A Study on Drug Repositioning for Rare Diseases based on Biological Pathways

학위논문 (석사) -- 서울대학교 대학원 : 치의학대학원 치의과학과, 2020. 8. 김홍기.서론: 본 연구는 생물학적 패스웨이를 활용하여 희귀질환의 신약재창출 방법론 연구를 목적으로 한다. 전 세계적으로 7,000여 개의 희귀질환이 존재하지만, 치료제는 약 5% 정도만 존재해 더 많은 연구가 필요하다. 희귀질환 치료제 연구에는 전통적인 신약개발 연구보다는 이미 승인된 약물의 새로운 의학적 용도를 찾는 신약재창출이 시간과 비용이 줄어 대안이 될 수 있다. 생물학적 패스웨이는 생체요소 간의 상호작용을 상세히 설명해둔 생물학적 심층 지식으로 유전자들의 정보를 유기적으로 바라볼 때 사용된다. 따라서 신약재창출을 위해 생물학적 패스웨이는 활용하기에 적합하다. 희귀질환의 신약재창출 약물 후보를 찾기 위해 약물관련 유전자들의 정보와 희귀질환 관련 유전자정보를 분석하여 공통 생물학적 패스웨이 목록을 활용한다. 공통 생물학적 패스웨이로 만들어진 희귀질환과 약물의 유사도를 계산하여 희귀질환-약물 후보목록을 만든다. 방법: 희귀질환 유전자정보 데이터베이스 Panel의 희귀질환 309개와 유의미한 관련성을 가진 유전자정보를 활용하였다. 약물 데이터베이스로 DRUGBANK를 사용하였으며, 1888개의 승인된 약물과 관련된 유전자정보를 사용하였다. 패스웨이 데이터베이스로 Reactome에서 제공하는 분석 도구를 사용하여 희귀질환과 약물에 관련된 유전자 목록의 생물학적 패스웨이를 FDR 값을 기준으로 각각 수집하였다. 수집한 생물학적 패스웨이들 중 희귀질환과 약물에 공통으로 관련된 생물학적 패스웨이는 1883개로, 희귀질환과 약물의 유사도를 확인을 위해 활용된다. 희귀질환과 약물의 유사도는 FDR값을 벡터화하여 유클리디안 유사도로 계산하였다. 결과: 본 연구 방법을 통해 희귀질환-약물 후보목록을 만들었다. 희귀질환–약물의 유사도 결과로 나온 값이 작은 값일수록 서로 가까운 거리에 존재한다고 설명할 수 있다. 따라서 유사도 값은 희귀질환의 신약재창출 후보가 될 가능성을 나타낸다. 이를 확인하기 위해 FDR 승인되어 희귀질환 치료제로 쓰이는 약 정보와 값을 비교하였다. Lomitapide 약물은 Homozygous familial hypercholesterolemia 질병 치료제로, 유사도 값이 2.8로 309개의 희귀질환 중 34번째로 약물-희귀질환 목록에 Familial hypercholesterolaemia targeted panel의 이름으로 존재했다. 희귀질환-패스웨이-약물로 분석한 결과가 희귀질환-유전자-약물의 관계보다 유의미한 결과라는 것을 Thalidomide 약을 통해 비교해보았다. 희귀질환-패스웨이-약물에서 Thalidomide 치료제가 어떤 희귀질환에 관련성이높은지를 순서대로 볼 수 있었고, Bladder cancer pertinent cancer susceptibility라는 희귀질환이 가장 가까운 것으로 확인되었고, 관련 연구가 진행되었었음을 확인하였다. 결론: 희귀질환-약물 목록이 희귀질환 치료제와의 비교를 통해 연관성이 있다는 것을 확인하였고, 우선순위목록을 통해 얼마나 연관성이 있는지 알 수 있었다. 또한, 희귀질환-유전자-약물의 관계보다 희귀질환-패스웨이-약물이 더 많은 신약재창출 가능성을 가진 정보라는 것을 알 수 있었다. 따라서 희귀질환 뿐만 아니라, 다른 질병의 관련 유전자 정보를 활용하여 생물학적 패스웨이를 추출하고, 이를 신약재창출 후보를 정렬하여 알 수 있도록 기대해 볼 수 있다.Introduction: The purpose of this study is to utilize biological pathway data for rare disease drug repositioning. There are more than 7,000 rare diseases worldwide, but there is only treatment for 5% of these diseases. While there is a great need for treatments, traditional drug development is a very time consuming and costly process. For rare disease treatment, drug repositioning can potentially be a quicker and cheaper alternative. Biological pathway data describe the interaction between biological elements in detail and can be used to analyze gene data from a wider perspective. Therefore, it is hypothesized that they are suitable to use in drug repositioning. In this study, a common biological pathway list is generated from drug-related and rare disease-related gene data to find new drug candidates for rare diseases. Using the common pathway list and rare disease-drug similarity, a rare disease-drug candidate list is generated. Methods: 309 rare diseases from the Genomics England PanelApp is utilized with the relevant genes. 1,888 approved drugs and related genetic information is used from DrugBank. Using analysis tools provided by Reactome, biological pathways relevant to the rare disease-gene and drug-gene lists were collected based on FDR values. Among the collected biological pathways, there are 1,883 biological pathways commonly associated with the rare diseases and drugs, which are then used to calculate the similarity between the rare diseases and drugs. The Euclidean similarity of the rare diseases and drugs are calculated by vectorizing the FDR values. Results: Through this study, a rare disease-drug candidate list was generated. In the list, it can be interpreted that the smaller the value between a rare disease and drug is the more similar they are. Therefore, the more similar a rare disease and drug is, the more likely it is to be a candidate for rare disease drug repositioning. The results were compared with existing approved drugs used to treat rare diseases, for evaluation. Lomitapide is a drug used to treat Homozygous familial hypercholesterolemia. In the drug-rare disease list it has a similarity value of 2.8 with its PanelApp equivalent disease, which is rank 34 out of 309 rare diseases. The rare disease-pathway-drug results were also compared with the rare disease-gene-drug results with the drug, Thalidomide. In the rare disease-pathway-gene results, it is observed that Bladder cancer pertinent cancer susceptibility was the closest disease to Thalidomide, which coincides with recent literature. Discussion: From the results, it can be confirmed that the rare disease-drug list was relevant with existing rare disease treatments and that this relevance can also be measured. In addition, it is found that rare disease-pathway-drug associations are more applicable to drug repositioning than rare disease-gene-drug associations. Finally, it is believed that biological pathways can be used not just for rare diseases but also for finding drug repositioning candidates in common diseases.Ⅰ. 서 론 1 1. 연구의 필요성 1 2. 신약재창출 4 3. 연구 목적 9 Ⅱ. 연구재료 및 방법 10 1. 연구 재료 10 1) 희귀질환 데이터베이스 10 2) 약물 데이터베이스 11 3) 생물학적 패스웨이 데이터베이스 12 2. 방법 15 1) 전처리과정 15 2) 희귀질환약물의 공통 생물학적 패스웨이 21 3) 거리계산법 23 Ⅲ. 결론 27 1. 희귀질환-약물 유사도 27 2. 신약재창출 약물 후보 28 Ⅳ. 고찰 39 참고문헌 41 Abstract 45Maste

