Highly conserved non-coding sequences and the 18q critical region for short stature: a common mechanism of disease?

Abstract

Background: Isolated growth hormone deficiency (IGHD) and multiple pituitary hormone deficiency (MPHD) are heterogeneous disorders with several different etiologies and they are responsible for most cases of short stature. Mutations in different genes have been identified but still many patients did not present mutations in any of the known genes. Chromosomal rearrangements may also be involved in short stature and, among others, deletions of 18q23 defined a critical region for the disorder. No gene was yet identified.

Methodology/principal findings: We now report a balanced translocation X;18 in a patient presenting a breakpoint in 18q23 that was surprisingly mapped about 500 Kb distal from the short stature critical region. It separated from the flanking SALL3 gene a region enriched in highly conserved non-coding elements (HCNE) that appeared to be regulatory sequences, active as enhancers or silencers during embryonic development.

Conclusion: We propose that, during pituitary development, the 18q rearrangement may alter expression of 18q genes or of X chromosome genes that are translocated next to the HCNEs. Alteration of expression of developmentally regulated genes by translocation of HCNEs may represent a common mechanism for disorders associated to isolated chromosomal rearrangements.

Figure 1. Breakpoint map of the 263/96 patient.

a. Map of the 18q breakpoint region. The position of the breakpoint at 74.8 Mb of chromosome 18 is indicated by a vertical arrow. Below is a schematic representation of the deletion map and of the short stature critical region , . The position of the markers used is indicated; the critical region boundaries are indicated by dotted lines. Under the map are the genes in the region: horizontal arrows represent transcription orientation. b. Map of the Xq breakpoint region. The position of the breakpoint at 109.7 Mb of chromosome X is indicated by a vertical arrow. Under the map are the genes in the region: horizontal arrows represent transcription orientation. c. Southern Blot analysis of the 18q breakpoint: DNA from the patient and of two normal DNAs was digested with BamH1 and fractionated by PFGE as described previously . The Blot was hybridized with the probe 263/5. d. Southern Blot analysis of the Xq breakpoint: DNA from the patient and of two normal DNAs was digested with KpnI and fractionated by PFGE as described previously . The Blot was hybridized with the probe 263×1. Probes are described in Materials and Methods. Map positions are from NCBI Release 36.1

Figure 2. Comparative analysis of the HCNE rich region in 18q.

The portion of the analysis from the VISTA Genome Browser (http://genome.lbl.gov/vista/index.shtml) from 73,5 to 75 Mb of human chromosome 18 is reported to show conservation between human and mouse, chicken or fugu. The HCNEs are numbered above the peaks. The position of the 263/96 breakpoint is indicated by a vertical arrow, that of the genes SALL3 and ATP9B by a horizontal arrow. The homology between human and the species indicated is shown on the right (%). Map positions in human correspond to NCBI Release 35.

Figure 3. Some of the HCNEs have enhancer or silencer function.

Luciferase report assays of the cell lines indicated, transfected with constructs containing promoter only vector (P), a conserved elements from a different genomic region, showing enhancer activity (EN), an unrelated negative control (UR: human/mouse non-conserved element), HCNE1, 9 and E as in Fig. 2. All values are expressed as % of the luciferase activity of the promoter vector (P). *: p value<0.001.

Figure 4. ChIP of the HCNE on total E11.5 chromatin and adult mouse brain.

Embryonal (a) and adult brain chromatin preparations (b) were immunoprecipitated with the antibodies to the histone modifications indicated on the X axis and the DNA was PCR amplified with primers specific for each HCNE. INP: PCR from total chromatin. AcH3 and acH4: ChIP with antibodies to acetylated histone H3 or H4. 2MK4H3: ChIP with antibodies to 2-methyl K4 of histone H3; 3MK27H3: ChIP with antibodies to 3-methyl K27 of histone H3. All values are fold increases, compared to an equal amount of INP. Myc, Xist (positive controls), Atp9b, Sall3 and Chrdl1 indicate primers in the 5′UTR region of each gene. Only HCNEs positive for at least one modification were reported in the figure.

Figure 5. Real time RT-PCR of the Chrdl1, Sall3 and Atp9b genes in mouse tissues.

RNAs from the tissues and the developmental stages indicated on the X axis were reverse transcribed and PCR amplified as described in Materials and Methods. Each value was normalized as described in Materials and Methods and the ratio with the histone H3 gene expression was calculated.

Figure 6. Chromatin modifications of genes flanking the 18q breakpoint, in the LB263/96 patient.

Chromatin of the patient 263/96 and two normal lymphoblastoid cell lines was IP with antibodies to acH3, acH4 or to 2MK4H3 and the DNA was amplified by real time PCR with primers in the promoter regions of each gene. One or two sets of primers were utilized for each gene, as indicated. Modifications were calculated as described in Materials and Methods. In the figure, the ratio between acH3/acH4 (black) and acH4/2MK4 (gray) in chromatin from the LB263/96 patient and two normal controls (MA and LB696) is shown.