Nucleotide Polymorphism Genotyping Research Articles

A preliminary mitochondrial DNA SNP genotyping assay for inferring genealogy

The description of phenotypic traits as a result of eyewitness accounts at crime scenes is a standard police practice. Traditional facial composites have been enhanced more recently by computer-aided photofitting techniques. The ‘silent witness’ of DNA evidence also has the potential to provide ‘molecular photofits’. Ancestry as a phenotypic trait is particularly amenable to molecular photofitting as the establishment of human origins through mitochondrial DNA (mtDNA) and Y chromosome haplotyping has been the subject of much recent interest. In this study, a multiplex mtDNA single nucleotide polymorphism (SNP) genotyping assay was devised to assign maternally inherited mtDNA to one of 15 haplogroups using 11 defining SNPs and a deletion. 145 DNA samples were unambiguously assigned to one of 12 haplogroups (three haplogroups were not present) and these were associated with the self-declared genealogy of the donor and that of their mother and maternal grandmother. The assay demonstrated the potential to discriminate between self-declared African, Asian or ‘non-indigenous Australian’ (European) ancestry with 71%, 88% and 90% accuracy, respectively. Larger DNA sample populations that include Y chromosome and autosomal markers have the potential to predict ancestry with finer detail.

A preliminary study on the role of inherited prothrombotic risk factors in Taiwanese patients with sudden sensorineural hearing loss

Sudden sensorineural hearing loss (SSHL) is a disease with unknown etiology. Recently, several studies revealed that some inherited prothrombotic risk factors are associated with SSHL in western populations. The objective of this study was to investigate the roles of the two most common genetic prothrombotic factors, the factor V Leiden G1691A and prothrombin G20210A in Taiwanese patients with SSHL. Twenty-four patients diagnosed with SSHL of more than 30 dB on average pure tone audiometry (PTA) and thirty-six healthy subjects without a history of hearing loss were enrolled in this study. Genomic DNA was isolated from peripheral blood leukocytes and the single nucleotide polymorphisms (SNPs) genotyping of factor V Leiden G1691A and prothrombin G20210A were analyzed using the TaqMan genotyping assays. Neither factor V Leiden G1691A nor prothrombin G20210A was detected in SSHL patients or in the control subjects. Both the patient group and the control group exhibited wild-type V Leiden 1691GG and wild-type prothrombin 20210GG. In conclusion, the factors V Leiden G1691A and prothrombin G20210A do not seem to play any role in Taiwanese patients with SSHL. Further studies with a large series of patients are needed to identify other possible candidate genes in order to elucidate the pathogenesis of SSHL.

Confronting two-pair primer design for enzyme-free SNP genotyping based on a genetic algorithm

BackgroundPolymerase chain reaction with confronting two-pair primers (PCR-CTPP) method produces allele-specific DNA bands of different lengths by adding four designed primers and it achieves the single nucleotide polymorphism (SNP) genotyping by electrophoresis without further steps. It is a time- and cost-effective SNP genotyping method that has the advantage of simplicity. However, computation of feasible CTPP primers is still challenging.ResultsIn this study, we propose a GA (genetic algorithm)-based method to design a feasible CTPP primer set to perform a reliable PCR experiment. The SLC6A4 gene was tested with 288 SNPs for dry dock experiments which indicated that the proposed algorithm provides CTPP primers satisfied most primer constraints. One SNP rs12449783 in the SLC6A4 gene was taken as an example for the genotyping experiments using electrophoresis which validated the GA-based design method as providing reliable CTPP primer sets for SNP genotyping.ConclusionsThe GA-based CTPP primer design method provides all forms of estimation for the common primer constraints of PCR-CTPP. The GA-CTPP program is implemented in JAVA and a user-friendly input interface is freely available at http://bio.kuas.edu.tw/ga-ctpp/.