PLCE1 Regulates the Migration, Proliferation, and Differentiation of Podocytes

PLCE1 encodes phospholipase C epsilon, and its mutations cause recessive nephrotic syndrome. However, the mechanisms by which PLCE1 mutations result in defects associated with glomerular function are not clear. To address this, we investigated the function of PLCE1 in podocytes called glomerular epithelial cells, where the pathogenesis of nephrotic syndrome converges. PLCE1 colocalized with Rho GTPases in glomeruli. Further, it interacted with Rho GTPases through the pleckstrin homology domain and Ras GTP-binding domains 1/2. Knockdown or knockout of PLCE1 in podocytes resulted in decreased levels of GTP-bound Rac1 and Cdc42, but not those of RhoA, and caused a reduction in cell migration. PLCE1 interacted with NCK2 but not with NCK1. Similar to the PLCE1 knockout, NCK2 knockout resulted in decreased podocyte migration. Knockout of PLCE1 reduced the EGF-induced activation of ERK and cell proliferation in podocytes, whereas knockout of NCK2 did not affect proliferation. Further, the knockout of PLCE1 also resulted in decreased expression of podocyte markers, including NEPH1, NPHS1, WT1, and SYNPO, upon differentiation, but the knockout of NCK2 did not affect the expression of these markers. Therefore, our findings demonstrate that PLCE1 regulates Rho GTPase activity and cell migration through interacting with NCK2 and that PLCE1 also plays a role in the proliferation and differentiation of podocytes, regardless of the presence of NCK2.ope

