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Introduction: Microdeletions or contiguous gene syndromes are characterized by variable complex clinical phenotypes caused by hemizygosity of contiguous genes, defined mainly by a common deletion region, or of a major causal gene locus. Delineation of the pathogenic genes within these CGS regions is a major challenge. Identification of breakpoints at nucleotide resolution of balanced chromosomal rearrangements localized within these regions constitutes a key strategy for definition of the phenotypically important genes. The aim of this study is the identification of molecular alterations responsible for an extremely complex clinical phenotype resembling 1q41q42 microdeletion syndrome (coarse facial features, severe developmental delay, congenital heart disease and congenital microcephaly) presented by an individual with the t(1;3)(q42.11;p25.3)dn. FCT (HMSP-ICT/0016/2013) info:eu-repo/semantics/publishedVersion

Genotype-phenotype correlations were initially used to identify the minimal region of overlap between differently sized rearrangements in association with a specific phenotypic feature. This approach enabled the molecular dissection of the disease. However, the detection of the genotype by low resolution cytogenetic techniques hampered accurate genotype-phenotype correlations and the search for causative genes. With the advent of micro-array technology, the genotypes were more easily obtained, previously known imbalances were molecularly delineated and novel, previously unknown imbalances could be identified. This genome wide approach enabled the collection of patients with similar genotypes and subsequent identification of common clinical features in order to detect novel microdeletion/microduplication syndromes. This latter design is referred to as the genotype-first approach. In the first part of the thesis, this genotype-first approach was applied to a large group of patients with developmental disabilities and multiple congenital anomalies and enabled the identification and characterization of a microdeletion/microduplication syndrome at chromosome 16p13.1. Association studies have shown a significant overrepresentation of the 1.55 Mb microdeletion in the patient versus the control population and thus indicates a causative role in the etiology of developmental delay. Furthermore, the genome wide approach enabled the identification of uncommon submicroscopic rearrangements and further delineation of known imbalances on chromosome 4p16. Taken together, this research has enabled I) the identification and further delineation of minimal regions of overlap associated with certain manifestations typical for WHS, II) the characterization of a submicroscopic deletion outside of both currently accepted critical regions in a patient with mild representation of WHS and III) the identification of a submicroscopic duplication including the critical region in a patient with developmental delay and multiple congenital anomalies. In the second part of the thesis, terminal deletions on chromosome 4p16 were characterized at the sequence level to gain more insight into the origin of constitutional breakage and the mechanism of telomere rescue at broken chromosomes leading up to terminal constitutional deletions. We developed a combined strategy of high resolution micro-array analysis and telomere anchored PCR followed by Sanger sequencing. This strategy enabled us to characterize 9 terminal breakages at basepair level. Detailed analysis revealed a direct addition of telomere sequence adjacent to the breaks and a microhomology of 2 to 5 bp in phase with the telomere repeat at nearly all broken chromosomes. Additional in silico analysis has shown an enrichment of DNA polymerase arrest sites nearby the breakpoint. Those data suggest that an arrest in DNA replication leads to broken chromosomes followed by a template dependent healing via telomerase. In the third part of the thesis, we investigated the role of position-effects at chromosome 4p16 deletion syndrome since it is known from studies in yeast and human cancer cells that regions flanking the relocated telomere can be subjected to aberrant gene expression. To this end, 21 stably expressed genes from the tip to 4.5 Mb away from the telomere were studied in lymphoblastic cell lines of patients with interstitial and terminal deletions. Several genes flanking both interstitial and terminal deletions revealed variable expression patterns compared to normal individuals. In particular, telomere flanking gene inactivation was reversible and methylation dependent. Those data strongly suggests that telomere position-effects (TPE) play a role in pathology and should be taken into account when performing genotype-phenotype correlations. Abbreviations 9 GENERAL INTRODUCTION AND AIMS OF THE STUDY 11 1. GENOTYPE-PHENOTYPE CORRELATIONS 13 1.1. Microscopically visible chromosomal rearrangements 13 1.2. Submicroscopic rearrangements 14 2. BROKEN CHROMOSOMES LEADING TO TERMINAL DELETIONS 21 2.1. Telomeres 21 2.2. Rescue of telomeres 22 2.3. Telomere length control 23 2.4. Position-effect variegation or PEV 24 3. AIMS OF THE STUDY 25 GENOTYPE-PHENOTYPE CORRELATIONS 27 Paper 1: Recurrent reciprocal deletions and duplications of 16p13.11 29 Paper 2: Genotype-phenotype correlation in 21 patients with Wolf-Hirschhorn syndrome 49 Paper 3: Duplication of the Wolf-Hirschhorn syndrome critical region causes neurodevelopmental delay 71 Paper 4: A familial microdeletion 600 kb proximally of the WHSCR reveals a mild WHS phenotype 83 Paper 5: Benign and pathogenic copy number variation on the short arm of chromosome 4 101 MECHANISM GENERATING 4P TERMINAL DELETIONS 113 Paper 6: Telomere healing following DNA polymerase arrest induced breakages is likely the main mechanism generating chromosome 4p terminal deletions 115 INVESTIGATING THE ROLE OF POSITION-EFFECTS IN 4p16 MICRODELETION SYNDROME 137 Paper 7: Silencing of telomere flanking genes on chromosome 4p16 suggests telomere position-effects 139 GENERAL DISCUSSION & CONCLUSION 157 1. Genetic dissection of common features in microdeletion syndromes 159 1.1. The advantage of molecular karyotyping 159 1.2. The use of animal models in dissecting CGS 161 1.3. The use of induced pluripotent stem cells 162 1.4. Monitoring for additional mutations 162 2. Origin of phenotypic variability 162 3. Telomere position-effects 163 3.1. Monitoring gene-expression outside the deletion boundaries 163 4. Overall conclusion 166 SUMMARY/SAMENVATTING 167 REFERENCES 173 CURRICULUM VITAE 187 List of publications 189 Acknowledgements-dankwoord 191 nrpages: 192 status: published

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International audience; Alagille syndrome (AGS) is a well defined genetic disorder characterised by five major features. An autosomal dominant mode of transmission with reduced penetrance has been suggested by the analysis of a limited number of families. However there has been no statistical analysis. We report here the first segregation analysis of AGS, using 33 families collected through 43 probands. Segregation analysis of these families allowed us to conclude that AGS is transmitted as a dominant disorder with 94% penetrance and 15% of cases are sporadic. The expressivity of the phenotype was variable and 26 persons (15 parents and 11 sibs) were identified as presenting minor forms of the disease. These results are valuable for genetic counselling.