Analysis of the relationship between hidden inherited germline factor and cancer risk

학위논문(박사)--서울대학교 대학원 :의과대학 협동과정 종양생물학전공,2019. 8. 윤성수.Introduction: Previous cancer-associated germline mutation studies have highlighted tumor suppressor genes such as TP53, APC, BRCA2, NF1, PMS2, and RB1. Many studies on tumor suppressor genes have been conducted because of the high penetration rate of pathogenic mutations. For this reason, cancer-related germline studies have been focused on tumor suppressor genes, and the role of germline genes other than tumor suppressor genes remains unclear. However, the possibility of presenting non-tumor suppressor genes that affect tumor development is not negligible. The mechanism of tumorigenesis by these non-tumor suppressor genes that exist as germline mutations contribute differently to tumor development. In this study, we will discuss the relationship between two different types of non-tumor suppressor genes and tumorigenesis. The first is the germline mutation of oncogenes in Acute Myeloid Leukemia(AML) patients, and the second is the correlation between primary immunodeficiency-associated gene mutations and tumor risk. Methods: To determine the oncogenic germline mutation of AML, WES data of 76 individuals from Seoul National University were analyzed. The correlation between immunodeficiency disease and cancer development was assessed using ICGC WGS data from 2566 individuals. For the normal control, WGS data of 2504 individuals were obtained from the 1000 Genome Project, and tumor susceptibility was examined by comparing the cancer and control groups. Results: We focused on the germline FLT3 as an oncogenic gene that increases the incidence of AML. An FLT3 germline mutation in four loci was identified in 76 patients with AML, and the cancer incidence was confirmed to increase in the locus corresponding to D358V. The overall survival of patients with D358V was significantly decreased, indicating that the mutation had a negative effect on the prognosis of patients. Regarding the association between PID and cancer, it has been confirmed that the PID-related genes increase cancer risk by 1.5 fold. Seven carcinomas were also associated with increased cancer risk, and the age of onset was significantly reduced in hepatocellular carcinoma and bladder cancer patients with PID. Conclusions: We revealed the association between two germline mutations and increased cancer risk, which was previously undefined owing to the rare frequency. The first germline mutation gene was the FLT3 mutation known as the oncogene. FLT3, which exists as a germline mutation, is presumed to contribute to the development of tumor cells by reducing FLT3 activity and turning on alternative cell signal transduction, unlike the FLT3 somatic mutation that induces cell division of tumor cells. The second germline mutation is a primary immunodeficiency disease-associated gene. Loss-of-function in immunodeficiency-associated genes does not directly induce cancer development but is assumed to promote tumorigenesis owing to a diminished immune surveillance effect. In addition to germline factors identified in this study, more novel germline factors are expected to be revealed. These efforts will contribute to the development of prophylactic treatments to prevent cancer patients from experiencing a relapse after treatment.서론: 이전의 암 관련 생식선 돌연변이 연구는 TP53, APC, BRCA2, NF1, PMS2 및 RB1과 같은 종양 억제 유전자에 집중되어 연구 되어 왔다. 종양 억제 유전자의 경우 병원성 돌연변이의 질병 침투도가 높기 때문에 연구가 용이하여 많은 연구자로 하여금 수행되었다. 이 때문에 암 관련 생식선 연구는 종양 억제 유전자에 초점을 맞추었고 이러한 이유로 종양 억제 유전자 이외의 유전자의 생식선 돌연변이의 역할은 여전히 불분명하다. 그러나, 종양 발달에 영향을 미치는 비 종양 억제 유전자의 존재 가능성은 무시할 수 없다. 생식선 돌연변이로 존재하는 이러한 비 종양 억제 유전자에 의한 종양 형성의 메커니즘은 종양억제 유전자의 종양 발달메커니즘과 다른 방식으로 종양생성에 기여할 것으로 생각된다. 이 연구에서는, 비 종양 억제 유전자의 생식선 돌연변이의 두 가지 다른 유형에 대해 논의 하고자 한다. 첫 번째는 AML 환자의 종양 유전자(Oncogene)의 생식선 돌연변이이고, 두 번째는 선천성 면역 결핍 유전자 돌연변이와 종양 위험 사이의 상관 관계이다. 연구방법: 서울대병원을 내원한 76명의 급성골수성백혈병 환자의 엑솜유전체 데이터가 종양 유발 유전자(Oncogene)의 생식세포 돌연변이 분석에 사용되었다. 2566명의 ICGC 전장 유전체 데이터가 선천성 면역 결핍 유전자와 암 발생의 상관관계를 확인하는데 사용되었으며, 2504명의 1000 게놈 프로젝트 전장 유전체 데이터가 비교 군으로 사용되어 비교 분석 되었다. 결과: 급성골수성백혈병의 발병을 증가시키는 종양 유발 유전자의 생식세포돌연변이 분석을 위해 FLT3 유전자를 확인 하였다. 76명의 급성골수성백혈병 환자에서 4개 좌위의 FLT3 생식세포 돌연변이가 발견되었으며 p.D358V 좌위에 해당하는 돌연변이가 급성골수성백혈병 발병을 증가시키는 것이 확인 되었다. 또한 이 돌연변이를 가지고 있는 환자의 경우 좋지 않은 예후를 보였다. 선천성 면역결핍질환의 경우 선천성 면역결핍 질환 유전자는 암 발병을 1.5배 증가 시켰다. 특히 7개 임종에서 암 위험이 증가하였으며 방광암과 간암의 경우 선천성 면역결핍 질환 유전 변이를 보유한 암환자의 암 발병이 유의하게 일찍 발병하는 것이 확인 되었다. 결론: 우리는 이 논문에서 전에 밝혀지지 않은 두가지 종류의 생식세포 돌연변이와 암 발병위험간의 상관관계를 밝혔다. 첫번째 생식선 돌연변이 유전자는 종양 유전자로 알려져 있는 FLT3돌연변이 이다. 생식선 돌연변이로 존재하는 FLT3의 경우 종양세포의 세포분열을 무한정 촉진 시키는 FLT3 체세포 돌연변이와는 다르게 FLT3기능 저하를 유도하고 대안 세포 신호전달계를 활성 시켜 종양세포의 발달에 기여하는 것으로 판단된다. 두번째 생식선 돌연변이는 선천성 면역 결핍 질환과 관련된 유전자와의 상관관계이다. 선천성 면역결핍과 관련된 유전자의 결핍은 종양 유전자 혹은 종양 억제 유전자들 처럼 직접적인 세포 분열을 촉진 하지 않지만, 면역 감시 효과의 저하로 인해 종양 발생을 촉진 하는 것으로 판단 된다. 이 논문에서 확인된 생식세포 돌연변이뿐만 아니라 밝혀져야 하는 더 많은 알려지지 않은 생식세포 돌연변이가 존재할 것으로 예상된다. 이와 같은 노력은 치료 후 재발하는 암을 극복하기 위한 예방적 치료법을 개발하는데 기여할 것으로 생각된다.Abstract 1 Contents 3 List of Tables and Figures 4 General Introduction 6 Chapter 1 9 Recurrent germline FLT3 pathogenic variant (D358V) is associated with a high prevalence of hematopoietic malignancy and poor prognosis in acute myeloid leukemia Introduction 9 Material and Methods 12 Results 15 Discussion 32 Chapter 2 35 Association between mild primary immunodeficiency disease and cancer risk Introduction 36 Material and Methods 40 Results 57 Discussion 103 References 108 국문요약 117Docto

