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66 A DIRECT HAPLOTYPING METHOD OF PUTATIVE FUNCTIONAL VARIANTS IN PROMOTER REGIONS OF THREE CANDIDATE GENES IN ACUTE LUNG INJURY.
  1. M. M. Kalscheur1,
  2. C. Flores2,
  3. S. F. Ma2,
  4. M. Burke3,
  5. W. J. Buikema3,
  6. J. G.N. Garcia2
  1. 1Pritzker School of Medicine
  2. 2Pulmonary and Critical Care
  3. 3Cancer Research Center DNA Sequencing Facility, The University of Chicago, Chicago, IL

Abstract

Rationale Due to their functional relevance, single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) have been used in association studies of complex diseases. The combination of both types of polymorphisms in short genomic regions (< 500 bp), termed SNPSTRs, constitutes a powerful tool as it allows for the empiric determination of gametic phase, or haplotype, thus adding a new level of complexity over single variants. Our aim is to select a set of SNPSTR pairs in candidate genes in acute lung injury (ALI) and to develop a multiplex method to directly determine the haplotypes of each of these SNPSTR pairs. Methods: Candidate genes identified by microarray analysis of animal models of ALI were selected. Functional relevance of SNPs and STRs in the promoter region was established for the selected candidate genes using PubMed. Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi>) was used to design SNPSTR primers as well as the complimentary fluorescently labeled primers for SNP alleles. A three-step direct haplotyping method was performed as follows: (1) coamplification of the genomic regions of interest by multiplex PCR, (2) linear amplification of the amplicons with FAM- and HEX-labeled fluorescent primers, and (3) identification of haplotypes by GeneMapper software v3.7.

Results Three candidate genes (IL-10, HMOX-1, and CXCL2) were identified with reported functionally relevant SNPSTRs and were arranged into a multiplex protocol. Genotyped variants were -1087 G/A SNP and the -1121 CAn STR for IL-10, -413 A/T SNP and the -199 GTn for HMOX-1, and -437 A/G SNP and the -665 CAn STR for CXCL2. A Coriell CEPH DNA panel, consisting of individuals of European and African American decent (23 and 24 individuals, respectively), was then used to develop our haplotyping method. SNP alleles and STR repeats were confirmed with direct DNA sequencing.

Conclusion This SNPSTR method represents an efficient and robust approach to obtain direct haplotypes in three candidate genes, which will advantage association studies of genetic variants in ALI.

Funding: Specialized Centers of Clinically Oriented Research P50 HL-073994 and Fundacion Canaria Dr. Manuel Morales.

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