분자 도치 프로브 (MIP) 기반 다중 타깃 포획을 이용한 약물유전체 분석

의과대학/박사As the target capture platform for the pharmacogenomics analysis, we designed a panel of molecular inversion probes to cover 80 genes which can affect exposure and response to drugs. By stepwise rebalancing, probes were optimized to capture targets with high coverage (96%) and accuracy (99.8%). The panel features the efficient probe preparation as initial microarray-based synthesis of duplex MIPs (microDuMIPs) is applied. Target capture and next-generation sequencing (NGS) of 191 individuals with this panel identified a number of unreported low-frequency genetic variants in targeted genes. In silico prediction showed genetic variants at low frequency enriched for ones with deleterious functional impacts. By further in vitro investigation, selected rare or low-frequency variants were validated to alter functions in influx/efflux transporters (SLC22A1/OCT1, SLCO1B1/OATP1B1, ABCB1/MDR1 and a metabolizing enzyme (CYP2C19). This implicates genetic variants at low frequency, which might have been overlooked in past pharmacogenetic studies, may bear relevance to the inter-individual difference in response to drugs. As a follow-up, we applied the platform to capture DNA sequences of 90 participants in phase 1 clinical trials for Tacrolimus, an immunosuppressive drug known for large inter-individual variance in pharmacokinetics (PK). Besides CYP3A5*3, an established index variant for the PK variability, POR*28 and rare variants in UGT1A4 were revealed to be the account for the part of the PK variability. We verified that the POR*28 allele decreased exposure to drug, distinctively in CYP3A5 full expressers. Rare variants in UGT1A4 may cause variable extent of glucuronidation as phase II metabolism of secondary importance, given that this genetic effect is conspicuous in phenotypic extremes with impaired CYP3A5 function (*1/*3 and *3/*3). To the best of our knowledge, this is the first time to implicate the association of genetic variations in UGT1A4 with variable responses to Tacrolimus. Collectively, POR*28 and rare variants in UGT1A4 can explain the PK variance approximately 6% more than solely by CYP3A5*3. These results suggest that not only common but also rare variants are worth being considered for pharmacogenomic analysis and testing. Our platform also presents a proof of concept that NGS-based pharmacogenomic testing at earlier phase of clinical trials can provide the foundation for understanding metabolic pathways of drugs by extensive genetic screening including rare variants scan. Moreover, this shows that MIP-based multiplexed target capture can be one of effective tools to investigate individualized metabolic pathways of drugs.ope

microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes

Molecular inversion probe (MIP)-based capture is a scalable and effective target-enrichment technology that can use synthetic single-stranded oligonucleotides as probes. Unlike the straightforward use of synthetic oligonucleotides for low-throughput target capture, high-throughput MIP capture has required laborious protocols to generate thousands of single-stranded probes from DNA microarray because of multiple enzymatic steps, gel purifications and extensive PCR amplifications. Here, we developed a simple and efficient microarray-based MIP preparation protocol using only one enzyme with double-stranded probes and improved target capture yields by designing probes with overlapping targets and unique barcodes. To test our strategy, we produced 11 510 microarray-based duplex MIPs (microDuMIPs) and captured 3554 exons of 228 genes in a HapMap genomic DNA sample (NA12878). Under our protocol, capture performance and precision of calling were compatible to conventional MIP capture methods, yet overlapping targets and unique barcodes allowed us to precisely genotype with as little as 50 ng of input genomic DNA without library preparation. microDuMIP method is simpler and cheaper, allowing broader applications and accurate target sequencing with a scalable number of targets.ope

ZMYND10 stabilizes intermediate chain proteins in the cytoplasmic pre-assembly of dynein arms

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.ope

Genetic testing of Korean familial hypercholesterolemia using whole-exome sequencing

