A simple, bead-based approach for multi-SNP molecular haplotyping.

Single nucleotide polymorphisms (SNPs) within a gene region have often been studied to determine their effect on phenotype. Although a single base pair change can produce a phenotypic change, phenotype is often influenced by the presence of multiple polymorphisms and their relative positions within a given region. For example, if multiple changes occur in a promoter region, how they influence gene expression will depend on their cis/trans configuration. As such, it is essential to consider the haplotype, or the alignment of multiple SNP alleles on each chromosome when attempting to associate genomic changes with phenotype. Unfortunately, no method of high-throughput molecular haplotyping of multiple SNPs currently exists. In response to this unmet need, we have developed an inexpensive, reliable bead-based capture-based haplotyping (CBH) assay to determine the phase, or haplotype, of multiple SNP alleles in a high-throughput manner. The CBH assay requires minimal setup and handling, requires no centrifugation steps and can be performed in <1 h. Data collection is performed via flow cytometry and the assay yields plus/minus results allowing for automated calling by a simple computer application. We will present data demonstrating the molecular haplotyping of 11 SNPs within exon 2 of the N-acetyltransferase-2 (NAT2) gene, which expresses an important drug-metabolizing enzyme. This assay has applications in diagnostic testing, promoter analysis, association studies and pharmacogenetic analysis.

[1]  R S Judson,et al.  Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Doll,et al.  Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. , 2000, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[3]  Brian Staats,et al.  SNP500Cancer: a public resource for sequence validation and assay development for genetic variation in candidate genes , 2004, Nucleic Acids Res..

[4]  Susanne Knapp,et al.  Interleukin-10 promoter polymorphisms and the outcome of hepatitis C virus infection , 2003, Immunogenetics.

[5]  J. Goedert,et al.  Genetic acceleration of AIDS progression by a promoter variant of CCR5. , 1998, Science.

[6]  D. Grant,et al.  Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. , 1993, Carcinogenesis.

[7]  D. Grant,et al.  Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression. , 1990, DNA and cell biology.

[8]  B. Leventhal,et al.  Genomic organization of the SLC1A1/EAAC1 gene and mutation screening in early-onset obsessive-compulsive disorder , 2001, Molecular Psychiatry.

[9]  K K Kidd,et al.  Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence. , 2000, Human molecular genetics.

[10]  Charles R Cantor,et al.  Direct molecular haplotyping of long-range genomic DNA with M1-PCR , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  D. Grant,et al.  Human acetyltransferase polymorphisms. , 1997, Mutation research.

[12]  M. Weiner,et al.  Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry. , 2000, Cytometry.

[13]  A. Alderborn,et al.  Molecular haplotype determination using allele-specific PCR and pyrosequencing technology. , 2003, Genomics.

[14]  L. Lazzeroni,et al.  Association of IL4R haplotypes with type 1 diabetes. , 2002, Diabetes.

[15]  M. Krajinovic,et al.  Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukemia , 2004, The Pharmacogenomics Journal.

[16]  P. Carmeliet,et al.  VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death , 2003, Nature Genetics.

[17]  K K Kidd,et al.  Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[18]  W M Barnes,et al.  PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Y. Kashi,et al.  Direct micro-haplotyping by multiple double PCR amplifications of specific alleles (MD-PASA). , 2002, Nucleic acids research.

[20]  E. Boerwinkle,et al.  DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene , 1998, Nature Genetics.

[21]  Russell Higuchi,et al.  Effective amplification of long targets from cloned inserts and human genomic DNA. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Richard J. Mural,et al.  Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  R. Minchin,et al.  N-and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. , 1992, Biochemical and biophysical research communications.

[24]  M. Rieder,et al.  Sequence variation in the human angiotensin converting enzyme , 1999, Nature Genetics.

[25]  S. Humphries,et al.  A functional haplotype in the 5′ flanking region of the factor VII gene is associated with an increased risk of coronary heart disease , 2003, Journal of thrombosis and haemostasis : JTH.

[26]  K. Lindblad-Toh,et al.  SBE-TAGS: an array-based method for efficient single-nucleotide polymorphism genotyping. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Little,et al.  N-acetyltransferase polymorphisms and colorectal cancer: a HuGE review. , 2000, American journal of epidemiology.

[28]  D. Nickerson,et al.  PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. , 1997, Nucleic acids research.

[29]  A. Papp,et al.  Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to duchenne muscular dystrophy carrier testing , 1994, Human mutation.

[30]  I. Roots,et al.  Pitfalls in N-acetyltransferase 2 genotyping. , 1999, Pharmacogenetics.

[31]  R. Minchin,et al.  Pharmacogenetics of the arylamine N-acetyltransferases , 2002, The Pharmacogenomics Journal.

[32]  T. Rustan,et al.  Human N-acetylation of benzidine: role of NAT1 and NAT2. , 1996, Cancer research.