Deletion size analysis of 1680 22q11.2DS subjects identifies a new recombination hotspot on chromosome 22q11.2

Abstract

Recurrent, de novo, meiotic non-allelic homologous recombination events between low copy repeats, termed LCR22s, leads to the 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome/DiGeorge syndrome). Although most 22q11.2DS patients have a similar sized 3 million base pair (Mb), LCR22A-D deletion, some have nested LCR22A-B or LCR22A-C deletions. Our goal is to identify additional recurrent 22q11.2 deletions associated with 22q11.2DS, serving as recombination hotspots for meiotic chromosomal rearrangements. Here, using data from Affymetrix 6.0 microarrays on 1680 22q11.2DS subjects, we identified what appeared to be a nested proximal 22q11.2 deletion in 38 (2.3%) of them. Using molecular and haplotype analyses from 14 subjects and their parent(s) with available DNA, we found essentially three types of scenarios to explain this observation. In eight subjects, the proximal breakpoints occurred in a small sized 12 kb LCR distal to LCR22A, referred to LCR22A+, resulting in LCR22A+-B or LCR22A+-D deletions. Six of these eight subjects had a nested 22q11.2 deletion that occurred during meiosis in a parent carrying a benign 0.2 Mb duplication of the LCR22A-LCR22A+ region with a breakpoint in LCR22A+. Another six had a typical de novo LCR22A-D deletion on one allele and inherited the LCR22A-A+ duplication from the other parent thus appearing on microarrays to have a nested deletion. LCR22A+ maps to an evolutionary breakpoint between mice and humans and appears to serve as a local hotspot for chromosome rearrangements on 22q11.2.

Figure 1.

Identification of LCR22A+-D deletion by qPCR and haplotype analyses. (A, Top left) Trio of the KD23 pedigree in which a female proband is affected with 22q11.2DS (black circle), while the parents are normal (open shapes). (Top right) Illustration of the position of the LCR22A+-D deletion and the LCR22A-A+ interval with respect to the 22q11.2 region, shown in bp coordinates (hg19 assembly; the position of the chromosome breakpoints is estimated). Illustration of haplotypes of the 22q11.2 region in the trio in which individual alleles are depicted in different colors. The two coding genes, DGCR6 and PRODH as well as the non-coding gene, DGCR5 is indicated as gray filled ovals, distal to LCR22A and proximal to the LCR22A+-D deleted region, illustrated as an open box. The position of the DGCR5 gene in the illustration of the haplotypes in the trio is shown as a gray filled oval. The mother carries an LCR22A-A+ duplication CNV, indicated as a dark gray box. Below is a zoomed in snapshot from the UCSC Genome browser (hg19 assembly) showing the segmental duplication (LCR) track in dense format with LCR22A+ as indicated. Neighboring genes are shown along with a subset of splice variants. The DNA from the fosmid clone used as a probe for FISH mapping (Fig. 2) is indicated by a red star. The position of primer pairs used for qPCR assays is shown. The two primer sets flanking LCR22A+, where the breakpoints occur, are indicated by red color arrows (see primers in Supplementary Material, Table S2). The position of the microsatellite marker, D22S1638 used to identify haplotypes is indicated in blue font. (B) Bar graph of qPCR results for the proband (KD23), mother and father is shown. The y-axis indicates the relative quantity with respect to the control DNA, HapMap sample, GM12878. The x-axis indicates the primer pairs used for qPCR assay using primers illustrated above. Error bars are shown. More details are shown in Supplementary Material, Figure S2 and Table S3. (C) Table listing the sample name, family relation, qPCR results and size in bp of each allele of each microsatellite marker for the proband (Pro), mother (M) and father (F). The colors indicate from which parent (pink from mother and blue from father) had the particular sized PCR product in bp for a given microsatellite marker. Unfilled cells indicate that the alleles for a particular microsatellite marker could not be assigned to be derived from one or the other parent, and was therefore uninformative. (D) Chromatogram profiles of D22S1638 are shown as a representative example. The peaks identify the size, in bp of the PCR products from 1C. Adjacent stutter peak artifacts for each PCR product are typical for microsatellite markers. Details of all results for all samples are shown in Supplementary Material, Figure S4 and summarized in Supplementary Material, Table S4.

Figure 2.

