Genome-wide analysis of Ollier disease: Is it all in the genes?

Abstract

Background: Ollier disease is a rare, non-hereditary disorder which is characterized by the presence of multiple enchondromas (ECs), benign cartilaginous neoplasms arising within the medulla of the bone, with an asymmetric distribution. The risk of malignant transformation towards central chondrosarcoma (CS) is increased up to 35%. The aetiology of Ollier disease is unknown.

Methods: We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis using Affymetrix SNP 6.0 array on 37 tumours of 28 Ollier patients in combination with expression array using Illumina BeadArray v3.0 for 7 ECs of 6 patients.

Results: Non-recurrent EC specific copy number alterations were found at FAM86D, PRKG1 and ANKS1B. LOH with copy number loss of chromosome 6 was found in two ECs from two unrelated Ollier patients. One of these patients also had LOH at chromosome 3. However, no common genomic alterations were found for all ECs. Using an integration approach of SNP and expression array we identified loss as well as down regulation of POU5F1 and gain as well as up regulation of NIPBL. None of these candidate regions were affected in more than two Ollier patients suggesting these changes to be random secondary events in EC development. An increased number of genetic alterations and LOH were found in Ollier CS which mainly involves chromosomes 9p, 6q, 5q and 3p.

Conclusions: We present the first genome-wide analysis of the largest international series of Ollier ECs and CS reported so far and demonstrate that copy number alterations and LOH are rare and non-recurrent in Ollier ECs while secondary CS are genetically unstable. One could predict that instead small deletions, point mutations or epigenetic mechanisms play a role in the origin of ECs of Ollier disease.

Figure 1

Genome-wide copy number alterations in all 22 chromosomes. A) Copy number alterations in controls, Ollier enchondromas (ECs) and chondrosarcoma (CS) grade I, II, III. The upper panel shows genome-wide frequency plots of gains and losses in 29 controls and 32 Ollier tumors. Gains are plotted in green above 0% baseline and losses are plotted in red below 0% baseline. The X-axis corresponds to the genomic region from chromosomes 1 to 22 and the Y-axis represents the percentage of gains and losses of all selected samples at the specific location in genome. The lower panel shows frequency plot of 29 controls, 14 ECs, 12 CS I, 4 CS II and 2 CS III. The number and size of genomic alterations increases with increasing tumor grade. Enchondromas and control samples show a comparable number and size of genomic alterations, which can be attributed to common copy number variation. B) An example of copy number alterations in Ollier enchondroma (L206). This figure shows copy number alterations in all 22 chromosomes of enchondroma (L206). The black band indicates the number of copies of the chromosomes. The blue bands show the unpaired LAIR value. The lower band of this contains the originally uninformative homozygous SNPs. The top band indicates heterozygous informative SNPs. With LOH or imbalances between the alleles, the position of this band will decrease. As a loss of chromosome 6 these heterozygous SNPs are becoming homozygous showing the LOH. C) An example of copy number alterations in Ollier chondrosarcoma grade III (L810). Copy number loss with LOH is present at chromosome 4, 5q, 6q, 9p, 12p, 13 and 14q.

Figure 2

FAM86D. A) Homozygous loss of FAM86D in L206. A 200 Mb region containing the FAM86D gene at 3p12.3 is shown (X-axis). The gene lies in a ~100 Mb homozygous deleted region, within a larger area of hemizygous deletion. The individual copy number probes are shown as the log ratio of the intensity and zero is two copies. The horizontal lines are segments with identical copy number as identified by the HMM SNP-FASST algorithm. B) MLPA for 38 controls, 14 Ollier enchondromas (ECs) and 23 Ollier chondrosarcomas (CS). The Y-axis shows ratio profile and 1.0 indicates two copies of a given chromosomal locus. Homozygous copy loss of the FAM86D as shown by SNP array was confirmed in L206 and L910. Most of the controls and tumors show either two copies or hemizygous loss of this gene. Note that only outliers are displayed with ID numbers.

Figure 3

NIPBL protein expression. A) Example of nuclear expression of NIPBL in Ollier enchondroma (400 times magnification). B) Number of Ollier and solitary tumors with nuclear NIPBL expression.