Targeted breakage of a human chromosome mediated by cloned human telomeric DNA.

Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

Characterization of three de novo derivative chromosomes 16 by "reverse chromosome painting" and molecular analysis.

We have analyzed three de novo chromosome 16 rearrangements--two with a 16p+ chromosome and one a 16q+--none of which could be fully characterized by conventional cytogenetics. In each case, flow karyotypes have been produced, and the aberrant chromosome has been isolated by flow sorting. The origin of the additional material has been ascertained by amplifying and labeling the DNA of the abnormal chromosome by degenerate-oligonucleotide-primer-PCR and hybridizing it in situ to normal metaphase spreads (reverse chromosome painting). Both 16p+ chromosomes contain more than 30 Mb of DNA from the short arm of chromosome 9(9p21.2-pter), while the 16q+ contains approximately 9 Mb of DNA from 2q37. The breakpoints on chromosome 16 have been localized in each case; the two breakpoints on the short arm are at different points within the terminal band, 16p13.3. The breakpoint on the long arm of chromosome 16 is very close to (within 230 kb of) the 16q telomere. Determination of the regions of monosomy and trisomy allowed the observed phenotypes to be compared with other reported cases involving aneuploidy for these regions.

A large duplicated area in the polycystic kidney disease 1 (PKD1) region of chromosome 16 is prone to rearrangement.

An area of 500 kb at the proximal end of the polycystic kidney disease 1 (PKD1) region has been mapped in detail, with 260 kb cloned in cosmids. The area cloned from normal individuals contains two homologous but divergent regions each of 75 kb, including the previously described marker 26-6. Pulsed-field gel electrophoresis identified a duplication of 75 kb of this region, referred to as the OX duplication (OXdup), in three patients with PKD1. The OXdup probably arose by an unequal exchange promoted by misalignment of partially homologous areas. Study of the OXdup in a large PKD1 family showed that it segregated with PKD1 in just one-half of the family, indicating that a recent crossover had occurred between the OXdup and PKD1 and showing that it was not a PKD1 mutation. Further analysis identified an OXdup breakpoint fragment: the OXdup was subsequently identified in 2 normal individuals of 110 assayed. The finding of the OXdup and in other individuals an 11-kb deletion (OXdel) at a similar point within this duplicated area indicates that this is an unusually unstable genomic region.