Genetic variants of PNPLA3, TM6SF2, SAMM50 and metabolomic profiles

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2021.8. 이경재.배경 일부 유전자 변이와 다른 대사체 양상이 비알콜성 지방간질환과 관련되어 있다고 보고되고 있지만 유전자 변이와 대사체 분석을 동시에 시행한 연구는 거의 없으며 소아청소년 집단에서는 시행된 연구가 없다. 목적 본 연구의 목적은 소아 비알콜성 지방간 질환에 미치는 유전적 변이의 영향을 파악하고, 더불어 환자와 대조군의 대사체 차이와 유전자변이와 연관성을 분석하여 질병의 병인을 보다 명확히 규명하고자 함이다. 방법 유전자 분석을 위하여 228명의 소아 환자와 225명의 대조군 (과체중 69, 정상체중 156명)이 등록되었고 그중 105명의 환자와 61명의 대조군 (과체중 118명, 정상체중 48명)을 대사체 분석을 위해 포함하였다. 다음과 같은 PNPLA3 (rs738409), TM6SF2 (rs58542926) and SAMM50 (rs2073080, rs3761472) 4개의 유전자 변이를 분석하였다. 혈장 대사체는 Biocrates AbsoluteIDQ p400 kit와 Thermo Q Exactive Plus orbitrap mass spectrometer를 통해 분석하였다. 모든 참가자는 신체계측을 시행하였고 일반 혈액검사와, 간기능검사를 받았으며, 과체중집단에서는 복부둘레와 혈압, 공복 혈당, 인슐린, 지방분석을 시행하였다. 간 초음파를 통해 지방간 유무와 지방간 단계를 평가하였으며 비침습적 섬유화 점수를 따라 섬유화 정도를 평가하였다. 비알콜성 지방간질환에 영향을 미치는 유전자 변이와 대사체 차이는 모두 일반소아 집단과 과체중집단에서 분석하였다. 결과 PNPPLA3 (rs738409), TM6SF2 (rs58542926), SAMM50 (rs2073080, rs3761472) 변이는 일반 소아집단 (승산비: 1.99~ 3.26, P <0.05)과 과체중소아집단 (승산비: 2.22~ 22.94, P <0.05에서 독립적으로 NAFLD위험을 높였다. 다른 독립적인 위험인자는 체질량지수-Z 점수와, 남성이었으며, 과체중집단에서는 공복 인슐린이 추가적인 위험 인자였다. 이 유전자변이들은 나이, 성별, 체질량지수-Z점수와 관련없이 알라닌 아미노 전이효소와, 초음파상의 지방간 정도, 섬유화 수치를 증가시켰다. 이러한 유전적영향은 전체 소아 집단보다 과체중집단에서 더욱 컸다. 과체중집단과, 정상체중 집단 모두에서 비알콜성 지방간질환 환자는 대조군에 비해 더 높은 혈장 류신, 아이소류신, 발린 같은 branched chain 아미노산과, 글루타메이트, 타이로신, 포스파티딜콜린, 스핑고마이엘린, 다이글리세라이드, 중성지방 농도를 보였으며 이것들중 branched chain 아미노산, 글루타메이트 등은 HOMA-IR과 양성상관관계를 보였다. 글라이신, 글루타민, 세린 농도는 비알콜성 지방간환자에서 낮았으며 글라이신, 글루타민은 HOMA-IR과 음의 상관관계를 보여 환자와 대조군간의 대사체 차이는 인슐린저항성과 연관이 있음을 시사하였다. TM6SF2 변이가 있는 환자에서는 변이가 없는 환자보다 더 낮은 혈장 포스파티딜콜린, 스핑고마이엘린, 중성지방 농도를 나타냈으며 이것은 비알콜성지방간 유무와 반대되는 분포였다. 결론 본 연구는 소아청소년 연령에서 최초로 다양한 유전자 변이와 혈장 대사체를 동시에 분석한 연구이다. PNPLA3, TM6SF2, SAMM50 은 소아청소년 일반집단과 과체중집단 모두에서 비알콜성 지방간질환의 발생과 중증도에 독립적으로 영향을 미쳤으며 그 영향은 과체중집단에서 컸다. 101개의 대사체가 비알콜성 지방간질환과 대조군 사이에 차이가 있었으며 그중 49개는 인슐린 저항성과 관련이 있었다. TM6SF2 변이를 가진 사람은 혈장 지방농도가 더 낮았으며, 이것은 일반적인 NAFLD환자와는 반대되는 양상이었으며 다른 유전자 변이에 따른 대사체 변화는 후속 연구가 필요하겠다.Background Some genetic variants and different metabolomic profiles have been reported to be associated with nonalcoholic fatty liver disease (NAFLD). Few studies have reported various genetic variants and associated metabolites simultaneously, and no study has been conducted in pediatric populations. Objective The aim of this study was to investigate the effects of genetic variants on pediatric NAFLD and analyze metabolic differences between NAFLD patients and controls in a pediatric population. In addition, other risk factors for pediatric NAFLD were aimed to investigate. Methods NAFLD was defined if hepatic steatosis was shown on ultrasound. A total of 228 NAFLD patients (body mass index-Z [BMI-Z] = 2.51 ± 1.01) and 225 controls (BMI-Z = 0.22 ± 1.48) were included. All participants underwent examination by anthropometry and blood cell count and liver function analysis. Four variants of PNPLA3 (rs738409), TM6SF2 (rs58542926) and SAMM50 (rs2073080, rs3761472) were genotyped by TaqMan allelic