Detection Of Single Nucleotide Polymorphisms In The ANPEP Gene By Denaturing High Performance Liquid Chromatography

IntroductionSingle nucleotide polymorphisms or SNPs are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered. They can occur in both coding and noncoding regions of the genome. They are the most common form of DNA sequence variations. SNPs, which make up about 90% of all human genetic variation, occur every 100 to 300 bases along the 3-billion-base human genome. For a variation to be considered as a mutation, it must occur in less than 1% of the population. SNPs have a major impact in the field of human genetics. They have been successfully shown to be useful polymorphic genetic markers for the identification of genes associated with complex diseases, the analysis of individual susceptibility to specific diseases, and the research in pharmacogenomics. In addition, SNP maps are helpful to i-dentify thousands of additional markers along the genome, thus simplifying navigation of the much larger genome map generated by researchers in the HGP.Aminopeptidase N, encoded by the ANPEP gene, has been identified as the cellular receptor for human coronavirus HCoV-229E and was suggested as a putative receptor for the spike glycoprotein encoded by the SARS-associated coronavirus ( SARS-CoV). Identification of SNPs within the ANPEP gene will be much useful to study the host genetic susceptibility to infections by human coronaviruses. We have screened the whole coding region of the ANPEP gene u-sing denaturing high performance liquid chromatography ( DHPLC ) and found 21 SNPs within the gene.Materials and Methods1. Samples and Genomic DNA ExtractionSamples of whole blood specimens were obtained from 110 normal unrelated individuals. Genomic DNA was extracted according to the standard proteinase K - SDS - phenol/chloroform protocol.2. SNP Screening and SequencingAll ANPEP exons and their flanking intronic sequences were amplified from genomic DNA samples by PCR. The amplified fragments were subjected to DH-PLC screening. Fragments showing altered DHPLC chromatogram profiles were sequenced.2. 1 Primer Design and PCR Amplification: Specific primer sets were designed for each exon and flanking intronic sequences according to the genomic sequence of the ANPEP gene. PCR amplifications were performed on a PCR thermocycler ( UNO II 48 Biometra, Germany). The quality and quantity of PCR products were determined on 1.5% agarose gels by standard procedures.2.2 DHPLC Analysis: A Transgenomic WAVE庐 DNA Fragment Analysis System and associated WAVE-Maker鈩?. 1 software were used. The optimum conditions were determined empirically, based upon the Tm and melting profile of each individual fragment.2.3 DNA Sequencing: PCR products (50jxl) were first purified using the PCR Purification Kit ( TaKaRa Biotechnology Co. , Ltd. ). The subsequent products were sent to TaKaRa Biotechnology Co. , Ltd. and directly sequenced from both directions.3. Bioinformatic AnalysisAPN protein motif analysis was performed using ExPASy proteomic server ( http://us. expasy. org/prosite/). APN protein homology search was performed using FASTA version 3 of EBI (http;//www. ebi. ac. uk/fasta 33/) and BLAST of NCBI ( http ://www. ncbi. nlm. nih. gov/BLAST/).ResultsA total of 21 SNPs were identified in the ANPEP gene. They were: Q86R (257A > G), 1321M (962C > T), I603M (1809A > G), S651L (1952C > T), S752N (2255G>A), G764R (2290G>A), P227P (681G>A), 17951 (2385C > T), I871I (2613A > G) , IVS7 + 17G > A, IVS13 +41A > C, IVS14 -16A > G, IVS17 + 12C > G, IVS17 + 44C > T, IVS17 -21G > C, IVS19 + 37T>A, IVS19 +75A>G, IVS19 +126C>T, IVS19 + 64A>G, IVS19 + 137G > T and IVS20 + 114C > A. Nine of the variants were in the coding region, among which 6 were non-synonymous with amino acid changes and 3 synonymous without amino acid changes. The remaining 12 ones were in the noncoding intronic region. Except Q86R (257A >G) , I871I (2613A >G) and IVS17 -21G > C, the remaining 18 SNPs were newly found and, to our knowledge, have never been reported before.Conclus...