Abstract 935: Genetic variants in chromosome 6p21.3 and risk of non-Hodgkin lymphoma

Abstract Background. Non-Hodgkin lymphoma (NHL) case-control studies consistently demonstrate the G-308A promoter polymorphism in the tumor necrosis factor gene on chromosome 6p21.3 to be associated with increased risk of the NHL subtype, diffuse large B-cell lymphoma (DLBCL). Initial results from a genome-wide association study have implicated a polymorphism located on 6p21.33 with increased risk of the follicular lymphoma subtype. In the present analysis, we evaluated 488 candidate gene regions (defined as 20kb 5’, 10kb 3’) and their relation to risk for NHL and the DLBCL and follicular lymphoma subtypes with a particular focus on genes within the 6p21 chromosomal region. Methods. We genotyped 6679 tag single nucleotide polymorphisms (SNPs) in 947 cases and 826 population-based controls from a multicenter U.S.-based case-control study. Genotyping was conducted on a custom-designed Illumina Infinium assay at the NCI Core Genotyping Facility. We obtained a gene region-level summary of association by computing the minimum p-value (“minP test”). We used logistic regression to evaluate the association between single nucleotide polymorphism (SNP) genotypes and haplotypes with NHL (adjusted for sex, race and age). For NHL subtypes, we conducted polytomous multivariate unconditional logistic regression. We evaluated tests for trend under the co-dominant model used a three-level ordinal variable for each SNP. Results. We identified 34 gene regions significantly associated with all NHL (p <0.05). The three most significant gene regions were all found on chromosome 6p21.3 (RING1/RXRB; AIF1/BAT2; BAT4, p=0.001). Within those gene regions, the most significant SNPs (p<0.0002) were rs2855429 and rs213213 (RING1/RXRB), rs2857597 and rs3130071 (AIF1/BAT2), and rs3115667 (BAT4). All five SNPs were statistically significantly associated with both DLBCL and follicular lymphoma. Additional gene regions on 6p21.3 associated with NHL include MSH5 (p=0.009), FAM46A (p=0.01) and TNXB/CREBL1 (p=0.04). Stratification by age and gender did not alter our results; consistent results were observed in analyses restricted to non-Hispanic Caucasians. Haplotype analyses yielded the same regions and SNPs of significance. Detailed analysis of the 6p21.3 region will be presented. Conclusions. Our results further implicate gene variants within the 6p21.3 chromosomal region in NHL etiology. Full characterization of the 6p21.3 region and identification of functional variants within this region are warranted for understanding lymphomagenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 935.

Abstract 3585: Taqman genotyping assays can be used on decalcified and paraffin embedded tissue from patients with osteosarcoma

Abstract Insight in genetic variants predicting treatment outcome and/or side effects related to cancer medication will contribute to optimize treatment for the individual patients. However, most clinical trials performed so far in cancer treatment did not routinely collect and store patient germ line DNA. Formalin-fixed paraffin-embedded tissues (FFPE) from archived clinical samples could provide a source of germ line DNA for genetic research. Specially, in cancers like osteosarcoma with a 5-year overall survival of approximately 50-60%, archived histological specimens are often the only accessible source of biological material. This material, however, generally has been decalcified and paraffin-embedded for many years. DNA from formalin-fixed and paraffin embedded (FFPE) tissues, specially, in decalcified FFPE tissues has a higher frequency of non-reproducible sequence alterations compared with germ line DNA. In our study, we investigated whether DNA obtained from these tissues can be used for reliable single nucleotide polymorphism (SNP) genotyping. Therefore, we analyzed whether the genotypes obtained from DNA of these preserved tissues, were similar as those found in freshly DNA isolated from peripheral blood cells from the same patients. We studied two SNPs in the drug transporter MDR1 (ABCB1) using Taqman® SNP genotyping assays. Genotypes of the germ line DNA derived from freshly isolated DNA of 20 surviving patients with osteosarcoma were compared with genotypes obtained from archived material from decalcified FFPE blocks of the same patients. Decalcified FFPE-derived DNA yielded smaller PCR fragments compared to DNA extracted from peripheral blood cells, with a maximum reliable size of ∼300bp. We were able to evaluate each SNP in 19 of the 20 cases included in this study. All successfully genotyped samples showed 100%-concordance between genotypes obtained from DNA of FFPE tissue and the genotypes obtained from DNA of blood. In conclusion, we have demonstrated that decalcified FFPE tissue can be used for genetic polymorphism analysis using Taqman® allelic discrimination assays. This forms an unique opportunity to combine new insights in genetic research with historical patient cohorts, for instance of patients with osteosarcoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3585.

