Aberrant interchromosomal exchanges are the predominant cause of the 22q11.2 deletion

Abstract

Chromosome 22q11.2 deletions are found in almost 90% of patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS). Large, chromosome-specific low copy repeats (LCRs), flanking and within the deletion interval, are presumed to lead to misalignment and aberrant recombination in meiosis resulting in this frequent microdeletion syndrome. We traced the grandparental origin of regions flanking de novo 3 Mb deletions in 20 informative three-generation families. Haplotype reconstruction showed an unexpectedly high number of proximal interchromosomal exchanges between homologs, occurring in 19/20 families. Instead, the normal chromosome 22 in these probands showed interchromosomal exchanges in 2/15 informative meioses, a rate consistent with the genetic distance. Meiotic exchanges, visualized as MLH1 foci, localize to the distal long arm of chromosome 22 in 75% of human spermatocytes tested, also reflecting the genetic map. Additionally, we found no effect of proband gender or parental age on the crossover frequency. Parental origin studies in 65 de novo 3 Mb deletions (including these 20 patients) demonstrated no bias. Unlike Williams syndrome, we found no chromosomal inversions flanked by LCRs in 22 sets of parents of 22q11 deleted patients, or in eight non-deleted patients with a DGS/VCFS phenotype using FISH. Our data are consistent with significant aberrant interchromosomal exchange events during meiosis I in the proximal region of the affected chromosome 22 as the likely etiology for the deletion. This type of exchange occurs more often than is described for deletions of chromosomes 7q11, 15q11, 17p11 and 17q11, implying a difference in the meiotic behavior of chromosome 22.

Figure 1

The 22q11.2 region including the locations of the LCR-22 segmental duplications A–D is depicted. Cosmid probes used for FISH are shown below the line. The names and relative locations of genes and STS markers are noted above the line. The figure is not drawn to scale. Results of deletion sizing experiments by FISH in a cohort of 277 patients is given in the lower part of the figure. The LCRs coinciding with the deletion endpoints are given with the relative percentage of patients that were found to be deleted for that given region.

Figure 2

(A) Four markers from within the 3 Mb deleted region that were used for parental origin determination are depicted in a sample pedigree. The names of the markers are listed to the left. Dashes represent a marker that did not amplify. The proband shows haploinsufficiency for alleles from her mother, noted by zeroes (0). (B) Genotyper data presented in the pedigree for marker D22S264 is given. Note the absence of commonly shared peaks, representing alleles, between mother and proband.

Figure 3

(A) Markers flanking the typical deletion endpoints that were used for haplotype reconstruction are shown in a three-generation pedigree consistent with an intrachromosomal exchange. Dashes depict a marker that did not amplify. (B) Sizes and intensity of allele peaks are shown from Genotyper analysis for a single marker (D22S427).

Figure 4

(A) Haplotype reconstruction depicting an interchromosomal exchange. The regions flanking the deletion (shown by a thin bar) are derived from different grandparents, indicating the exchange. (B) Genotyper data from this family for a single proximal flanking marker.

Figure 5

Immunostaining and FISH signals for a spermatocyte nucleus in pachynema. Anti-SCP3 antibodies (red), anti-MLH1 antibodies (green), CREST antisera (yellow) and c87f 9 (violet). The enlarged region is of chromosome 22. The chromosome 22 crossover site, as indicated by the anti-MLH1 focus (green), is at the distal end of the synaptonemal complex. The cosmid signal of c87f 9 (violet) is close to the centromere (yellow). The sex body containing the X and Y chromosomes is indicated by an arrowhead. In all cases, the CREST foci are significantly larger than the MLH1 foci.

Figure 6

FISH analysis of interphase nuclei to detect an inversion involving the standard 3 Mb region is shown. Probes were labeled in the order of the map (centromere to telomere) depicted in Figure 1, using a green, red, yellow order. All nuclei showed this color/probe order as demonstrated above, consistent with the map.