Treatment and Management of Sexually Transmitted Diseases in Sexually Assaulted Women

The evaluation and management of the sexually assaulted women is a complex, multifaceted task. The purpose of the medical examination after a sexual assault is to assess the patient for physical injuries, and to collect evidence for forensic evaluation and possible legal proceedings. Laboratory samples should be obtained at the initial visit and should include testing for pregnancy, syphilis, and hepatitis B and human immunodeficiency virus infections. Treatment should address physical injuries, pregnancy prophylaxis, sexually transmitted diseases and psychosocial sequelae. Appropriate referral services should be initiated during the initial visit. Victims of sexual assault require appropriate care, follow-up, and information regarding their legal rights. The history should be confined to medically relevant facts and should be conducted in a safe and quiet environment. In many emergency departments, however, the emergency room doctors is responsible for the initial evaluation and management. Therefore, it is imperative that the emergency room doctors be familiar with the process and issues specific to the management of the victim of a sexual assault.ope

Disease modeling of ADAMTS9-related nephropathy using kidney organoids reveals its roles in tubular cells and podocytes

Introduction: Mutations in ADAMTS9 cause nephronophthisis-related ciliopathies (NPHP-RC), which are characterized by multiple developmental defects and kidney diseases. Patients with NPHP-RC usually have normal glomeruli and negligible or no proteinuria. Herein, we identified novel compound-heterozygous ADAMTS9 variants in two siblings with NPHP-RC who had glomerular manifestations, including proteinuria. Methods: To investigate whether ADAMTS9 dysfunction causes NPHP and glomerulopathy, we differentiated ADAMTS9 knockout human induced pluripotent stem cells (hiPSCs) into kidney organoids. Single-cell RNA sequencing was utilized to elucidate the gene expression profiles from the ADAMTS9 knockout kidney organoids. Results: ADAMTS9 knockout had no effect on nephron differentiation; however, it reduced the number of primary cilia, thereby recapitulating renal ciliopathy. Single-cell transcriptomics revealed that podocyte clusters express the highest levels of ADAMTS9, followed by the proximal tubules. Loss of ADAMTS9 increased the activity of multiple signaling pathways, including the Wnt/PCP signaling pathway, in podocyte clusters. Conclusions: Mutations in ADMATS9 cause a glomerulotubular nephropathy in kidney and our study provides insights into the functional roles of ADMATS9 in glomeruli and tubules.ope

LRRC6 regulates biogenesis of motile cilia by aiding FOXJ1 translocation into the nucleus

Background: LRRC6 is an assembly factor for dynein arms in the cytoplasm of motile ciliated cells, and when mutated, dynein arm components remained in the cytoplasm. Here, we demonstrate the role of LRRC6 in the active nuclear translocation of FOXJ1, a master regulator for cilia-associated gene transcription. Methods: We generated Lrrc6 knockout (KO) mice, and we investigated the role of LRRC6 on ciliopathy development by using proteomic, transcriptomic, and immunofluorescence analysis. Experiments on mouse basal cell organoids confirmed the biological relevance of our findings. Results: The absence of LRRC6 in multi-ciliated cells hinders the assembly of ODA and IDA components of cilia; in this study, we showed that the overall expression of proteins related to cilia decreased as well. Expression of cilia-related transcripts, specifically ODA and IDA components, dynein axonemal assembly factors, radial spokes, and central apparatus was lower in Lrrc6 KO mice than in wild-type mice. We demonstrated that FOXJ1 was present in the cytoplasm and translocated into the nucleus when LRRC6 was expressed and that this process was blocked by INI-43, an importin α inhibitor. Conclusions: Taken together, these results hinted at the LRRC6 transcriptional regulation of cilia-related genes via the nuclear translocation of FOXJ1. Video Abstractope

