Polymorphisms of the 22q11.2 breakpoint region influence the frequency of de novo constitutional t(11;22)s in sperm

Abstract

The constitutional t(11;22) is the most frequent recurrent non-Robertsonian translocation in humans, the breakpoints of which are located within palindromic AT-rich repeats on 11q23 and 22q11 (PATRR11 and PATRR22). Genetic variation of the PATRR11 was found to affect de novo t(11;22) translocation frequency in sperm derived from normal healthy males, suggesting the hypothesis that polymorphisms of the PATRR22 might also influence the translocation frequency. Although the complicated structure of the PATRR22 locus prevented determining the genotype of the PATRR22 in each individual, genotyping of flanking markers as well as identification of rare variants allowed us to demonstrate an association between the PATRR22 allele type and the translocation frequency. We found that size and symmetry of the PATRR22 affect the de novo translocation frequency, which is lower for the shorter or more asymmetric versions. These data lend support to our hypothesis that the PATRRs form secondary structures in the nucleus that induce genomic instability leading to the recurrent translocation.

Figure 1.

Analysis of the allelic origin of de novo t(11;22) translocations. (A) Nested PCR strategy for determining allelic origin of the der(22) of the t(11;22). PATRRs are shown by thick arrows, and the AT-rich region flanking the PATRR22 is indicated by a hatched box. PCR primers are shown by thin arrows to indicate the location and orientation. (B) Representative results of the nested PCR. For an A/C heterozygote, the first PCR can distinguish the allelic origin of the PCR products based on product size (upper panel). For a B/C heterozygote, the size of the first PCR product is similar to one another (middle panel). The second PCR can clearly distinguish between the allelic origins (lower panel). Two bands were observed in lane 2, suggesting that the template DNA includes two or more translocations. M, size markers; P, DNA from t(11;22) translocation carrier served as positive controls.

Figure 2.

Screening and genotyping of rare short PATRR22 variants. (A) Genomic structure of the PATRR22 region. LCR-specific primers are designed to amplify the PATRR22 (arrows). Proximal region represents the ‘AT-rich region-HSAT1-Alu’ cassette repeated three or four times (6,29). Sites for the forward primer (F) carry sequence variations among the AT-rich region polymorphic alleles. In the B and C alleles, the PCR product originates from the closest F site, whereas it can anneal with the second closest site in the A allele. (B) LCR-specific PCR for amplifying the PATRR22. Upper panel represents the LCR-specific PCR for the PATRR22, whereas the lower panel indicates the genotyping of the AT-rich region flanking the PATRR22. Genotypes of the AT-rich region are given at the bottom. The PATRR22-specific PCR product from the A allele is much longer than the others for the reason described above. M, size markers. (C) Rare variants. Lane 1, standard B/C heterozygote; lane 2, compound heterozygote for B/C rare variants; lane 3, C/C homozygote; lane 4, compound heterozygote for C rare variants.

Figure 3.

M-fold analysis of the secondary structure of rare short variants of the PATRR22 and proximal AT-rich region flanking the PATRR22 (A allele). For the AT-rich region flanking the PATRR22, only the central 229 bp region is shown.

Figure 4.

Rare der(22) junction derived from an A allele. (A) Schematic representation of the organization of junction from the der(22) from an A/C heterozygote. The A/C heterozygote produces three types of der(22); a standard der(22) with the breakpoint at the center of the PATRR22 of the A allele (a), a rare der(22) with the breakpoint at the center of the AT-rich region of the A allele (b), and a standard der(22) with the breakpoint at the center of PATRR22 of the C allele (c). (B) The der(22)-specific PCR. The allelic origin of the der(22) can be determined by its size. Lanes 1 and 5, a der(22) derived from a C allele; lanes 9 and 13, a der(22) derived from an A allele; lane 2, a rare der(22) with the breakpoint at the center of AT-rich region of the A allele. M, size markers; P, DNA from a t(11;22) translocation carrier served as a positive control.