Enhancer deletions of the SHOX gene as a frequent cause of short stature: the essential role of a 250 kb downstream regulatory domain

Abstract

Background: Mutations and deletions of the homeobox transcription factor gene SHOX are known to cause short stature. The authors have analysed SHOX enhancer regions in a large cohort of short stature patients to study the importance of regulatory regions in developmentally relevant genes like SHOX.

Methods: The authors tested for the presence of copy number variations in the pseudoautosomal region of the sex chromosomes in 735 individuals with idiopathic short stature and compared the results to 58 cases with Leri-Weill syndrome and 100 normal height controls, using fluorescence in situ hybridisation (FISH), single nucleotide polymorphism (SNP), microsatellites, and multiplex ligand dependent probe amplification (MLPA) analysis.

Results: A total of 31/735 (4.2%) microdeletions were identified in the pseudoautosomal region in patients with idiopathic short stature; eight of these microdeletions (8/31; 26%) involved only enhancer sequences residing a considerable distance away from the gene. In 58 Leri-Weill syndrome patients, a total of 29 microdeletions were identified; almost half of these (13/29; 45%) involve enhancer sequences and leave the SHOX gene intact. These deletions were absent in 100 control persons.

Conclusion: The authors conclude that enhancer deletions in the SHOX gene region are a relatively frequent cause of growth failure in patients with idiopathic short stature and Leri-Weill syndrome. The data highlights the growing recognition that regulatory sequences are of crucial importance in the genome when diagnosing and understanding the aetiology of disease.

Figure 1

Schematic representation of the PAR1 region showing the position of evolutionarily conserved non-coding DNA elements (CNEs), microsatellites and cosmids that were used for deletion mapping. The SHOX gene is indicated in red and resides between 505.1 and 540.1 kb from the telomere (NCBI build 125, March 2006). The CNEs and microsatellites used for deletion mapping are shown above the scale bar. CNEs are indicated in green; CNE4, CNE5 and CNE9 with a star symbol have shown enhancer activity in the developing chicken limb bud. CNE3, CNE4, CNE5, CNE7 and CNE9 have previously shown enhancer activity in the chicken neural tube. Microsatellites are indicated in blue. The SHOX exons and the cosmids contig used for deletion mapping are shown below the scale bar. Cosmid 34F05 (LLN0YCO3′M34F05) includes the SHOX gene; Cosmid 51D11 (LLN0YCO3'M'51D11) includes CNE6, CNE7 and markers DXYS10085; Cosmid 61E05 (LLN0YCO3'M'61E05) includes CNE8 and markers DXYS10087 and DXYS10096; Cosmid 10D05 (LLN0YCO3'M'10D05) includes CNE9. TEL, telomere; CEN, centromere.

Figure 2

Mapping of 63 PAR deletions in patients with idiopathic short stature (ISS) and Leri–Weill syndrome (LWS) using the SALSA P018C and P018D-1 SHOX MLPA kit. Maximum deletion sizes are according to Ensemble Genome Browser coordinates (in kb) and are given on the right side. Blackened areas indicate the presence of two copies of the MLPA probe; white areas indicate the deletion of one allele; green areas indicate the presence of more than two copies of the MLPA probe; grey areas indicate the common minimal deletion interval in patients, and solidus areas indicate that the analyzed marker was non-informative. The ISS 24-33 and LWS 50-63 patients present cases with an intact SHOX region and downstream deletions. In the control population, two deletions of the locus at 685 Xptel, a duplication of locus at 818 Xptel, as well as two duplications of the ASMT gene at 1711 Xptel were found. Note also that in one patient (no.9) a deletion of the STS gene was identified.

Figure 3

FISH analyses of patients with idiopathic short stature. The CNE9-containing cosmid 10D05 (LLN0YCO3'M'10D05) was used as a probe for FISH analysis. (A) Metaphase chromosomes from a female control with FISH signals on both X chromosomes. (B) Metaphase chromosomes from a female patient (no. 24) with a deletion on one of the chromosomes (arrows). (C) Metaphase from a male control with FISH signals on both X and Y chromosomes. (D) Metaphase from a male patient (no. 33) with a deletion on the Y chromosome (arrows) but not on the X chromosome. A reference probe residing on the long arm of the X-chromosome (cosmid P9) and a Y centromeric probe (DYZ3) were used as controls. The green signals indicate cosmids 10D05 and P9 and hybridised to opposite ends of the X chromosome; the red signal indicates probe DYZ3.

Figure 4

Summary of the spectrum of different SHOX mutations in patients with idiopathic short stature (ISS) and Leri–Weill syndrome (LWS). From all studied 735 unrelated patients with ISS, 31 are caused by SHOX-related deletions. Eight presented deletions proximal to SHOX (black) and 23 presented a SHOX gene deletion (hatched). A further five patients with ISS presented an intragenic mutation (white; 4 missense, 1 frameshift mutation; data not shown), which were initially excluded from the deletion study (total number of patients with ISS: 740). In 67 unrelated LWS, 38 are caused by SHOX- related defects: 13 presented a deletion downstream of SHOX (black), 16 presented a SHOX gene deletion (hatched) and nine presented an intragenic mutation (white; 5 missense, 3 nonsense, 1 frameshift mutation). The nine patients with intragenic mutations were initially excluded from the deletion study. In total, 4.9% of patients with ISS and 56.7% of patients with LWS are caused by point mutations or deletions of SHOX or its regulatory regions.