A Study On The Genome-wide Detection Of Copy Number Variation And Loss Of Heterozygosity Of Human Non Small Cell Squamous Lung Cancer Using Single Nucleotide Polymorphism Microarray

Object In the last few years,reports have demonstrated that copy number gain/or loss (copy number variation CNV) influences gene expression and result in phenotypic variation by disrupting genes and altering gene dosage.Loss of heterozygosity (LOH) of chromosomal regions bearing tumor suppressor genes is a key event in the oncogenesis and development of epithelial and mesenchymal tumors. But the functions of LOH and CNV have not been fully understood. Previous studies attempting to identify genetic changes occurring in non small cell lung cancers have relied on cytogenetic studies, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH).LOH can be analysed through allelotyping of tumors with polymorphic genetic markers.Restriction fragment length polymorphism (RFLP) or microsatellites are reliable genetic markers, but the genotyping procedure is rather laborious and time-comsuming on a genome-wide scale.Genome-wide detection of LOH, as well as CNV in cancer genomes, has drawn recent attention in the field of cancer genetics.SNPs are the most common form of sequence variation in the human genome, occurring approximately every～1000 bp.Affymetrix Single Nucleotide Polymorphism Microarray was originally developed for large-scale genome-wide association studies, provide a powerful platform for both genome-wide LOH analysis and CN detection. In this platform, the use of large numbers of SNP-specific probes showing linear hybridization kinetics allows not only high-resolution(<10Kb) LOH analysis at 906,600 SNP loci but also accurate determination of the CN status at each LOH region compared with the low resolution of array-CGH. The aim of this study is to obtain the genetic changes of non small cell squamous lung cancer by using Affymetrix genome-wide SNP 6.0 array for screening out genes that are related to their phenotypes, and ultimately try to study the melocular mechanisms of non small cell squamous lung cancer.Method Genomic DNA extracted from 15 non small cell squamous lung cancer tissues and matched normal lung tissues were used to be analysed by genome-wide SNP 6.0 arrays.The hybridization intensity of each probe was analyzed using Affymetrix proprietary software for genotyping and copy number at each locus and inorder to obtain global profiles of CNV and LOH of the tumors.Result 1.The SNP call rate of 15 arrays ranged from 95.07% to 99,26%,with average call rate of 97.49%. QC call rate of the 15 arrays ranged from 2.08 to 3.05,averaged 2.73.All the call rates of the arrays were in excess of 93%,the cardinal quality control standard.2.Of the 15 arrays,There are 7837 regions which CN=1,accounted for 54.6% of all the CNV regions; There are 6018 regions which CN=3,accounted for 41.9% of all the CNV regions;There are 226 regions which CN=4,accounted for 1.57% of all the CNV regions; The majority of the chromosome loss alterations were located on chromosome 4,3,5,1,10;The majority of the chromosome gain alterations were located on chromosome 7,2,8,1,12.3.LOH was detected in all the 15 arrays with different degrees,a total number of 1,921,845 were detected in the 15 arrays.The minimum number of 54,274 LOH was detected in sample C08-0142 and the maximum number of 400,078 LOH was detected in sample C08-0027.The percentage of LOH incidence rate ranged from 0.03% to 28.1%.4.92 homozyous deletion regions were detected in the 15 arrays,which were located on chromosome1,2,3,4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,21,X,Y.The percentage of HD occurred among all the chromosome were chromosome 4(21.7%), chromosome 5(11.9%),chromosome 3(9.8%),chromosome Y(8.7%),chromosome X(7.6%),chromosome 19(6.5%),chromosome 17(5.4%),chromosome 1(4.3%), chromosome 8,10,15,18(3.3%),chromosome 7(2.2%),chromosome 2,6, 9,12,13,14,16,21(1.1%).The shortest HD region was located on q13.1 of chromosome 19,covering 101kb;the longest HD region was located on p12.3～p12.2 of chromosome 3,covering 7,146kb.Conclusion(1)Genome-wide chromosone copy number variation and LOH can be detected by using high resolution Affymetrix Genome-wide SNP 6.0 array simultaneousl.(2) We could identify narrow amplified and deleted region to find new oncogenes and tumor suppressor genes by using the Affymetrix Genome-wide SNP 6.0 array and software Genome Typing Console 3.0.(3).The novel amplified or deleted genes discovered here might beeome targets for the early diagnosis and therapy for non small cell squmous lung cancer.