Breast Cancer Risk Single Nucleotide Polymorphism (SNP) Genotypes Do Not Correlate with Risk Estimates from Existing Prediction Models.

Abstract Background:Breast cancer risk assessment professionals employ models to estimate individual probabilities of developing breast cancer. The Gail, Claus and BRCAPro models integrate personal, reproductive and family history variables to generate absolute risk estimates. A number of common DNA variants (single nucleotide polymorphisms, SNPs) are associated with modest elevations (relative risk < 2) of breast cancer risk. We investigated the correlation between genotype at 18 published risk SNPs and model-derived breast cancer risk estimates.Methods:Subjects were 475 unaffected, BRCA mutation-negative women who were evaluated for a family history of breast or ovarian cancer. At the time of testing, subjects provided information that was used to estimate absolute breast cancer risk by Gail, Claus, and BRCAPro models. Subjects were genotyped by Sequenom iPLEX MassArray (Sequenom, Inc., San Diego, CA) at loci reported to be associated with elevations in breast cancer risk (1p11.2, 2q35, 5p12, 6q22.33, 8q24, CASP8, COX11, FGFR2, LSP1, MAP3K1, RAD51L1, SLC4A7, TNRC9). Kruskal-Wallis and Spearman correlation tests were used to compare risk estimates for individuals by genotype and number of risk alleles.Results:The median age at sample donation was 46 years (range 24-83). Most subjects (94.9%) were white, and 65.5% were of Ashkenazi Jewish (AJ) ancestry. Median breast cancer risk scores risk estimates were significantly lower from BRCAPro (5 yr: 1.1%, lifetime (LT): 11.6%) than those derived from Gail (5 yr: 1.8%, LT: 18.5%) or Claus (5 yr: 1.5%, LT: 11.8%) models. No significant differences (p>0.05) in distribution of estimated breast cancer risk scores were found between women carrying 0, 1, or 2 risk alleles at any locus except 6q22.33, where AJ women homozygous for the risk allele rs2180341 were noted to have higher LT Claus (p=0.001) and BRCAPro (p=0.01) estimates. When comparing subjects with no risk alleles to those with 1-2 risk alleles for each SNP, no significant differences in distribution of estimated risk were noted at any loci, including 6q22.33. There was no significant correlation between the total number of risk alleles carried by a subject and her estimated lifetime Gail (ρ=-0.052, P=0.27), Claus (ρ=0.051, P=0.32), or BRCAPro (ρ=0.024, P=0.60) risk.Conclusions:In this analysis, risk allele genotype did not correlate with absolute breast cancer risk estimated by standard models. This suggests that the contribution to risk of genotype at these SNPs is insufficient to impact the modeled estimate. Further studies are necessary to evaluate whether incorporation of genotypic information can improve the calibration or discriminatory accuracy of existing models. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 901.

Robust QTL fine mapping by applying a quantitative transmission disequilibrium test to the Mendelian sampling term

In many farm animal populations, high-density single nucleotide polymorphism (SNP) genotypes are becoming available on a large scale, and routine estimation of breeding values is implemented for a multiplicity of traits. We propose to apply the basic principle of the quantitative transmission disequilibrium test (QTDT) to estimated Mendelian sampling terms. A two-step procedure is suggested, where in the first step additive breeding values are estimated with a mixed linear model and the Mendelian sampling terms are calculated from the estimated breeding values. In the second step, the QTDT is applied to these estimated Mendelian sampling terms. The resulting test is expected to yield significant results if the SNP is in sufficient linkage disequilibrium and linkage with quantitative trait loci (QTL). This principle is illustrated with a simulated data set comprising 4665 individuals genotyped for 6000 SNP and 15 true QTL. Thirteen of the fifteen QTL were significant on a genome-wide 0.1% error level. Results for the empirical power are derived from repeated samples of 1000 and 3000 genotyped individuals, respectively. General properties and potential extensions of the methodology are indicated. Owing to its computational simplicity and speed, the suggested procedure is well suited to scan whole genomes with high-density SNP coverage in samples of substantial size and for a multiplicity of different traits.

