An Investigation of Genetic Variation in Complex Disorders of the Pituitary Gland Emanuela Spadoni UCL MPhil 1 Declaration I, Emanuela Spadoni, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Congenital hypopituitarism can be triggered by environmental insults, such as viral infections, vascular or degenerative damage and exposure to alcohol and drugs. Genetic mutations have been identified in genes responsible for pituitary development and function, however, only up to 10% of patients affected by hypopituitarism have recognised mutations in known genes. Clinical phenotypes may arise from gene dosage imbalance, but routine cytogenetic and molecular techniques can be insufficiently sensitive to detect chromosome rearrangements that are either submicroscopic in size or limited to a specific genomic locus or both. In the present study, ten patients with a complex pathology of the pituitary gland of unknown aetiology were clinically pre-selected according to a criteria checklist, to undergo a genome-wide screening of copy number changes with high-resolution 250K SNP array. Genomic sequencing of three candidate genes, BARX2, OTX2 and BMP4, was carried out in larger cohorts of selected patients. Three pathogenic genomic imbalances, chromosome 6q terminal duplication, chromosome 11q terminal deletion, and an interstitial deletion of chromosome 22q were detected in two patients, and the breakpoints were defined at high resolution. A submicroscopic rearrangement in the chromosome region 1p36.33 was found in a patient presenting with the association of hypopituitarism and tetralogy of Fallot. Two CNVs were found at the location of the breakpoints of a cytogenetically visible translocation between chromosomes 11q and 22q in one patient. One region of genomic imbalance, identified in one patient, contained the whole length of the ENG gene. A novel amino acid change was identified in BARX2 in a patient with isolated hypopituitarism. A novel nonsense mutation was found in OTX2 in a patient with pathology of the pituitary gland associated wth severe eye defects. These findings contribute to the understanding of the genetic bases of congenital hypopituitarism. 3 Table of Contents 1. Introduction ................................................................................................................. 15 1.1. Pituitary Gland ......................................................................................................... 15 1.1.1. Anatomy and Physiology of the Pituitary Gland .............................................. 15 1.1.2. Pathology of the Pituitary Gland ....................................................................... 18 1.1.3. Embryology of the Pituitary Gland ................................................................... 20 1.1.4. Molecular Basis of Pituitary Development and Disorders ................................ 23 1.2. Copy Number Variations (CNVs) ............................................................................ 30 1.2.1. Definition and Origin of CNVs ......................................................................... 31 1.2.2. Effects of CNVs in Complex Disorders ............................................................ 32 1.3. Single Nucleotide Polymorphisms (SNPs) .............................................................. 34 1.3.1. Definition of SNPs ............................................................................................ 35 1.3.2. Role of SNPs in Disease Gene Discovery......................................................... 35 1.4. Array Techniques ..................................................................................................... 38 1.4.1. Bacterial Artificial Chromosome (BAC) Based Arrays .................................... 39 1.4.2. Oligonucleotide Based Arrays .......................................................................... 40 1.4.3. Single Nucleotide Polymorphism (SNP) Arrays .............................................. 41 1.4.4. Known and Emerging Syndromes .................................................................... 45 1.4.5. Disease Gene Mapping ..................................................................................... 46 1.5. Hypothesis and Major Outcomes ............................................................................. 48 2. Methods ....................................................................................................................... 49 2.1. Patients ..................................................................................................................... 49 2.1.1. Patients Selected for High Resolution Genome-wide Copy Number Change Screening ..................................................................................................................... 49 2.1.2. Patients Selected for Mutation Analysis. .......................................................... 50 2.2. Affymetrix GeneChip 250K Sty1 Mapping Array................................................... 50 2.2.1. Quality Control of DNA.................................................................................... 51 2.2.2. Sample Processing with Affymetrix GeneChip 250K Sty1 Mapping Array .... 51 2.2.3. Copy Number Analysis ..................................................................................... 52 2.2.4. Reference Subsets of the International HapMap Project Dataset ..................... 52 2.2.5. Batch Analysis Workflow Parameters .............................................................. 53 2.3. Genomic Sequencing ............................................................................................... 54 2.3.1. Primer Design.................................................................................................... 54 2.3.2. Suspension and Aliquots of Primers ................................................................. 55 2.3.3. DNA Aliquots ................................................................................................... 55 2.3.4. PCR Protocol ..................................................................................................... 55 2.3.5. Purification of PCR Products ............................................................................ 58 2.3.6. Sequencing Reaction ......................................................................................... 59 2.4. Restriction Digestion ................................................................................................ 63 2.4.1. Selection of Restriction Enzyme ....................................................................... 63 2.4.2. Restriction Digestion Protocol .......................................................................... 64 3. Results - Patient Selection for CNV Analysis............................................................. 66 3.1. Checklist of Inclusion Criteria ................................................................................. 66 3.2. Database of Endocrinological Patients..................................................................... 69 3.3. Association of Hypopituitarism with Tetralogy of Fallot ........................................ 71 3.4. Patient 6089 with Chromosome 11q Deletion Syndrome ........................................ 73 3.5. Patient 18905640 with Karyotype 46,XY,t(11q;22q) .............................................. 74 4 3.6. Clinical Details of the Patients Included in the CNV Analysis ............................... 75 4. Results - CNV Analysis .............................................................................................. 79 4.1. 250K SNP Array to Perform Genome-wide Copy Number Change Analysis ........ 80 4.2. Ethical Approval and Informed Consent.................................................................. 80 4.3. Analytical Workflow for the Screening of Multiple CNVs ..................................... 81 4.4. SNP Genotype Call Rates ........................................................................................ 84 4.5. Detection of Multiple CNVs .................................................................................... 85 4.5.1. Distribution of CNVs per Patient ...................................................................... 85 4.5.2. Distribution of CNVs per Chromosome ........................................................... 86 4.6. Distribution of Unique CNVs per Patient ................................................................ 87 4.7. Pathogenic Chromosome Imbalances ...................................................................... 90 4.7.1. Chromosome 6q Terminal Duplication and Chromosome 11q Terminal Deletion ....................................................................................................................... 91 4.7.2. Chromosome 22q11.21 Interstitial Deletion ..................................................... 93 4.8. Potentially Pathogenic CNVs ................................................................................... 95 4.8.1. Chromosome 22q12.3 Interstitial Deletion ....................................................... 96 4.8.2. Chromosome Region 11q24.2 Imbalance and KIRREL3 Gene ...................... 100 4.8.3. Chromosome 1p36.33 Deletion ...................................................................... 104 4.8.4. Chromosome 9q34.11 Interstitial Deletion ..................................................... 109 4.9. Risk