Familial hypercholesterolemia (FH) is a genetic disorder with an increased risk of early-onset coronary artery disease. Although some clinically diagnosed FH cases are caused by mutations in LDLR, APOB, or PCSK9, mutation detection rates and profiles can vary across ethnic groups. In this study, we aimed to provide insight into the spectrum of FH-causing mutations in Koreans. Among 136 patients referred for FH, 69 who met Simon Broome criteria with definite family history were enrolled. By whole-exome sequencing (WES) analysis, we confirmed that the 3 known FH-related genes accounted for genetic causes in 23 patients (33.3%). A substantial portion of the mutations (19 of 23 patients, 82.6%) resulted from 17 mutations and 2 copy number deletions in LDLR gene. Two mutations each in the APOB and PCSK9 genes were verified. Of these anomalies, two frameshift deletions in LDLR and one mutation in PCSK9 were identified as novel causative mutations. In particular, one novel mutation and copy number deletion were validated by co-segregation in their relatives. This study confirmed the utility of genetic diagnosis of FH through WES.ope

Development of high speed confocal line scanning microscope for imaging of biological tissues

의공학과/석사[한글] 1960년대 Minsky에 의해 개발된 공초점 현미경은 안구검사, 안질의 조기 진단, 치료중의 질병의 추이나 발전 정도를 관측하는데 상당히 유용하다. 특히 각막 내피 세포는 감염으로 인한 손상시, 재생 불가능한 조직으로 안과에서의 조기 진단이 중요하다. 살아있는 사람의 눈은 항상 움직이고 있다. 이것을 고정 관련 빠른 안구 움직임(fixation-related saccadic eye movement)이라 하며 상당히 빠른 안구 운동을 한다. 눈의 빠른 움직임 때문에 각막 내피 세포의 고속, 고해상도 영상을 얻을 수 있는 공초점 현미경 시스템이 필요하다.본 연구에서 하나의 음향 광학 편향기(Acousto optic deflector)와 한 개의 검출기인 선주사 카메라를 사용하여 선주사 공초점 레이저 현미경 시스템을 제안하고 구현하였다. 본 시스템에서 고속 스캐닝을 위해 광탄성 작용에 따라 기계적 움직임이 없는 음향 광학 편향기를 이용하여 초당 50 프레임 (1024 X 1024 pixel image) 초당 100 프레임 (1024 X 512 pixel image), 초당 200 프레임 (512 X 512 pixel image) 의 이차원 공초점 영상을 획득하였다. 50배 대물 렌즈 사용시, 본 시스템의 횡축 해상도는 공초점 방향 0.6um, 비 공초점 방향 1.07um이었으며 종축 해상도는 3.33um으로 측정되었다. 기존 상용화된 공초점 현미경 시스템들보다 더욱 빠른 속도로 이차원 영상을 재생하고, 삼차원 영상을 재구성할 수 있었다. 본 고속 선주사 공초점 레이저 현미경 시스템을 통하여 임상이나 생물학 분야에서 고속으로 일어나는 현상에 대한 분석에 유용할 것으로 예상되며 본 연구에서는 빠른 안구 운동을 하는 안구 조직 영상의 획득을 목적으로 개발하였다 [영문]Since it is developed by Minsky in 1960s, the confocal microscope is predicted to have increasing importance in the examination of the eye, the early diagnosis of eye disease, as well as the monitoring and clinical evaluation of its treatment.In particular, an early diagnosis of ophthalmology is very important because endothelial cell is non regeneration cell in case of injury by infection. Eyes of live human beings are always moving. It called fixation-related saccadic eye movement and quite fast ocular movements. Confocal microscope system is needed to get high resolution and speed images of endothelial cell due to fast ocular movements.In this system, we adopted the acousto-optic deflector that is a non-mechanical device by photo-elastic interactions for high-speed scanning system. It generates two-dimensional images with speeds of 50 frames per second (1024 X 512 pixel image), 100 frames per second (1024 X 1024 pixel image) and, 200 frames per second (512 X 512 pixel image).The measured axial resolution of the system is 0.6？m for confocal axis (Y-axis) and 1.07？m for non-confocal axis (X-axis) with a 50X objective. The measured lateral resolution of the system is 3.33？m with a 50X objective.To the best of our knowledge, it is capable of producing three-dimensional images by accumulating 2-D images within less time than any other devices. High speed slit-scanning confocal laser microscope (HS-CLM) might be useful for analyzing fast interactions in the clinical and biological applications.ope

Targeted Next-Generation Sequencing for Comprehensive Genetic Profiling of Pharmacogenes