Clinical Heterogeneity Associated with MYO7A Variants Relies on Affected Domains

Autosomal dominant hearing loss (ADHL) manifests as an adult-onset disease or a progressive disease. MYO7A variants are associated with DFNA11, a subtype of ADHL. Here, we examined the role and genotype-phenotype correlation of MYO7A in ADHL. Enrolled families suspected of having post-lingual sensorineural hearing loss were selected for exome sequencing. Mutational alleles in MYO7A were identified according to ACMG guidelines. Segregation analysis was performed to examine whether pathogenic variants segregated with affected status of families. All identified pathogenic variants were evaluated for a phenotype-genotype correlation. MYO7A variants were detected in 4.7% of post-lingual families, and 12 of 14 families were multiplex. Five potentially pathogenic missense variants were identified. Fourteen variants causing autosomal dominant deafness were clustered in motor and MyTH4 domains of MYO7A protein. Missense variants in the motor domain caused late onset of hearing loss with ascending tendency. A severe audiological phenotype was apparent in individuals carrying tail domain variants. We report two new pathogenic variants responsible for DFNA11 in the Korean ADHL population. Dominant pathogenic variants of MYO7A occur frequently in motor and MyTH4 domains. Audiological differences among individuals correspond to specific domains which contain the variants. Therefore, appropriate rehabilitation is needed, particularly for patients with late-onset familial hearing loss.ope

Microbiome analysis reveals that Ralstonia is responsible for decreased renal function in patients with ulcerative colitis

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Assessment of Bone Turnover Markers and Serum Cartilage Marker in Postmenopausal Women with Bisphosphonate

Objectives: This study was conducted to assess whether treatment with bisphosphonate affects the levels of bone turnover markers (urine deoxypyridinoline, serum osteocalcin) and cartilage turnover marker (serum cartilage oligomeric matrix protein, COMP). Methods: 30 postmenopausal women within 1 year from the diagnosis of menopause were enrolled in our study. These patients were divided into bisphosphonate, or control groups according to the treatment they received. Blood samples for turnover markers were taken before the initiation of medication, after 3 and 6 months. Known bone markers such as serum osteocalcin, urine deoxypyridinoline and cartilage marker such as serum cartilage oligomeric matrix protein (COMP) were evaluated. Results: 20 without severe hot flushes, but with osteoporosis or osteopenia received alendronate sodium 70 mg weekly regimen (BPN group) and 10 patients received no medication (control group). The mean osteocalcin levels and urine deoxypyridinoline levels were suppressed according to the treatment period in BPN group but not in control group. In BPN group, mean COMP levels remained similar at 0, 3 and 6 months with no statistical significance. However, mean COMP levels of BPN group were significantly decreased in comparison to the control after 6 months (19.08 ± 7.44 μg/mL vs. 22.50 ± 6.93 μg/mL, P=0.022). There existed significant correlations between osteocalcin, UDP and COMP (r=0.596 between osteocalcin and UDP, r=0.431 between UDP and COMP, r=0.341 between osteocalcin and COMP). Conclusion: Bisphophonate significantly suppressed the excretion of bone turnover marker in the patients, but tended to decrease cartilage marker. These results indicate that the treatment with bisphosphonate not only favorably affect bone metabolism but also decrease cartilage turnover, with possible implication for improving osteoporosis and osteoarthritis in postmenopausal womenope

Overlooked KCNQ4 variants augment the risk of hearing loss

Pathogenic variants of KCNQ4 cause symmetrical, late-onset, progressive, high-frequency-affected hearing loss, which eventually involves all frequencies with age. To understand the contribution of KCNQ4 variants to hearing loss, we analyzed whole-exome and genome sequencing data from patients with hearing loss and individuals whose hearing phenotypes were unknown. In KCNQ4, we identified seven missense variants and one deletion variant in 9 hearing loss patients and 14 missense variants in the Korean population with an unknown hearing loss phenotype. The p.R420W and p.R447W variants were found in both cohorts. To investigate the effects of these variants on KCNQ4 function, we performed whole-cell patch clamping and examined their expression levels. Except for p.G435Afs*61, all KCNQ4 variants exhibited normal expression patterns similar to those of wild-type KCNQ4. The p.R331Q, p.R331W, p.G435Afs*61, and p.S691G variants, which were identified in patients with hearing loss, showed a potassium (K+) current density lower than or similar to that of p.L47P, a previously reported pathogenic variant. The p.S185W and p.R216H variants shifted the activation voltage to hyperpolarized voltages. The channel activity of the p.S185W, p.R216H, p.V672M, and p.S691G KCNQ4 proteins was rescued by the KCNQ activators retigabine or zinc pyrithione, whereas p.G435Afs*61 KCNQ4 proteins were partially rescued by sodium butyrate, a chemical chaperone. Additionally, the structure of the variants predicted using AlphaFold2 showed impaired pore configurations, as did the patch-clamp data. Our findings suggest that KCNQ4 variants may be overlooked in hearing loss that starts in adulthood. Some of these variants are medically treatable; hence, genetic screening for KCNQ6 is important.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