Characterization of human chromosome 22 : Cloning of breakpoints of the constitutional translocation t(11;22)(q23;q11) and detection of small constitutional delections by microarray CGH

Chromosome 22 is the second smallest human chromosome, composing approximately 1.5% of the genome. The short arm of this acrocentric chromosome harbors ribosomal genes and the long arm contains the protein coding genes. This chromosome is gene-rich in comparison to the majority of other chromosomes, containing approximately 600 so far characterized genes. Many of these are involved in the etiology of a wide spectrum of diseases such as congenital and psychiatric disorders as well as cancers. The constitutional translocation t(11;22) is the most common reciprocal translocation in humans. This translocation is often found in families but can also occur de novo. Translocation carriers are normal and usually become diagnosed in connection with infertility problems or a birth of a genetically unbalanced child. In addition, an increased risk to breast cancer has been reported in some carriers, which suggests that the translocation might have an effect on a gene(s) involved in the etiology of breast cancer. We characterized the breakpoints of this translocation and found that the breakpoint region on chromosome 22 lies within an unclonable gap. The breakpoint on chromosome 11 is also located within an unstable region, as all BACs containing this segment are rearranged. We identified one BAC from chromosome 11 spanning the translocation breakpoint and two BAC clones from chromosome 22, which contain sequences similar/identical to the sequences mediating the translocations breakpoints on chromosome 22. A cosmid library from one translocation carrier was also constructed and chimeric cosmids from both derivative chromosomes were isolated. Their analysis revealed that no gene(s) seems to be disrupted by the translocation breakpoints. We also show that the breakpoints on both chromosomes occur at the tip of hairpins, which are formed due to the presence of long inverted repeats/palindromes. The formation of these structures is the likely reason behind "unclonability" of this region on chromosome 22 and the instability of BACs derived from chromosome 11. Furthermore, based on fiber-FISH experiments we conclude that the breakpoints of the translocations are highly conserved among carriers. The second aspect of the thesis is related to detection of micro-deletions and micro- gains, which cause a large number of genetic disorders. In order to improve the detection of such rearrangements, we applied and further developed the microarray-CGH methodology. We constructed three microarrays: one covering 7 Mb region in the vicinity of the NF2 gene in 22q12; the second is a full coverage chromosome 22 array; and the third is an array covering 6 Mb from the 22q11 region, including the typically deleted region in DiGeorgeVelo-Cardio-Facial syndrome. The latter region is particularly challenging, due to the presence of low copy repeats, high content of common repeats and unclonable sequences. Three types of targets were used in the arrays: i) genomic clones; ii) non-redundant, repeatfree pools of genomic DNA amplified by PCR; and iii) cDNA-based targets, single as well as in pools. We used the arrays to study neurofibromatosis type 2, acral melanoma, dermatofibrosarcoma, and DiGeorge/Velo-Cardio-Facial syndrome. We were able to detect homozygous/heterozygous deletions, amplifications, IGLV/IGLC locus instability and the breakpoints of an imbalanced translocation. Using the novel approach with repeat-free, PCRgenerated sequences, we detected heterozygous deletions using as little as 11.5 kb of genomic target sequence. We conclude that the array-CGH is a powerful method for the detection of gene-dosage imbalances. Our results also suggest that most, if not all, medically important segments of our genome will be accessible for analysis using high-resolution microarraybased CGH