis by smoking status was able to be used as its susceptibility marker and further as basic data to prevent the risk factors or rheumatoid arthritis. But the larger sample studies, including a variety of SNPs of DNA repair gene will be needed.

A single nucleotide polymorphism alters the sequence of SP1 binding site in the adiponectin promoter region and is associated with diabetic nephropathy among type 1 diabetic patients in the Genetics of Kidneys in Diabetes Study

Objective The adiponectin promoter single nucleotide polymorphism (SNP) −11391G/A is found to be associated with nephropathy in type 1 diabetic (T1D) patients among Danish, but not French, Finnish, and Swedish populations. In the present study, we identified the binding sites for transcriptional factors in the adiponectin promoter region and also evaluated the association between adiponectin promoter polymorphisms and diabetic nephropathy (DN) in T1D patients. Materials and Methods Three adiponectin promoter SNPs, including −11377C/G, −11391G/A, and −11426A/G, were genotyped with dynamic allele-specific hybridization. The subjects included 1177 American T1D patients (622 females/555 males) with or without DN. All patients are of European descent and selected from the Genetics of Kidneys in Diabetes (GoKinD) study. Results We identified four binding sites of transcriptional stimulatory protein (SP1) in the adiponectin putative promoter and found that the G allele of SNP −11377C/G altered the sequence for one of the SP1 binding sites. This polymorphism was significantly associated with DN in female T1D patients ( P=.022, OR=1.352, 95% CI=1.044–1.752). Further analyses indicated the common diplotype (haplotypic genotype) H1/H1, constructed with SNPs −11377C/G and −11391G/A, was significantly associated with DN in females ( P=.013), while the association of another diplotype H1/H2 with DN in females was of borderline significance ( P=.071). Conclusions The present study thus provides the first evidence that SNP −11377C/G alters the sequence in one of the SP1 binding sites in the adiponectin promoter region. This polymorphism, together with another promoter SNP −11391G/A, may confer susceptibility to the development of DN in T1D patients among the GoKinD population.

Genetic variation in receptor protein tyrosine phosphatase σ is associated with type 2 diabetes in Swedish Caucasians

Previously, it has been demonstrated that receptor protein tyrosine phosphatase sigma (RPTPsigma) is involved in glucose homeostasis and insulin signaling in several animal models. The aim of this study was to evaluate whether polymorphisms in this gene influence the development of type 2 diabetes (T2D) in humans. We investigated how genetic variations in the RPTPsigma gene influence susceptibility to impaired glucose tolerance (IGT) and T2D, in Swedish men and women. Genotyping of single nucleotide polymorphisms (SNPs) was performed by dynamic allele-specific hybridization in a total of 1057 Swedish Caucasians including 497 subjects with normal glucose tolerance (NGT), 262 subjects with IGT, and 298 patients with T2D. SNPs rs1143699, rs4807015, and rs1978237 were found to be associated with T2D. SNP rs1143699 was associated with male T2D patients when compared with NGT controls (odds ratio; OR = 1.57; P = 0.029). SNP rs4807015 showed association with T2D patients when compared with NGT controls (OR = 1.32; P = 0.025). Finally, SNP rs1978237 was associated with T2D patients when compared with NGT controls (OR = 1.59; P = 0.002). Logistic regression analysis demonstrated that for SNP rs1143699 in men, C/C homozygosity conveys an increased risk of T2D (OR = 2.19; P = 0.035), while SNP rs4807015 was associated with an increased risk of T2D in both men and women (OR = 1.74; P = 0.029). SNP rs1978237 also demonstrated a risk of T2D in men and women (OR = 1.59; P = 0.026). This study provides evidence for association of SNPs in the RPTPsigma gene with T2D in Swedish Caucasians. SNPs rs1143699, rs4807015, and rs1978237 confer an increased risk of developing T2D.

DNA Diagnostics by Surface-Bound Melt-Curve Reactions

Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allele-specific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First, for mutation scanning (resequencing), a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps, providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were correctly revealed in 16 individuals. Second, to score multiple different mutations in parallel, 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type- and mutation-specific probes. Both experiments were performed in a blinded manner, and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample, with no false-positive or false-negative signals across all of the investigations. In conclusion, DASH performs accurately and robustly when applied to DNA diagnostic challenges, including mutation scoring and mutation scanning.

Genetic association analysis of the adiponectin polymorphisms in type 1 diabetes with and without diabetic nephropathy