discrimination assay. Metabolic profiles were checked in children with overweight. The pediatric NAFLD fibrosis score (PNFS), the AST/platelet ratio index (APRI) and fibrosis-4 (FIB-4) were used to evaluate the degree of hepatic fibrosis. Genetic risk factors for NAFLD in all participants were analyzed by adjusting for age, sex and BMI-Z. Subgroup analysis was conducted in children with overweight with more metabolic adjustments. The genetic risk score was calculated to evaluate the synergetic effects of 4 genetic variants. Among them, 166 (105 NAFLD and 61 control) children were enrolled for metabolomic analysis. The plasma metabolome was quantified using a Biocrates AbsoluteIDQ p400 kit and Thermo Q Exactive Plus Orbitrap mass spectrometer. Results The four genetic variants (rs738409, rs58542926, rs2073080 and rs3761472), male sex and BMI-Z independently increased susceptibility to NAFLD. These variants remained significant risk factors with higher odds ratio in children with overweight in addition to fasting insulin and triglyceride. These variants increased the alanine aminotransferase level, PNFS, APRI, and FIB-4 independently. As the genetic risk score increased, aspartate transaminase, alanine aminotransferase, PNFS, APRI, and FIB-4 increased independently suggesting synergetic effects of these 4 variants. NAFLD patients showed a higher plasma levels of branched chain amino acids (BCAAs, leucine, isoleucine, valine), tyrosine, phosphatidylcholines (PCs), sphingomyelins (SMs), diglyceride, triglycerides (TGs) than control. Some of these metabolites including BAAA, glutamate, PCs, SMs had positive association with homeostasis model assessment-estimated insulin resistance (HOMA-IR). Plasma levels of glutamine, glycine and serin were lower in NAFLD patients than control. Glutamine and glycine showed negative correlation with HOMA-IR. The carries of TM6SF2 variants significantly showed lower plasma PCs, SMs and TG compared to wild type and the distribution of metabolites was reversed to the NAFLD. Conclusion The effects of genetic variants and metabolomic profiles in children with NAFLD was first demonstrated in this study. Genetic variants of PNPLA3, TM6SF2 and SAMM50 are associated with the development and severity of pediatric NAFLD and their effects are greater in children with overweight than normal weight. These variants have synergetic effects on severity of pediatric NAFLD. A total of 49 metabolites showed significant differences between subjects with NAFLD and control, are associated with insulin resistance. While variants of TM6SF2 results in lower plasma lipids, other variants did not show significant differences in metabolome.Chapter 1. Introduction 1 Chapter 2. Method 4 Chapter 3. Results 15 Chapter 4. Discussion 46 Chapter 5. Conclusion 61 Chapter 6. Abbreviations 63 Chapter 7. Bibliography 65 Chapter 8. Supplementary tables 79 Chapter 9. Abstract in Korean 84 Chapter 10. Aknowledgements 87박

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