Applying Forward Genetic Approaches to Rare Mendelian
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APPLYING FORWARD GENETIC APPROACHES TO RARE MENDELIAN DISORDERS AND COMPLEX TRAITS By ANLU CHEN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. David Buchner Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ANLU CHEN Candidate for the degree of Doctor of Philosophy*. Committee Chair Hung-Ying Kao Committee Member David Buchner Anna Mitchell Anthony Wynshaw-Boris Eckhard Jankowsky Date of Defense July 5th, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein TABLE OF CONTENTS TABLE OF CONTENTS……………………………………………...…………………i LIST OF TABLES…………………………………………………….………………...iv LIST OF FIGURES…………………………………………………………….…..…...vi LIST OF ABBREVIATIONS…………………………………………………………viii ACKNOWLEDGEMENT……………………………………………………………...ix ABSTRACT…………………………………………………………………………......xi Chapter 1. Background and Significance……………………………………….……...1 Background………………………………………………………………………………2 1. Forward genetics and reverse genetics…………………………………………………2 1.1. History of genetic studies…………………………………………………….2 1.2. New era of forward genetics using next-generation sequencing……………..5 2. Complex traits and human diseases…………………………………………………….6 2.1. Genome-wide association study (GWAS)…………………………………...6 2.2. Missing heritability…………………………………………………………..7 3. Rare Mendelian disorders……………………………………………………………..10 3.1. Rare disorders……………………………………………………………….10 3.2. Consanguineous families……………………………………………………10 Significance……………………………………………………………………………..12 Chapter 2. Mutations in the Mitochondrial Ribosomal Protein MRPS22 Lead to Primary Ovarian Insufficiency………………………………………………………...14 (Adapted from Chen A. et al. Human Molecular Genetics 2018) i Abstract…………………………………………………………………………………15 Introduction…………………………………………………………………………….16 Results…………………………………………………………………………………..18 1. Identification of mutations in MRPS22 in patients with POI…………………………18 2. Cellular studies of POI patient-derived fibroblasts…………………………………...33 3. Embryonic lethality of Mrps22 deficient mice………………………………………..38 4. mRpS22 in Drosophila germ cells is required for fertility……………………………40 Discussion…………………………………………………………………………….....44 Materials and Methods……………………………………………………………….. 50 Chapter 3. Mutations in PIK3C2A Cause Syndromic Short Stature Associated with Cataracts and Skeletal Abnormalities……………………….………………………..58 (Manuscript in preparation) Abstract…………………………………………………………………………………59 Introduction…………………………………………………………………………….60 Results…………………………………………………………………………………...61 1. Identification of mutations in PIK3C2A in patients with syndromic short stature……61 2. Identification of cellular defects in patient-derived fibroblasts……………………….76 3. Pik3c2a deficiency causes cataracts in zebrafish model………………………………79 Discussion……………………………………………………………………………….83 Materials and Methods………………………………………………………………...87 ii Chapter 4. Widespread Epistasis Regulates Glucose Homeostasis and Gene Expression………………………………………………………………………………97 (Adapted from Chen A. et al. PLoS Genet. 2017) Abstract…………………………………………………………………………………98 Introduction…………………………………………………………………………….99 Results………………………………………………………………………………….101 1. Contribution of epistasis to metabolic traits………………………………………....101 2. Regulation of gene expression by epistasis………………………………….………114 3. Context-dependent effects on gene expression………………………………………128 4. Significant contribution of epistasis to trait heritability……………………………..133 Discussion……………………………………………………………………………...136 Materials and Methods……………………………………………………………….142 Chapter 5. Summary and Future Direction…………………………………………157 Summary………………………………………………………………………………158 Future Directions……………………………………………………………………...159 1. Researchers are not alone in battles against genetic diseases………………………..159 2. Gene therapy to cure the diseases……………………………………………………161 3. Strategies to predict disease risk loci………………………………………………...162 4. Strategies to better under current data……………………………………………….163 5. What’s beyond genetic studies in understanding human disorders?...........................164 Reference……………………………………………………………………………....166 iii LIST OF TABLES Table 2.1. Hormone levels in four individuals with POI………………………………..22 Table 2.2. Adrenocorticotropic hormone stimulation test for patients with POI………..22 Table 2.3. List of gaps in WES coverage in Family I with POI and primers used for Sanger sequencing of these regions………………………………………………….......27 Table 2.4. Plasma adrenal steroid levels in two individuals with POI…………………..32 Table 2.5. Survival of offspring from a Mrps22 heterozygous knockout mouse (+/-) intercross…………………………………………………………………………………39 Table 2.6. Phenotypes of RNAi-mediated mRpS22 tissue-specific knockdown in