Combined inhibition of XIAP and BCL2 drives maximal therapeutic efficacy in genetically diverse aggressive acute myeloid leukemia.

Aggressive therapy-resistant and refractory acute myeloid leukemia (AML) has an extremely poor outcome. By analyzing a large number of genetically complex and diverse, primary high-risk poor-outcome human AML samples, we identified specific pathways of therapeutic vulnerability. Through drug screens followed by extensive in vivo validation and genomic analyses, we found inhibition of cytosolic and mitochondrial anti-apoptotic proteins XIAP, BCL2 and MCL1, and a key regulator of mitosis, AURKB, as a vulnerability hub based on patient-specific genetic aberrations and transcriptional signatures. Combinatorial therapeutic inhibition of XIAP with an additional patient-specific vulnerability eliminated established AML in vivo in patient-derived xenografts (PDXs) bearing diverse genetic aberrations, with no signs of recurrence during off-treatment follow-up. By integrating genomic profiling and drug-sensitivity testing, this work provides a platform for a precision-medicine approach for treating aggressive AML with high unmet need.

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population.

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48×10(-21), higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA. In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.

Association study of the polymorphisms on chromosome 12p13 with atherothrombotic stroke in the Japanese population.

Recent genome-wide association study using four prospective population-based cohorts identified two single-nucleotide polymorphisms (SNPs) on chromosome 12p13, rs12425791 and rs11833579, to be significantly associated with the incidence of atherothrombotic stroke. To examine the association of these SNPs with atherothrombotic stroke in the Japanese population, we carried out a case-control association study using a total of 3784 cases and 3102 controls. We also examined the effect of these SNPs on the subtypes of ischemic stroke. Association analysis was carried out using logistic regression model after adjustment of age, sex and cardiovascular risk factors. Rs12425791 was significantly associated with atherothrombotic stroke (P=0.0084, odds ratio (OR)=1.15). When we analyzed effects of rs12425791 on ischemic stroke subtypes, rs12425791 was significantly associated with both small-artery occlusion (P=0.015, OR=1.15) and large-artery atherosclerosis (P=0.024, OR=1.19). Rs11833579 showed no association with atherothrombotic stroke or its subtypes in our population. Our data suggest that rs12425791 on chromosome 12p13 is a genetic marker for atherothrombotic stroke in multiethnic population.

Functional SNP of ARHGEF10 confers risk of atherothrombotic stroke.

Although stroke is a common cause of death and a major cause of disability all over the world, genetic components of common forms of ischemic stroke are largely unknown. To identify susceptibility genes of atherothrombotic stroke, we performed a large case-control association study and a replication study in a total of 2775 cases with atherothrombotic stroke and 2839 controls. Through the analysis in 860 cases and 860 age- and sex-matched controls, we found that a single-nucleotide polymorphism (SNP), rs2280887, in the ARHGEF10 gene was significantly associated with atherothrombotic stroke even after the adjustment of multiple testing by a permutation test [unadjusted P = 1.2 x 10(-6), odds ratio = 1.80, 95% confidence interval (CI) = 1.42-2.28]. This association was replicated in independent 1915 cases and 1979 controls. Subsequent fine mapping found another three SNPs which showed similar association due to strong linkage disequilibrium to rs2280887 (r(2) > 0.95). In the functional analyses of these four highly associated SNPs, using luciferase assay and electrophoretic mobility shift assay we found that rs4376531 affected ARHGEF10 transcriptional activity due to the different Sp1-binding affinity. In small GTPase activity assay, we found that a gene product of ARHGEF10 specifically activated RhoA. A population-based cohort study revealed the subjects with rs4376531 CC or CG to increase the incidence of ischemic stroke (P = 0.033, hazard ratio = 1.79, 95% CI = 1.05-3.04). Our data suggest that the functional SNP of ARHGEF10 confers the susceptibility to atherothrombotic stroke.

CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection.

BACKGROUND: Cytochrome P450 2D6 (CYP2D6), one of the most important drug-metabolizing enzymes, has been reported to possess variation in the encoding CYP2D6 gene (cytochrome P450, family 2, subfamily D, polypeptide 6) that affects enzymatic activity. For the pharmacogenetic study of CYP2D6, accurate measurement of the dosage of the functional gene is essential; however, current genotyping techniques are insufficient because of their inability to provide the exact copy number of functional CYP2D6 genes. METHODS: We developed 3 quantitative real-time PCR (qPCR) assays for estimating the total copy number of the CYP2D6 gene, as well as 24-multiplex PCR-based real-time Invader assays (mPCR-RETINAs) for estimating the allele ratio at each variation locus. After determining the allele copy number at each locus, we estimated the frequencies of CYP2D6 alleles in a population and the diplotype in each individual by a CNVphaser (copy number variation phaser). The qPCR assays and RETINAs used for HapMap Japanese and Chinese samples were applied to 455 Japanese individuals. RESULTS: Forty-two individuals (9.2%) had one CYP2D6 gene copy, 207 (45.5%) had 2 copies, 161 (35.4%) had 3 copies, 40 (8.8%) had 4 copies, and 5 (1.1%) had 5 copies of the CYP2D6 gene. We found 16 different CYP2D6 alleles, with frequencies similar to those described in previous reports. In the diplotype analysis, we observed that CYP2D6*1/*1 and *1/*10-*36 were the most common diplotypes (approximately 20%) in our population. CONCLUSIONS: Our method is the first to determine the exact number of functional CYP2D6 gene copies. We believe our method will facilitate and accelerate the detailed pharmacogenetic analysis of CYP2D6.