263 PATERNAL ISODISOMY FOR CHROMOSOME 2 AS CAUSE OF BILE SALT EXPORT PUMP (BSEP) DEFICIENCY

Background: Bile salt export pump (BSEP) deficiency, a defect in a canalicular bile acid excretion, falls into the progressive familial intrahepatic cholestasis. It is an autosomal recessive disorder with several mutations being reported in the ABCB11 gene on chromosome 2q24−31. We describe a case of BSEP deficiency with a new mutation, where the patient received both mutated alleles from the father, through the rare phenomenon of uniparental disomy (UPD). Methods: A 4-year-old infant, underwent to orthotopic liver transplantation (OLT) at the age of 9 months because of persistent cholestasis and liver failure. Immunohistochemistry was performed with BSEP antibody. Genomic DNA was extracted from the baby and his parents. Molecular studies of ABCB11 were performed using denaturing high-pressure liquid chromatography (DHPLC) and direct sequencing. Gene copy number determination, segregation of the mutation and reconstruction of the flanking haplotype employed quantitative real time PCR (qPCR), single nucleotide polymorphisms (SNPs) genotyping, and polymorphic microsatellites. Results: Liver histology showed cirrhosis. Immunohistochemistal stain using anti-BSEP antibodies revealed wholly absent marking at canalicular margin of hepatocytes, consistent with the diagnosis of BSEP deficiency. DHPLC and sequencing analysis showed a novel, homozygous c.2620C>T transition in exon 20 of the ABCB11 gene. The mutation replaces arginine for cysteine at residue 833 (p. R833C). This change was absent in a large panel of normal chromosomes, and it was heterozygous in peripheral blood from the father, but not in several tissues obtained from the child’s mother. qPCR analyses did not disclose abnormal gene copy number in both the patient and his mother ruling out a microdeletion of ABCB11. Segregation analysis of seven intragenic SNPs and six flanking microsatellite markers showed data suggestive of UID of the region on chromosome 2q24−31 inherited from the father. Conclusion: In severe forms, infants with BSEP deficiency developed cholestasis and fibrosis, and end-stage liver disease requires OLT. Molecular studies are needed to offer measures as to avoid recurrence of the disease. The present case highlights the importance of full gene analyses to offer a more accurate genetic counselling.

SNP discovery based on CATS and genotyping in the finless porpoise (Neophocaena phocaenoides)

Single nucleotide polymorphisms (SNPs) discovery and genotyping were performed for the finless porpoises (Neophocaena phocaenoides). About 202 comparative anchor tagged sequence primers derived from genomes of human, mouse and some other mammals were used to screen the finless porpoise population. Of the 51 SNPs discovered, 25 were further characterized with ideal genotyping primers and using fragment length discrepant allele specific PCR assay. This is the first report of SNP loci for the finless porpoise, which is helpful to provide some novel molecular markers and new genetic information relevant to the conservation and management of this endangered species.

Newborn screening of G6PD variants using long term storage of dried blood spot

Aims To establish a rapid and simple DNA extraction method from dried blood spot (DBS) by using an automation system and to evaluate the use of DBS specimens for G6PD single nucleotide polymorphism (SNP) genotyping. Methods 100 DBS (stored up to 1 year) were subjected to DNA extraction using Qiagen Mini Kit with QIAcube automation system. Eluted DNA was subjected to OD reading to determine the A260/A280 ratio. DNA from each sample were subjected to sequencing and genotyping using Taqman MGB genotyping analysis for 1376 G>T, 1388 G>A, 95 A>G, 1024 C>T, 871 G>A, 487 G>A, and 563 C>T, 392 G>T of G6PD gene. Results Ratio A260/A280 of all samples ranged from 1.7 to 1.9. All samples were able to be amplified and the types of variants were able to be identified respectively. Conclusion Our study clearly shows that genomic DNA in dried blood spot in proper storage is stable and easily to extract. This approach could be utilised in newborn screening for other genetics diseases that are amenable to DNA analysis. This method provides a useful alternative to conventional DNA isolation, especially in clinical settings, as it enables easy storage and transport of samples from the point of acquisition.

