The Pennsylvania State University The Graduate School College of the Liberal Arts A CANDIDATE GENE STUDY AND A FULL GENOME SCREEN FOR MALE HOMOSEXUALITY A Thesis in Anthropology By Michael G. DuPree © 2002 Michael G. DuPree Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2002 We approve the thesis of Michael G. DuPree. Date of Signature ______________________________ _____________ Jeffrey A. Kurland Associate Professor of Anthropology and Human Development Chair of Committee Thesis Co-Adviser ______________________________ _____________ Kenneth M. Weiss Evan Pugh Professor of Anthropology and Genetics Thesis Co-Adviser ______________________________ _____________ Mark D. Shriver Assistant Professor of Anthropology and Genetics ______________________________ _____________ M. Beatrix Jones Assistant Professor of Statistics ______________________________ _____________ Dean R. Snow Professor of Anthropology Head of the Department of Anthropology ______________________________ _____________ Dean H. Hamer Chief, Gene Structure and Regulation Laboratory of Biochemistry National Cancer Institute National Institutes of Health Special Signatory ii ABSTRACT The causes of differences in sexual orientation are poorly understood. Although behavior genetic analyses have found that homosexuality is familial, candidate gene studies reveal no mechanisms that influence the development of the trait. Previous studies of a region of the X chromosome have shown a statistically significant excess of allele sharing at loci on Xq28 between pairs of homosexual brothers, but the locus explains only a portion of variance in the trait. Thus, there are potentially other loci throughout the genome that could influence the development and expression of sexual orientation. This thesis contains two reports on male homosexuality. The first considers whether differences in the gene encoding the aromatase enzyme (CYP19), a known factor in mammalian neural masculinization, influence sexual orientation in men. Results of the analyses suggest that variation in CYP19 does not wield such influence and likely undermines the candidacy of this pathway of development of male homosexuality. The second report and major portion of the thesis, consists of a full genome scan of a large collection of sibships containing brothers concordant for homosexuality. An allele-sharing linkage methodology is used to analyze the data. In the analyses, sexual orientation is considered both as a dichotomous phenotype as well as a quantitative trait and the Kinsey scales of sexual orientation are used to define the phenotype. The sample population consists of 144 sibships that comprise either two gay brothers (135 sibpairs) or three gay brothers (9 sibtrios). With available parents and heterosexual siblings, a total of 456 individuals are included in the study. The results suggest that several loci could influence this complex trait, although no conclusive evidence for a particular locus is obtained. Among the conclusions of the report is that a new candidate gene on 9q22, 17- β-Hydroxysteroid Dehydrogenase III (HSD17β3), may influence the development of homosexuality in some men. Other analyses justify continued interest in the Xq28 region but also indicate that separate developmental pathways might influence the trait as well. Dissertation Author: Michael G. DuPree Advisors: Jeffrey Kurland Ken Weiss iii TABLE OF CONTENTS List of Abbreviations ……………...……………………………………………………..vi List of Tables By Chapter ……………………………………………………………….vii List of Figures By Chapter ……………………………………………………………..viii Acknowledgements……………………………………………………………………….ix Introduction: The Research Focus of the Dissertation ………………………………..1 References………………………………………………………………………..10 A Candidate Gene Study of CYP19 (AROMATASE) and Male Homosexuality ….13 Abstract…………………………………………………………………………..14 Introduction………………………………………………………………………14 Materials and Methods…………………………………………………………...15 Subjects…………………………………………………………………..15 Genotyping……………………………………………………………….18 Results……………………………………………………………………………19 Discussion………………………………………………………………………..22 Acknowledgements………………………………………………………………23 References………………………………………………………………………..24 A Multiplex PCR Protocol for a Commercial Collection of Microsatellite Markers …………………………….27 Introduction………………………………………………………………………28 Materials and Methods…………………………………………………………...28 Experiment Series I………………………………………………………30 Experiment Series II……………………………………………………..31 Experiment Series III…………………………………………………….33 Discussion………………………………………………………………………..34 A Full Genome Screen for Male Homosexuality……………………………………...35 Abstract…………………………………………………………………………..36 Introduction………………………………………………………………………36 Methods…………………………………………………………………………..38 Sample Collection………………………………………………………..38 