Drosophila……………………………………………………………………………….41 Table 3.1. Phenotypic characteristics of patients in three families with Syndromic Short Stature……………………………………………………………………………………65 Table 3.2. Candidate variants identified by WES in patients with Syndromic Short Stature……………………………………………………………………………………69 Table 3.3. Survival of offspring from pik3c2a heterozygous knockout zebrafish (+/-) crosses……………………………………………………………………………………80 Table 3.4. List of primers used in the study of Syndromic Short Stature with PIK3C2A mutations………………………………………………………………………………...95 Table 3.5. List of antibodies used in the study of Syndromic Short Stature…………….96 Table 4.1. Number of mice used for analysis of body weight and plasma glucose……102 Table 4.2. Main and average effects on phenotypes…………………………………...109 Table 4.3. Main effects on gene expression……………………………………………118 Table 4.4. Summary of genes with mutliple meQTLs…………………………………118 iv Table 4.5. Genes exaimined by RNA-Seq and RT-qPCR for epistasis and additive interactions……………………………………………………………………………...122 Table 4.6. Interection effects on gene expression……………………………………...124 Table 4.7. Summary of genes with mutliple ieQTLs…………………………………..126 Table 4.8. Identification of fasting glucose QTLs using a combined linear model……153 Table 4.9. Identification of body weight QTLs using a combined linear model………154 Table 4.10. Primer sequences for RT-qPCR detection………………………………...155 v LIST OF FIGURES Fig. 2.1. Pedigrees of two consanguineous families with POI…………………………..19 Fig. 2.2. Absence of germ cells in the ovary of a female patient with the MRPS22 p.R202H mutation………………………………………………………………………..24 Fig. 2.3. Independent mutations in MRPS22 identified in two consanguineous families with POI………………………………………………………………………………….28 Fig. 2.4. Molecular analysis of fibroblasts from patients with the MRPS22 (p.R202H) mutation………………………………………………………………………………….34 Fig. 2.5. Oxidative phosphorylation is normal in fibroblasts from patients with the MRPS22 (p.R202H) mutation…………………………………………………………...36 Fig. 2.6. mRpS22 is required for female germ cell development in Drosophila………...43 Fig. 2.7. Structural analysis of disease-causing missense mutations in MRPS22……….48 Fig. 3.1. Pedigrees and phenotypic characteristics of patients with Syndromic Short Stature…………………....................................................................................................63 Fig. 3.2. Detailed phenotypic characteristics of individuals with PIK3C2A deficiency..........................................................................................................................66 Fig. 3.3. Loss-of-function mutations in PIK3C2A. ……………………………………..71 Fig. 3.4. Protein and mRNA levels of PIK3C2A in patient-derived cells……………….74 Fig. 3.5. Cilia defects in patient-derived PIK3C2A fibroblasts………………………….75 Fig. 3.6. PIK3C2A exon skipping in individual III-II-2 with Syndromic Short Stature……………………………………………………………………………………77 Fig. 3.7. Localization of ciliary markers in patient-derived PIK3C2A deficient fibroblasts………………………………………………………………………………...78 vi Fig. 3.8. Pik3c2a deficiency in zebrafish causes cataracts..……………………………..82 Fig. 4.1. Body weight and glucose levels in all CSS and control mice………………...103 Fig. 4.2. Schematic diagram of CSS and control crosses………………………………105 Fig. 4.3. Identification of 5 inter-chromosomal epistatic interactions that regulate fasting glucose levels in mice…………………………………………………………..111 Fig.4.4. Inter-chromosomal epistasis regulates fasting glucose levels…………………112 Fig. 4.5. Identification of meQTLs that regulate hepatic gene expression……………..116 Fig. 4.6. Positive correlation between cis-meQTLs and trans-meQTLs……………….117 Fig. 4.7. Identification of 5 trans-meQTLs that regulate the hepatic expression of Brca2……………………………………………………………………………………119 Fig. 4.8. Regulation of hepatic Zkscan3 expression by additive meQTLs……………..121 Fig. 4.9. Positive correlation between cis-ieQTLs and trans-ieQTLs………………….125 Fig. 4.10. Identification of 4 ieQTLs that regulate the hepatic expression of Agt……..127 Fig. 4.11. Schematic diagram illustrating the categorization of epistasis as either synergistic or antagonistic………………………………………………………………129 Fig. 4.12. Examples of synergistic and antagonistic ieQTLs…………………………..131 Fig. 4.13. Contribution of epistasis to the genetic regulation of hepatic gene expression……………………………………………………………………………....134 Fig. 4.14. No differences in mapping efficiency of RNA-Seq reads between B6 and CSSs…………………………………………………………………………………….148 vii LIST OF ABBREVIATIONS HD Huntington's disease ENU N-ethyl-N-nitrosourea RNAi RNA interference WGS whole genome sequencing WES whole exome sequencing GWAS Genome-wide association study POI Primary ovarian insufficiency LH luteinizing