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, 103 (12), 4534-9

High-resolution Mapping of DNA Copy Alterations in Human Chromosome 22 Using High-Density Tiling Oligonucleotide Arrays

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High-resolution Mapping of DNA Copy Alterations in Human Chromosome 22 Using High-Density Tiling Oligonucleotide Arrays

Alexander Eckehart Urban et al. Proc Natl Acad Sci U S A.

Abstract

Deletions and amplifications of the human genomic sequence (copy number polymorphisms) are the cause of numerous diseases and a potential cause of phenotypic variation in the normal population. Comparative genomic hybridization (CGH) has been developed as a useful tool for detecting alterations in DNA copy number that involve blocks of DNA several kilobases or larger in size. We have developed high-resolution CGH (HR-CGH) to detect accurately and with relatively little bias the presence and extent of chromosomal aberrations in human DNA. Maskless array synthesis was used to construct arrays containing 385,000 oligonucleotides with isothermal probes of 45-85 bp in length; arrays tiling the beta-globin locus and chromosome 22q were prepared. Arrays with a 9-bp tiling path were used to map a 622-bp heterozygous deletion in the beta-globin locus. Arrays with an 85-bp tiling path were used to analyze DNA from patients with copy number changes in the pericentromeric region of chromosome 22q. Heterozygous deletions and duplications as well as partial triploidies and partial tetraploidies of portions of chromosome 22q were mapped with high resolution (typically up to 200 bp) in each patient, and the precise breakpoints of two deletions were confirmed by DNA sequencing. Additional peaks potentially corresponding to known and novel additional CNPs were also observed. Our results demonstrate that HR-CGH allows the detection of copy number changes in the human genome at an unprecedented level of resolution.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Detection and analysis of a small heterozygous deletion in the β-globin locus. (A) The region flanking the β-globin gene, represented on the high-density HR-CGH array, and the signal obtained from probing the array with DNA from patient 05-029 (processed signal average of two probings). Blue, exons of known genes; orange, segmental duplication. (B) The signal indicating position and extent (indicated by dashed lines) of a ≈600-bp heterozygous deletion. (C) PCR analysis of the deletion locus yields an additional fragment from the patient sample ≈600 bp smaller in size than the only fragment from the control sample; its size was determined by DNA sequencing to be 622 bp.
Fig. 2.
Fig. 2.
Detection and analysis of a large deletion in chromosome 22q11 of patient 04-018. (A) Isothermal array interrogating the 22q11 region; breakpoint predictions resulted in an overall resolution (mean distance between predicted and verified breakpoints) of 620 bp. (B) Probe redundancy as determined by blastn (100,000-bp sliding window average; 80% minimum sequence identity) vs. chromosomal coordinates. Regions of high DNA repeat density coincide with one of the predicted breakpoints and with an additional region of less than clear deletion signal seemingly interrupting the 1.4 Mbp deletion at ≈19 Mbp (location of a known LCR that presumably causes crosshybridization). (C) Quantification of the contribution of crosshybridization originating from an LCR. Redundant oligomers were removed by applying blastn cutoff scores of varying stringency. Averaged log ratios are given for the entire LCR region from 18.7 to 19 Mbp. Using a threshold of 50 results in a log2 ratio of roughly −0.2, a value near the average log2 ratio of heterozygous deletions. This indicates that the region with less than clear deletion signal from ≈18.7 to 19.0 Mbp is due to crosshybridization, and that the deletion detected is contiguous. (D) Sequencing of the affected region verifies the 1.4-Mb deletion breakpoints. Pat, patient (green); CHR22, human reference genome (blue). The red sequence block corresponds to a 19-bp sequence framing the large deletion on both sides, suggesting a potential mechanism of deletion.
Fig. 3.
Fig. 3.
Various types of deletion as well as gains in copy number can be detected with the chromosome 22q isothermal HR-CGH array. (A) Deletion types typical for 22q11DS. (B) Deletion types atypical for 22q11DS. (C) Three different cases of gains in copy number in 22q11. Vertical red lines indicate position of breakpoint prediction. Arrows indicate additional putative CNPs [blue, known CNP; red, new CNP (see Table 2); orange, LCR regions].
Fig. 4.
Fig. 4.
The isothermal HR-CGH array covering chromosome 22q allows predicting substantially different deletion sizes for two 22Q11DS patients with typical A→D deletion that had not been distinguishable by conventional methods (breakpoint predictions are indicated by vertical red lines).

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