Objective Adiponectin [adipocyte C1q and collagen domain containing (ACDC)] is the most abundant adipose-specific protein. It is beneficial in that it improves insulin sensitivity and mitigates vascular damage, in addition to the possibility of it having anti-inflammatory properties. Clinical evidences demonstrate that serum adiponectin concentrations are increased in patients with type 1 diabetes (T1D) as well as in patients with microvascular complications. However, the genetic influence of the ACDC gene in T1D and diabetic microvascular complications is still unclear. The present study aims to evaluate the association of the ACDC genetic variation in T1D and diabetic nephropathy (DN). Materials and Methods Ten single nucleotide polymorphisms (SNPs) of the ACDC gene were genotyped in 432 T1D patients (of which, 196 had DN) and 187 nondiabetic control subjects, who were all Swedish Caucasians, by using dynamic allele specific hybridization. Results Single-marker association analysis demonstrated that SNPs +45G15G(T/G) and +276(G/T) were strongly associated with T1D [ P=.002, OR=1.855 (1.266–2.717) and P=.001, OR=1.694 (1.337–2.147)]. Further analysis for haplotypes of these two SNPs indicated that one of the common haplotype (T_G) was strongly associated with T1D [ P<.001, OR=1.769 (1.430–2.188)]. However, there was no significant difference in the allele frequencies of these two SNPs between the groups of T1D patients with nephropathy and the patients without nephropathy. Conclusions The present study thus suggests that SNPs +45G15G(T/G) and +276(G/T) in the ACDC gene are associated with T1D but not with DN among Swedish Caucasians.

Genetic influences of the intercellular adhesion molecule 1 (ICAM‐1) gene polymorphisms in development of Type 1 diabetes and diabetic nephropathy

AimThe intercellular adhesion molecule-1 (ICAM-1) gene is located on chromosome 19p13, which is linked to Type 1 diabetes (T1D). ICAM-1 expression is related to development of T1D and diabetic nephropathy. The present study aims to evaluate the genetic influence of ICAM-1 gene polymorphisms on the development of T1D and diabetic nephropathy.MethodsFive valid single nucleotide polymorphisms (SNPs) were genotyped in 432 T1D patients (196 patients had diabetic nephropathy) and 187 non-diabetic control subjects by using dynamic allele-specific hybridization (DASH) and pyrosequencing.ResultsSNPs rs281432(C/G) and rs5498 E469K(A/G) had high heterozygous indexes. They were significantly associated with T1D [P = 0.026, OR = 1.644 (95% CI 1.138–2.376) and P < 0.001, OR = 2.456 (1.588–3.8)]. Frequencies of the C allele in SNP rs281432(C/G) and the A allele in SNP rs5498 E469K(A/G) increased stepwise from non-diabetic control subjects to T1D patients without diabetic nephropathy and T1D patients with diabetic nephropathy. Further analysis for these two SNPs indicated that T1D patients had increased frequency of the common haplotype C-A, in comparison with non-diabetic control subjects (38.1 vs. 32.1%, P = 0.035).ConclusionThe present study provided evidence that SNPs rs281432(C/G) and rs5498 E469K(A/G) in the ICAM-1 gene confer susceptibility to the development of T1D and might also be associated with diabetic nephropathy in Swedish Caucasians.

Rapid Melting Curve Analysis on Monolayered Beads for High-Throughput Genotyping of Single-Nucleotide Polymorphisms

This report describes a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis is based on dynamic allele-specific hybridization (DASH). The DNA duplexes are conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. The method is based on random bead immobilization by microcontact printing. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Analyses that took more than 15 min could be performed in less that 1 min, enabling ultrarapid SNP analysis. In addition, an array version of the chip was implemented enabling the preparation of an array of bead arrays for high-throughput and rapid SNP genotyping.

Obesity and genetic polymorphism of ERCC2 and ERCC4 as modifiers of risk of breast cancer

To evaluate the relationship of genetic polymorphisms of ERCC2 and ERCC4 genes, both involved in nucleotide excision repair (NER), and the risk of breast cancer, a hospital-based case-control study was conducted in Korea. Histologically confirmed breast cancer cases (n = 574) and controls (n = 502) with no present or previous history of cancer were recruited from three teaching hospitals in Seoul during 1995-2001. Information on selected characteristics was collected by interviewed questionnaire. ERCC2 Asp(312)Asn (G>A) was genotyped by single-base extension assay and ERCC4 Ser(835)Ser (T>C) by dynamic allele-specific hybridization system. Although no significant association was observed between the genetic polymorphisms and the risk of breast cancer, women with both ERCC2 A allele- and ERCC4 C allele-containing genotypes showed a 2.6-fold risk (95% CI: 1.02-6.48) of breast cancer compared to women concurrently carrying the ERCC2 GG and ERCC4 TT genotypes. The breast cancer risk increased as the number of "at risk" genotypes increased with a borderline significance (P for trend = 0.07). Interactive effect was also observed between ERCC4 genotype and body mass idnex (BMI) for the breast cancer risk; the ERCC4 C allele containing genotypes posed a 1.7-fold (95% CI: 0.96-2.93) breast cancer risk in obese women (BMI>25 kg/m(2)) with a borderline significance. Our finding suggests that the combined effect of ERCC2 Asp(312)Asn and ERCC4 Ser(835)Ser genotypes might be associated with breast cancer risk in Korean women.

Genotyping by dynamic heating of monolayered beads on a microheated surface

A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for single-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4 degrees C-6 degrees C/min compared to earlier reported values of 2 degrees C-3 degrees C/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 microm was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.

Association analysis of the polymorphism T1128C in the signal peptide of neuropeptide Y in a Swedish hypertensive population.