Subjects…………………………………………………………………..39 Assessment of Sexual Orientation……………………………………….40 Genotyping……………………………………………………………….44 Microsatellite Markers…………………………………………...44 iv Genetic Analysis…………………………………………………………47 Assignment of Genotypes and Considerations of Allele Frequencies…...47 Analysis of Phenotypes: Sexual Orientation as a Dichotomous Trait ….48 Analysis of Phenotypes: The Kinsey Scales of Sexual Orientation ……49 Results……………………………………………………………………………50 Qualitative Analysis of Sexual Orientation……………………………...50 Sample Subset Considerations…………………………………………...56 Quantitative Analysis of Kinsey Phenotypes…………………………….60 Sample Subset Considerations…………………………………………...62 Discussion………………………………………………………………………..70 Qualitative Analysis of the Full Sample…………………………………70 Qualitative Analysis of Sample Subsets…………………………………71 Quantitative Analysis of the Full Sample………………………………..72 Quantitative Analysis of Sample Subsets………………………………..73 References………………………………………………………………………..75 Appendix: Qualitative Analyses of All Chromosomes………………………….82 Conclusion: Considering Complex Traits…………………………………………….86 References……………………………………………………………………….92 v LIST OF ABREVIATIONS ABI Applied Biosystems Incorporated CEPH Centre d’Etudes Polimorphisme Humane CGE capillary gel electrophoresis cM centimorgan DNA deoxyribonucleic acid DSM-III Diagnostic and Statistical Manual of Mental Disorders, edition III HIV Human Immunodeficiency Virus HSD hydroxysteroid dehydrogenase IBD identity by descent LMS linkage mapping set LOD logarithm of the odds ML maximum likelihood mRNA messenger ribonucleic acid NADPH nicotinamide-adenine dinucleotide phosphate (reduced) NCI National Cancer Institute OMIM Online Mendelian Inheritance in Man PCR polymerase chain reaction QTL quantitative trait locus rfu refraction fluorescence unit(s) sibpair sibling pair sibtrio sibling trio SD standard deviation SNP single nucleotide polymorphism vi LIST OF TABLES BY CHAPTER A Candidate Gene Study of CYP19 (Aromatase) and Male Homosexuality TABLE TITLE PAGE 2.1 Correlation Matrix: Sexual Orientation and Kinsey Components………17 2.2 Relative Risk Ratio For Full Siblings……………………………………20 A Full Genome Screen For Male Homosexuality TABLE TITLE PAGE 4.1 Marker Heterozygosity: Values Most Divergent From Published Sources………………………………………………..45 4.2 Least Successful Reactions………………………………………………46 vii LIST OF FIGURES BY CHAPTER A Candidate Gene Study of CYP19 (Aromatase) and Male Homosexuality FIGURE TITLE PAGE 2.1 Distribution of Kinsey Averages………………………………………...18 2.2 Chromosome 15 Exclusion Mapping…………………………………….21 A Full Genome Screen For Male Homosexuality FIGURE TITLE PAGE 4.1 Distribution of Kinsey Averages………………………………………...40 4.2 First Sexual Contact……………………………………………………...42 4.3 Self-Acknowledgement of Sexual Orientation…………………………..43 4.4 Data Collection: Valid Genotypes By Number of Markers……………..46 4.5 Chromosome 2: Qualitative Analysis…………………………………...51 4.6 Chromosome 7: Qualitative Analysis…………………………………...52 4.7 Chromosome 7: Exclusion Analysis…………………………………….53 4.8 Chromosome 9: Qualitative Analysis…………………………………...54 4.9 X Chromosome: Allele Sharing Proportions……………………………55 4.10 Chromosome 2: Qualitative Analyses Compared……………………….56 4.11 Chromosome 9: Qualitative Analyses Compared………………………..58 4.12 X Chromosome: Qualitative Analyses Compared……………………….59 4.13 Chromosome 3: Kinsey Subscales Compared…………………………..60 4.14 Chromosome 9: Quantitative Analyses of Kinsey Scales……………….61 4.15 Chromosome 1: Quantitative Analyses Compared……………………...62 4.16 Chromosome 5: Quantitative Analyses Compared………………………63 4.17 Chromosome 3: Quantitative Analyses Compared………………………64 4.18 Chromosome 9: Kinsey “Average” Compared………………………….65 4.19 Chromosome 9: Kinsey “Self-Identification” Compared……………….66 4.20 Chromosome 9: Kinsey “Attraction” Compared………………………..67 4.21 Chromosome 9: Allele Sharing Proportions…………………………….68 4.22 X Chromosome: Kinsey “Average” Compared…………………………69 viii ACKNOWLEDGEMENTS I would like to express my appreciation for a number of people who have made this research possible in various ways. I must first thank Jeffrey Kurland for being the one that was there from the beginning, there with encouragement, there with friendship. Secondly, Kenneth Weiss has never failed to remind me of what science is really all about when I needed it. He is singularly dedicated and one of the most sincere people I have had the priveledge to know. I owe an immense debt of gratitude to Dean Hamer who has been supportive both personally as well as financially since the beginning of these research projects. He nudged me into a scientific career and will always be an intellectual model. At NIH Stella Hu, Suping Sabol, Kira Leuders and Lev Sirota of our laboratory