第809回千葉医学会例会・第一外科教室談話会 64.

<p>RR, relative risk.</p

Advantage of Using Allele-Specific Copy Numbers When Testing for Association in Regions with Common Copy Number Variants

<div><p>Copy number variants (CNV) can be called from SNP-arrays; however, few studies have attempted to combine both CNV and SNP calls to test for association with complex diseases. Even when SNPs are located within CNVs, two separate association analyses are necessary, to compare the distribution of bi-allelic genotypes in cases and controls (referred to as SNP-only strategy) and the number of copies of a region (referred to as CNV-only strategy). However, when disease susceptibility is actually associated with allele specific copy-number states, the two strategies may not yield comparable results, raising a series of questions about the optimal analytical approach. We performed simulations of the performance of association testing under different scenarios that varied genotype frequencies and inheritance models. We show that the SNP-only strategy lacks power under most scenarios when the SNP is located within a CNV; frequently it is excluded from analysis as it does not pass quality control metrics either because of an increased rate of missing calls or a departure from fitness for Hardy-Weinberg proportion. The CNV-only strategy also lacks power because the association testing depends on the allele which copy number varies. The combined strategy performs well in most of the scenarios. Hence, we advocate the use of this combined strategy when testing for association with SNPs located within CNVs.</p> </div

The effects of a SNP in pre-miR-1206 on expression of mature miR-1206. A.

<p>Allelic forms of pre-miR-1206 were cloned into miRNA expression vector pEGP-miR. Graphs show analysis of mature miR-1206 expression in 293T cells, in relation to rs2114358 GG and AA genotypes. <b>B.</b> In prostate cancer cell lines. <b>C.</b> In colon cancer cell lines. <b>D.</b> In breast cancer cell lines.</p

SNPs in miRNA genes in 8q24.2 and 11q13.3 genotyped in SNP500 and HapMap samples.

a<p>allele 1/<u>allele 2</u>; allele frequencies are for underlined alleles. SNP500 samples:</p>b<p>Caucasian (n = 31),</p>c<p>African American (n = 24),</p>d<p>Hispanic (n = 23),</p>e<p>Pacific Rim (n = 24); HapMap samples: Hap CEU (n = 90), Hap CHB+JPT (n = 90), Hap YRI (n = 90).</p>f<p>miR-939 novel SNP at chr position 145590173;</p>g<p>miR-1237 novel SNP at chr position 63892701 (based on hg 18).</p><p>N.A. = not assessed.</p

The effects of two SNPs in pre-miR-612 on expression of mature miR-612. A.

<p>Allelic forms of pre-miR-612 were cloned into miRNA expression vector pEGP-miR. Graphs show analysis of mature miR-612 expression in 293T cells, in relation to combination of rs550894 and rs12803915 genotypes: CC/GG (reference), AA/GG and CC/AA. <b>B.</b> In prostate cancer cell lines. <b>C.</b> In colon cancer cell lines. <b>D.</b> In breast cancer cell lines.</p

Flowchart of the simulations.

<p>1) The allele-specific copy number states were simulated for controls given the expected frequencies. 2) The expected frequencies in the case population were calculated given the frequencies in the control population and the relative risks (RR) of both the allelic and the copy number effects (RR_allele and RR_CN respectively). 3) The allele-specific copy-number states were simulated for cases given the expected frequencies in cases. 4) We deducted the bi-allelic genotypes information from the allele-specific copy-number states, given the probabilities of each allele-specific copy-number state to be respectively called as AA, AB, BB or missing. Association tests were performed on both the allele-specific copy-number states that contain the complete information on allele and copy-number, and the bi-allelic genotypes that contain partial information on the allele. Classical criteria used in SNP analysis such as the Hardy-Weinberg equilibrium (HWE) departure in controls and the percentage (%) of missing data were computed on the bi-allelic genotypes.</p

Analysis of surface expression of Insulin Receptor (INSR) in miR-612 transfected cancer cell lines.

<p>Cell surface insulin receptor (INSR) expression was determined by anti-INSR staining and flow cytometry24 hours post-transfection of the indicated miR-612 expression vectors. The results are representative of four independent experiments.</p

Power of each of the five investigated strategies when only deletions were present.

<p>The power was computed for a sample of 1,000 cases and 1,000 controls under the different scenarios of risk and frequencies. On the X axis, f(B) and f(norm) refer respectively to the frequency of the allele B and to the frequency of normal chromosome carrying one copy of the CNV.</p

Schematic outline of experimental procedures. A.

<p>Co-transfection of allelic forms of miR-612 with a miR-1206 construct used as a normalization control. <b>B.</b> Co-transfection scheme for testing SNP effects on miR-612 processing. Three different combinations of allelic forms of miR-612 (#1∼#3) were co-transfected with control miR-1206 expression construct.</p

Power of each of the five investigated strategies when both deletions and duplications were present.

<p>The power was computed for a sample of 1,000 cases and 1,000 controls under the different scenarios of risk and frequencies. On the X axis, f(B) and f(norm) refer respectively to the frequency of the allele B and to the frequency of normal chromosome carrying one copy of the CNV.</p