Author:

Keywords:

contiguous gene syndromes, molecular dissection

Abstract:

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