"Sequencing-grade" screening for BRCA1 variants by oligo-arrays

The need for fast, efficient, and less costly means to screen genetic variants associated with disease predisposition led us to develop an oligo-nucleotide array-based process for gene-specific single nucleotide polymorphism (SNP) genotyping. This cost-effective, high-throughput strategy has high sensitivity and the same degree of accuracy as direct sequencing, the current gold standard for genetic screening. We used the BRCA1 breast and ovarian cancer predisposing gene model for the validation of the accuracy and efficiency of our strategy. This process could detect point mutations, insertions or deletions of any length, of known and unknown variants even in heterozygous conditions without affecting sensitivity and specificity. The system could be applied to other disorders and can also be custom-designed to include a number of genes related to specific clinical conditions. This system is particularly useful for the screening of long genomic regions with relatively infrequent but clinically relevant variants, while drastically cutting time and costs in comparison to high-throughput sequencing.

Classification of genetic profiles of Crohn’s disease: a focus on the ATG16L1 gene

Inflammatory bowel disease constitutes two related clinical entities, Crohn’s disease (CD) and ulcerative colitis (UC), both of which have increased in prevalence over the last decade. Family and twin studies have strongly indicated that genetic factors play a large role in an individual’s risk of developing inflammatory bowel disease. Despite this, it has proven difficult to isolate disease genes that confer susceptibility to this disease using classical candidate gene and linkage approaches, with the notable exception of the isolation of the caspase recruitment domain family, member 15 (CARD15) gene. However, over the last 2 years, genome-wide association (GWA) studies have become feasible, where modern high-throughput single nucleotide polymorphism (SNP) genotyping technologies can be applied to large and comprehensively phenotyped patient cohorts. Such approaches have enabled scientists to robustly associate specific variants with many complex diseases, including age-related macular degeneration, Type 2 diabetes, breast cancer and asthma. In the inflammatory bowel disease field, positive associations with CD and UC coming from GWA studies have been reported for an ever increasing number of genes. The most consistently and strongly associated variants have been in the CARD15, the interleukin 23 receptor (IL23R) and autophagy-related 16-like 1 (ATG16L1) genes. With respect to ATG16L1, the G allele of SNP rs2241880 has been shown in multiple association studies to confer strong risk for CD, although its association with UC remains more debatable. This SNP is in fact a common coding variant, specifically a threonine-to-alanine substitution at amino acid position 300 of the ATG16L1 protein (T300A), and appears to account for all of the disease risk conferred by this locus. This review addresses recent advances in GWA studies of inflammatory bowel disease, with specific focus on the growing evidence of the ATG16L1 gene’s role in CD and how its protein product operating within the autophagic pathway makes autophagy an attractive therapeutic target for this debilitating disorder.

Toward colloid-based biosensors for SNP genotyping and personalised medicine applications

Therapeutic drugs can assist some patients, however, other individuals may exhibit no response. Furthermore, a drug that is normally considered to be safe can have toxic effects in a small proportion of the population, occasionally causing death. Many altered drug responses depend on the genetic constitution of the recipient. The analysis of specific DNA markers within the genome of an individual [single nucleotide polymorphism (SNP) genotyping] could potentially identify subjects at risk in clinical trials and may enable clinicians to individualise medical therapy. The development of technology for personalised medicine relies on the ability to accurately and rapidly identify SNPs in genetic material. Some of the biotechnology tools for SNP identification are colloid-based biosensors, which are proving to possess several advantages over the traditional microarray format. Here, we survey the current technologies used for SNP genotyping and review colloid-based biosensors which are emerging as a higher throughput, potentially more accurate, technology.

Refinement of molecular approaches to improve the chance of identification of hematopoietic-restricted minor histocompatibility antigens

Minor histocompatibility antigens (mHAgs) constitute the target antigens of the T cell-mediated graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation (SCT). Several human mHAgs have been identified, but only a few are selectively expressed by hematopoietic cells representing potential targets for specific immunotherapy. Molecular approaches including cDNA library screening and genetic linkage analysis have been successfully applied to identify T cell-defined mHAgs, but each approach has its drawbacks which may lead to mis-identification of the mHAg of interest. We improved both molecular strategies to facilitate more robust identification of hematopoietic-restricted mHAgs. First, we adapted cDNA library cloning by using 293T cells with stable expression of the relevant MHC class I allele, CD80 and CD54. We demonstrated that cDNA library screening using this 293T expression system results in strong activation of cytotoxic T lymphocytes, which significantly contributes to improvement of the assay sensitivity. Second, we refined genetic linkage analysis using single nucleotide polymorphism (SNP) genotyping to narrow down the defined genetic region that holds the mHAg-encoding gene. We showed that SNP marker analysis provides additional information about the genetic position of the antigen-encoding gene. Application of these optimized molecular approaches will lead to more rapid and reliable molecular identification of hematopoietic-restricted mHAgs.

