STRUCTURAL GENETIC VARIATION AND DYSLIPIDEMIA AMONG MEN IN THE MULTICENTER AIDS COHORT STUDY by Rebecca Bosko Marino B.S., Slippery Rock University, 2003 Submitted to the Graduate Faculty of Graduate School of Public Health in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2014 1 UNIVERSITY OF PITTSBURGH Graduate School of Public Health This dissertation was presented by Rebecca Bosko Marino It was defended on August 15, 2014 and approved by Dissertation Advisor: Jeremy J Martinson DPhil Assistant Professor, Infectious Diseases and Microbiology Graduate School of Public Health University of Pittsburgh Committee Members: Lawrence Kingsley DrPH Professor Infectious Diseases and Microbiology Graduate School of Public Health University of Pittsburgh Charles Rinaldo PhD Chair and Professor Infectious Diseases and Microbiology Graduate School of Public Health University of Pittsburgh Robert Ferrell PhD Professor Human Genetics Graduate School of Public Health University of Pittsburgh ii Copyright © by Rebecca Bosko Marino 2014 iii Jeremy J Martinson DPhil STRUCTURAL GENETIC VARIATION AND DYSLIPIDEMIA AMONG MEN IN THE MULTICENTER AIDS COHORT STUDY Rebecca Bosko Marino, PhD University of Pittsburgh, 2014 ABSTRACT While highly active antiretroviral therapy has resulted in slowing the rate of progression to AIDS among individuals infected with human immunodeficiency virus, it has also resulted in detrimental metabolic lipid changes. As this dyslipidemia is not observed for all individuals receiving antiviral therapy, genetic factors likely influence the increased susceptibility for some. We designed this study to investigate the role of human gene copy number variation (CNV) in therapy associated dyslipidemia and risk assessment as well as investigating mRNA expression levels to identify new genetic variants associated with this lipid dysfunction. A custom multiplex ligation dependent probe amplification assay was developed to analyze CNV of reverse cholesterol transport pathway (RCT) genes within individuals (n=320) enrolled in the Multicenter AIDS Cohort Study (MACS). The resulting analysis demonstrated that CNV was present in extremely low levels within these genes as the only loss identified and verified was observed for CETP in one individual. To further identify lipid metabolism associated genes, blood-derived RNA from 437 MACS participants was analyzed using the Illumina Human HT- 12 microarray. Significant transcripts were present only for variation in HDL-C and Triglyceride levels with 4 differentially expressed transcripts (HDC, CPA3, GATA2 & SLC45A3) repeatedly identified. Finally, to determine if CNV can alter the functionality of single nucleotide iv polymorphism (SNP) genotyping, we analyzed SNPs in regions with/without CNV by Fluorescence Polarization, TaqMan SNP genotyping assays and Sanger Sequencing. SNPs in regions of no CNV were observed to have 3 distinct genotype groups but in the presence of CNV, this distinction was lost resulting in a continuous spread of allele values. These results show that CNV is not a major factor in the development of antiviral therapy- associated dyslipidemia. Other genetic variants, such as HDC, may explain some of the variability. Furthermore, when CNV is present it hinders the ability to SNP genotype when using the standard assumption of three genotype groups. As antiretroviral therapy is becoming more available for the over 35 million living with HIV-1, identification of factors leading to antiviral-associated dyslipidemia is important for Public Health. Here, we have identified genes that could serve as markers for lipid level changes helping physicians custom tailor therapy and care for these individuals. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIV 1.0 INTRODUCTION ........................................................................................................ 1 1.1 HUMAN IMMUNODEFICIENCY VIRUS ...................................................... 1 1.1.1.1 Types ...................................................................................................... 1 1.1.1.2 Global Burden ....................................................................................... 2 1.1.1.3 HIV-1 Life Cycle ................................................................................... 4 1.2 HIGHLY ACTIVE ANTI-RETROVIRAL THERAPY .................................. 5 1.3 HAART-ASSOCIATED DYSLIPIDEMIA ....................................................... 6 1.3.1 Infection Alone ................................................................................................. 6 1.3.2 HAART-Associated ......................................................................................... 7 1.4 GENETIC VARIATIONS ASSOCIATED WITH DYSLIPIDEMIA ............ 9 1.5 WHOLE-GENOME EXPRESSION ................................................................ 13 2.0 STUDY PREMISE ..................................................................................................... 15 2.1 HYPOTHESIS ................................................................................................... 15 2.1.1 Specific Aim 1: Copy Number Variation in Reverse Cholesterol Transport Genes ......................................................................................................... 15 2.1.2 Specific Aim 2: Transcriptome Analysis .................................................... 15 2.1.3 Specific Aim 3: Single Nucleotide Polymorphisms and CNV ................... 15 vi 3.0 MATERIALS AND METHODS .............................................................................. 16 3.1 SAMPLES .......................................................................................................... 16 3.1.1 MACS Sample Description ........................................................................... 16 3.1.1.1 Cross-Section from 2005 ..................................................................... 16 3.1.1.2 ARRA Cross-Section........................................................................... 17 3.1.1.3 Laboratory Control Samples ............................................................. 17 3.1.2 Nucleic Acid Extraction ................................................................................ 18 3.1.2.1 DNA ...................................................................................................... 18 3.1.2.2 RNA ...................................................................................................... 19 3.1.3 Nucleic Acid Quantification .......................................................................... 20 3.2 MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION ..... 21 3.2.1 Sample Selection ............................................................................................ 21 3.2.2 MLPA Probes ................................................................................................. 22 3.2.2.1 Reverse Cholesterol Transport Pathway .......................................... 22 3.2.2.2 Positive Control Copy Number Assay ............................................... 23 3.2.3 MLPA Procedure ........................................................................................... 24 3.2.3.1 MLPA Hybridization and Amplification .......................................... 24 3.2.3.2 MLPA Fragment Separation Conditions .......................................... 25 3.2.4 MLPA Analysis .............................................................................................. 26 3.2.5 Copy Number Calling ................................................................................... 26 3.3 POLYMERASE CHAIN REACTION (PCR) ................................................ 28 3.3.1 Primer Design ................................................................................................ 28 3.3.2 Primary Amplification .................................................................................. 28 vii 3.3.2.1 Optimization ........................................................................................ 28 3.3.2.2 Gel Electrophoresis ............................................................................. 30 3.3.3 Post-PCR Clean up ........................................................................................ 31 3.3.4 Sanger Sequencing ......................................................................................... 32 3.3.5 Ethanol Precipitation .................................................................................... 33 3.4 REAL-TIME QUANTITATIVE PCR ............................................................. 34 3.4.1 Single Nucleotide Polymorphism Assays ..................................................... 34 3.4.2 TaqMan Expression Analysis ....................................................................... 37 3.4.2.1 Synthesis of cDNA ............................................................................... 37 3.4.2.2 TaqMan Gene Expression Assays ..................................................... 38 3.5 TRANSCRIPTOME ANALYSIS..................................................................... 40 3.6 NANOSTRING CNV ANALYSIS ................................................................... 41 4.0 AIM 1: COPY NUMBER VARIATION AND RCT GENES ............................... 43 4.1 SAMPLE DEMOGRAPHICS .......................................................................... 47 4.2