FISH mapping of the 22q11.2 region. (A) FISH mapping was performed on Epstein-barr virus transformed lymphoblastoid cell lines from subjects BM1428.100 (normal), BM293 (3 Mb deletion/0.2 Mb LCR22A-A+ duplication), BM1024.001 (LCR22A+-D deletion) and BM1332.001 (LCR22A+-D deletion). Representative metaphase images of chromosome 22 and interphase nuclei are shown for each. The unique region on 22q11.1 near the pericentromeric region, containing CECR1 is shown by the green fluorescence signals (FITC), DGCR5 (within the LCR22A-A+ CNV) by the red fluorescence signals (S.O. dye) and TBX1-GNB1L by the aqua fluorescence signals. The appearance of yellow or white fluorescence in metaphase spreads indicates overlap of probe signals. White arrows in the metaphase spreads and interphase nuclei indicate the chromosome with the deletion. The fosmids and details of their map position are shown in Supplementary Material, Figure S1. (B) Illustration of the probes, metaphase (above) and interphase (below) images from the four different cell lines. There are two sets of red signals for the normal cell line and one very strong red signal from BM293 that has a 3 Mb deletion on one allele and a genomic duplication on one allele. There are red signals on both alleles for BM1024.001 and BM1332.001. For both samples, the aqua signals are absent from the deleted allele and present on the normal allele. Complete metaphase spreads and interphase nuclei are shown for seven cell lines used for FISH mapping in Supplementary Material, Figure S1.

Figure 3.

Summary of three types of recurrent 22q11.2 deletions. Illustration of three representative trios highlighting recurrent rearrangements based upon qPCR, microsatellite marker analysis and FISH mapping. The different alleles of chromosome 22q11.2 in each individual are shown in individual colors. Representative microsatellite markers are shown as small filled circles. For the type A deletion, the proband inherited the LCR22A-LCR22A+ duplication (two red filled circles marking D22S1638, surrounded by a black open box) from one parent and had a de novo 3 Mb LCR22A-D (or 1.5 Mb, LCR22A-B) deletion from the other parent. For the type B deletion, the proband had a nested LCR22A+-B or LCR22A+-D deletion, possibly on the opposite allele in the parent carrying the LCR22A-A+ duplication as shown. For the type C deletion, the proband had a nested LCR22A+-B or LCR22A+-D deletion from parents with normal alleles for 22q11.2.

Figure 4.

Genomic locus containing LCR22A+ and evolutionary breakpoint. (A) The position of the LCR22A+-D deletion and LCR22A-A+ duplication with respect to the 22q11.2 region with individual LCR22s are shown. The local region around LCR22A+ is illustrated as is the position of the ADU/VDU balanced translocation breakpoint within (coordinates are provided; hg19 assembly). Alternative splice variants of the non-coding gene, DGCR5 is shown in the snapshot from the UCSC Genome Browser. The lincRNA track for RNA-seq reads is shown for representative tissues. Dark blue indicates high expression in adult human tissues relative to other tissues with lower expression (light blue). The position of the Car15 gene in rodents is shown with respect to the 22q11.2 region, but this gene is not present in humans. The DGCR5 gene is not present in the mouse genome. (B) UCSC Genome Browser snapshot of the region of synteny in the mouse genome is shown (GRCm38/mm10, 2011). The right-hand side of the image maps to the LCR22A+ region, while the orange side maps 1.7 Mb away in the human region of synteny and contains genes Klhl22 and Scarf2. The evolutionary breakpoint between mouse and human maps between Scarf2 and Car15.

Figure 5.

Evolutionary breakpoints between human and mouse regions of synteny on 22q11.2. The known coding genes in the 22q11.2 region are aligned in order from the most centromeric end at the top to the most telomeric end. The region of synteny on mouse chromosome 16 is shown, with genes aligned in accordance to their map position. Note, that non-coding genes are not all predicted between humans and mice and are not shown (e.g. DGCR5 cannot be found in the mouse genome). Two genes in the region of synteny on mouse chromosome 16 map to human chromosome 2 (blue font). The USP18 gene maps to mouse chromosome 6 but not chromosome 16. Changes in the relative order of individual genes or sets of genes between mice and humans are indicated by different color-coded lines and arrows. The LCR22s are indicated as boxes in the human genome but they are not present in the mouse genome. The expected position in the mouse genome for the LCR22s, if they existed, are shown by the light gray dotted arrows. The thick black dotted line indicates the position of the Car15 gene in the mouse, which is within the LCR22A-LCR22A+ region, whose position is shown in the 22q11.2 region.