The enhancement of homogenous mass extension reaction: Comparison of two enzymes

Reliable and efficient PCR and extension reactions using standardized procedures are key elements for successful single nucleotide polymorphism (SNP) genotyping projects. To improve the cost efficiency and overall performance of SNP genotyping we evaluated two commercial thermostable DNA polymerases used for the extension reaction in the homogeneous mass extension MassARRAY genotyping system. The aim was to study whether the quality, accuracy, and expenses of a new TERMIPol DNA polymerase are competitive to the commonly used ThermoSequenase DNA polymerase. We compared the enzymes by testing 96 SNPs genotyped for DNA samples of 31 unrelated individuals and one water control. The success rates, congruence between the genotypes and completeness of extension reactions support the use of TERMIPol, especially when the amplification of the higher mass allele is difficult. Further, using TERMIPol enabled successful genotyping (>93%) of several SNPs that failed (<80% success) when using ThermoSequenase.

Purifying Genomic DNA from Plant Tissue on Automated High-Throughput and Moderate-Throughput Platforms

We describe automated methods for purifying genomic DNA from plant tissue. The Wizard Magnetic 96 Plant System uses MagneSil paramagnetic particles (PMPs) in a 96-well format to purify sufficient DNA for polymerase chain reaction (PCR)-based plant genotyping. The system can be scaled up for high yield to provide genomic DNA for large numbers of PCR-based tests or archiving. In contrast, the MagneSil ONE Fixed Yield System uses MagneSil ONE PMPs to purify a fixed amount of ultraclean DNA for high sensitivity in Third Wave Technology Invader single nucleotide polymorphism (SNP) Genotyping assays. These methods may be implemented on Beckman Coulter Biomek automated workstations.

2P135 ミスマッチ塩基対と金属イオンとの特異的な結合 : 1塩基多型の新規検出方法の開発に向けて(核酸 A) 構造・物性))

2P135 ミスマッチ塩基対と金属イオンとの特異的な結合 : 1塩基多型の新規検出方法の開発に向けて(核酸 A) 構造・物性))

Single Nucleotide Polymorphism discovery and genotyping within the chicken Tapasin gene

RiassuntoIdentificazione e genotipizzazione di Single Nucleotide Polymorphism nel gene Tapasin di pollo. Tapasin è una proteina transmembrana localizzata nel reticolo endoplasmatico la cui funzione è quella di permettere il caricamento del peptide antigenico sulla molecola MHC di classe I. Nel pollo il gene tapasin è costituito da 8 esoni ed è localizzato nell’MHC tra i due geni classici di classe II. L’obiettivo del presente lavoro è l’individuazione di polimorfismi SNP in linee commerciali e razze autoctone di pollo. L’analisi delle sequenze ottenute,che mostrano un’elevata omologia con le sequenze già depositate in GenBank, ha portato all’identificazione dell’omologia con 2 contig EST e con una sequenza che mappa sul cromosoma 16 del pollo. Inoltre, l’analisi mediante il programma AutoSNP ha permesso l’individuazione di 7 siti polimorfici putativi.

Linkages and associations to intermediate phenotypes underlying asthma and allergic disease.

Considerable effort and expense has been expended in attempts to detect specific polymorphisms in genetic loci contributing to susceptibility to allergic diseases. Increasingly, 'intermediate' phenotypes for asthma and other allergic diseases have become the focus of such investigations, due to disease heterogeneity and the difficulty of defining disease status. Together with meta-analysis of existing linkage datasets and the completion of many second-stage and specialized genome scans, the recent focus in asthma and atopy genetics has been on single nucleotide polymorphism discovery and genotyping in candidate genes.