Identification of a major recombination hotspot in patients with short stature and SHOX deficiency

Abstract

Human growth is influenced not only by environmental and internal factors but also by a large number of different genes. One of these genes, SHOX, is believed to play a major role in growth, since defects in this homeobox-containing gene on the sex chromosomes lead to syndromal short stature (Leri-Weill dyschondrosteosis, Langer mesomelic dysplasia, and Turner syndrome) as well as to idiopathic short stature. We have analyzed 118 unrelated patients with Leri-Weill dyschondrosteosis and >1,500 patients with idiopathic short stature for deletions encompassing SHOX. Deletions were detected in 34% of the patients with Leri-Weill dyschondrosteosis and in 2% of the patients with idiopathic short stature. For 27 patients with Leri-Weill dyschondrosteosis and for 6 with idiopathic short stature, detailed deletion mapping was performed. Analysis was performed by polymerase chain reaction with the use of pseudoautosomal polymorphic markers and by fluorescence in situ hybridization with the use of cosmid clones. Here, we show that, although the identified deletions vary in size, the vast majority (73%) of patients tested share a distinct proximal deletion breakpoint. We propose that the sequence present within this proximal deletion breakpoint "hotspot" region predisposes to recurrent breaks.

Figure 1

Deletion mapping of 27 patients with LWD (patients LWD1–LWD27) and 6 patients with ISS (patients ISS28–ISS33). Patients LWD2, LWD3, LWD4, LWD5, LWD8, LWD9, LWD19, LWD20, LWD22, and LWD28 have been described elsewhere as presenting a SHOX deletion (Schiller et al. 2000); however, the exact extent of the deletion sizes as well as the breakpoints were not defined. Blackened areas indicate the presence of the respective cosmid clone on both of the patient’s sex chromosomes; unblackened areas indicate the absence of a cosmid clone on one sex chromosome; shaded areas indicate the breakpoint region. The map positions of the cosmid clones are indicated by horizontal lines and the clone abbreviation (see the “Material and Methods” section). The approximate distance from the telomere is given in kb. Microsatellite markers are indicated with vertical lines at their respective map position. The genomic locus of the SHOX gene is indicated in gray and resides ∼555.1–582.3 kb from the telomere. The deletion hotspot is defined by the presence of cosmid P0117 and the absence of cosmid 29B11.

Figure 2

FISH of cosmids 29B11 and P0117 to metaphase (A) and interphase chromosomes (B) of patient LWD10. The absence of the signal of cosmid 29B11 and the presence of the signal of cosmid P0117 locates the breakpoint on cosmid P0117.

Figure 3

Results of SNP analysis of the critical breakpoint region of the overlap of cosmid clones P0117 and 29B11. This region—at ∼655–678 kb from the telomere on the X chromosome and at 57–80 kb on the BAC clone RP13-76L22 (GenBank accession number AL683871.15) (on the basis of the UCSC Genome Browser human May 2004 assembly and NCBI build 35)—harbors the VNTR AK1 (655,357–656,210 bp), as well as the microsatellite marker P117 (672,153–675,202 bp). SNPs within AK1, DXYS86 (P131), DXYS59 (dbSNP accession number rs6644571 at 666,749 bp), C7/SNP (dbSNP accession numbers rs1016964 at 671,526 bp and rs3995647 at 671,472 bp), and C8/SNP (dbSNP accession numbers rs2399945 at 676,936 bp and rs2399946 at 676,787 bp), as well as the few as-yet-unpublished SNPs within C9/SNP and C10/SNP, were checked for heterozygosity in seven families (patients LWD1, LWD6, LWD7, LWD10, LWD15, LWD16, and ISS30). The breakpoint of ∼5 kb (indicated in gray) could be defined by the heterozygosity of the SNPs within C7/SNP and the absence of heterozygosity of SNPs within DXYS59. SHOX maps to 555,079–582,318 bp, AK1 to 655,357–656,210 bp, DXYS86 to 665,323–665,477 bp, DXYS59 to 666,696–666,875 bp, C7/SNP to 671,103–671,885 bp, P117 to 672,153–675,202 bp, C8/SNP to 676,618–676,192 bp, C9/SNP to 679,663–681,329 bp, and C10/SNP to 683,363–684,552 bp (from the telomere).

Figure 4

Secondary-structure prediction, made by use of Mfold analysis, of the sequence harboring the hotspot region (B) and the 6-kb sequences distal (A) and proximal (C) to it. The free-energy values for each secondary-structure prediction are −902 kcal/mol (A), −617.9 kcal/mol (B), and −677.8 kcal/mol (C). High DNA bending and an extensive secondary-structure formation are observed only in the hotspot region (B).