The neuropeptide Y (NPY) signal peptide polymorphism T1128C has been linked to several risk factors for cardiovascular disease. The aim of the present study was to evaluate the significance of this polymorphism for cardiovascular and cerebrovascular disease outcome. In a prospective study cohort, 1032 hypertensive patients (174 myocardial infarction and 170 stroke patients and 688 matched controls) were analysed for the T1128C polymorphism in the NPY gene. The dynamic allele specific hybridization (DASH) method was used for genotyping. Serum from the same participants was analysed for total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides. The frequency of the NPY T1128C polymorphism was 8.4% among patients with a myocardial infarction or stroke, as compared to 5.1% in the control group (P = 0.040). The difference remained significant after adjustment for the cardiovascular risk factors age, sex, smoking status, body mass index, systolic and diastolic blood pressure, presence of diabetes, total cholesterol, HDL, LDL and triglycerides. The present study indicates that the NPY T1128C polymorphism is an independent predictor for myocardial infarction and stroke in a Swedish hypertensive population.

DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications.

Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele-biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow-on applications, we have developed DFold (http://dfold.cgb.ki.se)-a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele-specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole-genome analyses.

A nicotine C-oxidase gene (CYP2A6) polymorphism important for promoter activity.

In humans, several polymorphic variants have been described for the gene encoding the major nicotine C-oxidase, cytochrome P450 2A6 (CYP2A6), which is to a great extent responsible for the large interindividual differences seen at the enzymatic and activity levels. Hitherto, mainly polymorphic variants in the open reading frame have been identified. In the present study, we identified a novel single nucleotide polymorphism (SNP) located in the 5' flanking region of the CYP2A6 gene. Sequencing of 1.4 kb of the 5'-upstream region of the CYP2A6 gene from eight individuals revealed a c.-1013A>G polymorphism defining two new alleles, CYP2A6*1D and CYP2A6*1E, lacking or having also the CYP2A7 3'-UTR. Analysis of genomic DNA from 32 Swedish and 109 Turkish subjects by dynamic allele-specific hybridization (DASH) showed that, in both groups, the variants carrying the c.-1013A>G SNP represent approximately 70% of the total number of alleles. Transfection of HepG2 cells with luciferase reporter constructs containing 1019 bp of the CYP2A6 5'-regulatory sequence showed that the region between c.-1005 and c.-1019 elicited a strong enhancer effect and that the CYP2A6*1D promoter had significantly reduced expression as compared to CYP2A6*1A carrying c.-1013A. Electrophoretic mobility shift assays (EMSA) showed that nuclear proteins from HepG2 and B16A2 cells exhibited a higher binding affinity to the probe harboring c.-1013A as compared to the c.-1013G probe, although the transcription factor(s) responsible for this binding could not be identified. In conclusion, our results indicate the presence of a strong enhancer or promoter responsive element between c.-1005 and c.-1019 in the CYP2A6 gene and that a c.-1013A>G polymorphism in this region affects CYP2A6 transcription.

Scoring insertion-deletion polymorphisms by dynamic allele-specific hybridization.

Genome variation provides researchers with thousands of markers with which to study human demographic history and phenotypes. Insertion-deletion (indel) polymorphism is an important and abundant form of human genome variation, and convenient methods for genotyping indels are therefore needed. Here we evaluate dynamic allele-specific hybridization (DASH) for its ability to score indels. Evaluation of six model indel DASH assays based on synthetic oligonucleotides showed that length differences of 1-5 bp were accurately scored. Only single probes were required to assay indels of 3-4 bp or less, while longer indels tended to require the use of both allele probes serially. The best results were obtained by central placing of the probe over the indel. Model study findings were confirmed by running indel DASH assays upon PCR-amplified targets representing four polymorphisms from Alzheimer's disease candidate genes APBB1 and LRP1. These indels were genotyped in a set of 121 patients and 156 controls. While no disease association was found, the data quality confirmed that DASH is a robust and useful procedure for genotyping indels of the size range typically found in the human genome.

IFRET: an improved fluorescence system for DNA-melting analysis.

Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.

Novel and alternate SNP and genetic technologies.

There are many different genotyping technologies and chemistries. Other articles in this special issue focus on nanotechnology, bioinformatics, DNA chips and genotyping methods. This article focuses on four method categories not featured elsewhere in this, or the following special issue: (1) melting curve-based technologies such as dynamic allele-specific hybridization (DASH), melting curve single nucleotide polymorphism (McSNP), fluorescent resonance energy transfer (FRET), hybridization-based melting curves, and homogeneous assay formats based on melting curves; (2) non-PCR-dependent assays such as the oligonucleotide ligation assay and Invader, and isothermal amplification techniques such as rolling circle amplification; (3) rapid whole genome sequencing with methods such as the use of single molecule arrays and molecular resonance sequencing; (4) and other promising novel technologies.

SNP analysis by allele-specific extension in a micromachined filter chamber.

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides.

Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

Identification of a Single Nucleotide Polymorphism in the TATA Box of the CYP2A6 Gene: Impairment of Its Promoter Activity

Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5′ flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a −48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5′-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the −48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.