Phenotypic differences in drug responses have been associated with known pharmacogenomic loci, but many remain to be characterized. Therefore, we developed next-generation sequencing (NGS) panels to enable broad and unbiased inspection of genes that are involved in pharmacokinetics (PKs) and pharmacodynamics (PDs). These panels feature repetitively optimized probes to capture up to 114 PK/PD-related genes with high coverage (99.6%) and accuracy (99.9%). Sequencing of a Korean cohort (n = 376) with the panels enabled profiling of actionable variants as well as rare variants of unknown functional consequences. Notably, variants that occurred at low frequency were enriched with likely protein-damaging variants and previously unreported variants. Furthermore, in vitro evaluation of four pharmacogenes, including cytochrome P450 2C19 (CYP2C19), confirmed that many of these rare variants have considerable functional impact. The present study suggests that targeted NGS panels are readily applicable platforms to facilitate comprehensive profiling of pharmacogenes, including common but also rare variants that warrant screening for personalized medicine.restrictio

Evaluation of polygenic cause in Korean patients with familial hypercholesterolemia - A study supported by Korean Society of Lipidology and Atherosclerosis

BACKGROUND/OBJECTIVE: Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in LDLR, APOB, or PCSK9. Polygenicity is a plausible cause in mutation-negative FH patients based on LDL cholesterol (LDL-C)-associated single nucleotide polymorphisms (SNPs) identified by the Global Lipids Genetics Consortium (GLGC). However, there are limited data regarding the polygenic cause of FH in Asians. METHODS: We gathered data from 66 mutation-negative and 31 mutation-positive Korean FH patients, as well as from 2274 controls who participated in the Korean Health Examinee (HEXA) shared control study. We genotyped the patients for six GLGC SNPs and four East Asian LDL-C-associated SNPs and compared SNP scores among patient groups and controls. RESULTS: Weighted mean 6- and 4-SNP scores (0.67 [SD = 0.07] and 0.46 [0.11], respectively) were both significantly associated with LDL-C levels in controls (p = 2.1 × 10(-4), R(2) = 0.01 and p = 5.0 × 10(-12), R(2) = 0.02, respectively). Mutation-negative FH patients had higher 6-SNP (0.72 [0.07]) and 4-SNP (0.49 [0.08]) scores than controls (p = 1.8 × 10(-8) and p = 3.6 × 10(-3), respectively). We also observed higher scores in mutation-positive FH patients compared with controls, but the difference did not reach statistical significance. CONCLUSION: The present study demonstrates the utility of SNP score analysis for identifying polygenic FH in Korean patients by showing that small-effect common SNPs may cumulatively elevate LDL-C levels.ope

Clinical features of familial hypercholesterolemia in Korea: Predictors of pathogenic mutations and coronary artery disease - A study supported by the Korean Society of Lipidology and Atherosclerosis

BACKGROUND: Proper screening and diagnosis of familial hypercholesterolemia (FH) is of critical importance for cardiovascular prevention. However, the clinical diagnosis of FH remains difficult partly because its phenotype can vary between different ethnicities. The aim of this study was to determine the clinical features and the best diagnostic approach in Korean FH patients. The predictors of putative pathogenic mutations and coronary artery disease (CAD) were also identified. METHODS AND RESULTS: Ninety-seven patients with low-density lipoprotein-cholesterol >190 mg/dL and xanthoma or FH-compatible family history were included. Putative pathogenic mutations in LDLR, APOB, or PCSK9 genes were identified in 32% of the enrolled patients. The subjects were classified according to four sets of clinical criteria (Simon Broome, Dutch, MEDPED, Japanese). The mutation rates in definite type FH of Simon Broome or Dutch criteria were 35%-37% and lower in our patients than in those of other countries. The mutation detection rate by MEDPED criteria was 67%-75% and higher than those based on other criteria. The best low-density lipoprotein-cholesterol (LDL-C) threshold for predicting mutations was 225 mg/dL. LDL-C was found to be the only independent predictor of mutation carriers, while hypertension and low high-density lipoprotein-cholesterol were predictive of CAD. CONCLUSIONS: The conventional clinical criteria showed limited mutation detection power and low specificities in Korean FH patients, in whom the best LDL-C threshold for putative mutation was 225 mg/dL. Traditional cardiovascular risk factors were also significantly associated with CAD risk in this population.ope