Figure 1.
Breakpoint identification in individuals with deletions in the WS region and atypical phenotypes.
Chromosome 7 ideograms showing the region of interest, including the typical WS deletion (yellow boxes) relevant to rearrangements in probands 8399, 9061, and 9101. The LCR and CNV tracks show the location of these features. The copy number track for each individual shows the copy number data from the arrays (black dots), analyzed as described in Methods. The red box on the ideogram represents the extent of the deletion. The sequence tracks show the sequence of the junction fragment aligned with the same region from the non-deleted chromosome. The sequence highlighted in green for probands 9061 and 9101 is present at both breakpoints so the actual break cannot be known. The gel images to the right show the validation of the junctions by PCR amplification of the junction fragment from genomic DNA. (A). Proband 8399 has a 10.8 Mb deletion that includes the entire WS typical region and includes 91 genes. The parents of 8399 were lost to follow up but the junction fragment was absent from two control samples. (B) Proband 9061 has a deletion of 503 kb affecting 13 genes, which includes only part of the WS critical region. The gel image to the right shows the junction fragment was inherited from her mother and was absent from an unaffected relative. (C) Proband 9101 has a 4.4 Mb deletion affecting 71 genes including the entire WS critical region. Chromosome ideogram is the same as that for 9061. The gel image shows this was a de novo deletion in the proband because the junction fragment was absent in both parents. A detailed list of affected genes in these three probands is provided in Figure S2.
Figure 2.
Determination of a complex rearrangement on chromosome 5p15.2-5p14.3 required sequencing deletion breakpoints.
(A) Ideogram of chromosome 5 showing the 5p15.2‐5p14.3 deletion present in proband 9101. The copy number plot was derived from our microarray (black dots) and qPCR (Xs) results. The green arrowhead indicates an inherited CNV at the telomeric deletion breakpoint. Black and gray boxes depict regions of normal copy number, the red box indicates the extent of the deletion, and the gray box shows the inverted region. The sequence tracks show junction fragments along with genomic positions and orientations of flanking sequences. Eight base pairs of 5p14.2 sequence present at both breakpoints are highlighted in green. (B) Schematic of deletion and inversion including relative locations of PCR primers and FISH probes on reference and affected chromosomes 5. Telomeric is to the left. The gel image on the right shows amplification of the junction fragments present at the deletion/inversion breakpoints. The absence from both parents indicates this rearrangement occurred de novo. (C) Metaphase FISH analyses of cells from proband 9101, using BAC probes RP11‐91A5 (green), RP11‐91E20 (red), and RP11‐90G17 (yellow) confirming the presence of the inverted region of chromosome 5p14.5 (white arrowhead).
Figure 3.
Deletion breakpoints on chromosome 5 and 7 associated with ID/MCA.
Chromosome features for the region of interest are shown versus the physical position in Mb on the February 2009 human genome assembly. Red boxes on ideograms denote regions of detail. Gene and LCR tracks were adapted from the UCSC Genome Browser. CNV tracks show type and position as reported in the general population: red, loss; green, gain; blue, both. The copy number chart was derived from our SNP microarray (black dots) and qPCR (Xs) results. The red box on the copy number plots indicates the deletion. Sequence tracks from top to bottom show the junction fragment and the sequences flanking the centromeric and telomeric breakpoints. Regions of sequence identity at the breakpoints are highlighted in green. (A) Deletion of 2.57 Mb at 5q15 in proband 9239 that involves 15 genes. The gel image shows the presence of a PCR fragment amplified from the deletion junction in the proband but not in control samples (parental samples were not available). (B) Deletion of 2.13 Mb at 7q11.23 in proband 9152 that involves 34 genes. Genes shown in red are typically deleted in WS. This deletion includes a single gene, GTF2I, within the WS critical region. The GTF2I-ZP3 fusion transcript resulting from the deletion is shown below the genes track. The green arrowhead shows the location of a CNV at the telomeric breakpoint inherited on the deleted chromosome 7. The gel image shows presence of a PCR fragment amplified from the deletion junction in genomic DNA from the proband, but not in either parental sample, indicating the deletion occurred de novo.
Figure 4.
Identification of LCR mediated mosaic deletion using SNP arrays.
(A) Chromosome features for the region of interest in proband 8772 are shown versus the physical position in Mb on the February 2009 human genome assembly. Red box on ideogram denotes region of detail. LCRs, structural variants, the FISH probes used and the location of qPCR assays also are shown. The copy number section shows the data for the SNP arrays (black dots) and qPCR (Xs) assays. The tick marks represent 2 copies (red) and 1.5 copies (blue). Note the paucity of SNPs in the LCRs and that the putative deletion ends in the LCRs. (B, C) metaphase FISH using BAC probe RP11-554H10 (green) and a BAC probe (red) located within MYCN at 2p24.3, which is distal to the region of interest. (B) FISH analyses show images from a normal cell from 8872. (C) FISH analyses showing a cell with one chromosome deleted for BAC RP11-554H10. (B1, B2, C1, C2) Detailed views of both chromosomes 2 in each cell. Note absence of hybridization to RP11-554H10 in C2.
Figure 5.
Duplications in probands with ID/MCA.
Chromosome features for the regions of interest are shown versus physical position in Mb on the February 2009 human genome assembly. Red boxes on ideograms denote regions of detail. LCR and structural variant tracks were adapted from the UCSC Genome Browser. CNV and structural variation tracks show type and position of variants as reported in the general population: red, loss; green, gain; blue, both; grey, inversion. SNP copy number charts were derived from our microarray (black dots) and qPCR (Xs) results. Heat maps indicate p-values of observed copy number change for individual SNPs calculated using Partek Genomics Suite 6.3. Shading from blue to red represents probability from 0.0–1.0 of normal copy number. (A) Dup(1)(p36.11p35.3) in proband 8293. (B) De novo dup(2)(p22.1-2p16.1) in proband 9148. (C) De novo LCR-mediated dup(16)(p12.2-16p11.2) in proband 8464. (D1) and (D2) show detailed views of LCR architecture at the16p12.2 and 16p11.2 breakpoints, respectively. Directly oriented copies of UCSC segmental duplication 11963 are boxed in black.