Detection of FLT3 (FMS-like Tyrosine Kinase) Internal Tandem Duplication (ITD) Mutation using Next Generation Sequencing Technology and Nested PCR

학위논문 (석사)-- 서울대학교 대학원 : 협동과정 종양생물학전공, 2016. 2. 윤성수.Introduction: Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. Conventional PCR methods are not satisfactory in detecting relevant mutations in patients harboring the mutations. To increase detection sensitivity of FLT3-ITD, I developed new detection algorithm using next generation sequencing (NGS) data. I validated results using nested polymerase chain reaction (PCR) methods. I compared results of NGS data, nested PCR and conventional PCR methods. Methods: First, using whole exome sequencing data of 81 AML patients, I applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, I designed two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was designed to target previously reported regions in which ITD mutations were frequently found. PCR reactions of two steps were performed using the PCR primers sequentially. Results: In these 81 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, FLT3-ITD was positive in 11 patients (11/81, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all 11 patients. Nested PCR detected additional 4 patients positive for FLT3-ITD in this population. For 65 patients, FLT3-ITD was negative by both NGS and nested PCR method. One patient with FLT3-ITD by conventional PCR was not defined clearly by nested PCR and NGS method because of smear band and low depth coverage (coverage depth of exon 14 was 42). Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population compared with currently used conventional PCR method. The concordance rate of NGS method and nested PCR was 95% (77/81). Conclusions: FLT3-ITD is a very important genetic factor, guiding a therapeutic direction for AML patients. Here, I have developed more sensitive alternative detection methods for FLT3-ITD based on NGS. All of detection results were validated by nested PCR. NGS method is not only more sensitive than conventional PCR but also capable of determining FLT3-ITD size and amount in AML patients.Introduction 8 Material and methods 12 Results 19 Discussion 22 References 32 Abstract in Korean 35Maste