Identification of somatic chromosomal abnormalities in hypothalamic hamartoma tissue at the GLI3 locus

Abstract

Hypothalamic hamartomas (HH) are rare, benign congenital tumors associated with intractable epilepsy. Most cases are sporadic and nonsyndromic. Approximately 5% of HH cases are associated with Pallister-Hall syndrome (PHS), which is caused by haploinsufficiency of GLI3. We have investigated the possibility that HH pathogenesis in sporadic cases is due to a somatic (tumor-only) mutation in GLI3. We isolated genomic DNA from peripheral blood and surgically resected HH tissue in 55 patients with sporadic HH and intractable epilepsy. A genome-wide screen for loss of heterozygosity (LOH) and chromosomal abnormalities was performed with parallel analysis of blood and HH tissue with Affymetrix 10K SNP microarrays. Additionally, resequencing and fine mapping with SNP genotyping were completed for the GLI3 gene with comparisons between peripheral blood and HH tissue pairs. By analyzing chromosomal copy-number data for paired samples on the Affymetrix 10K array, we identified a somatic chromosomal abnormality on chromosome 7p in one HH tissue sample. Resequencing of GLI3 did not identify causative germline mutations but did identify LOH within the GLI3 gene in the HH tissue samples of three patients. Further genotyping of 28 SNPs within and surrounding GLI3 identified five additional patients exhibiting LOH. Together, these data provide evidence that the development of chromosomal abnormalities within GLI3 is associated with the pathogenesis of HH lesions in sporadic, nonsyndromic patients with HH and intractable epilepsy. Chromosomal abnormalities including the GLI3 locus were seen in 8 of 55 (15%) of the resected HH tissue samples. These somatic mutations appear to be highly variable.

Figure 1

Chromosomal Abnormalities on 2020 Blood and tissue pairs were assayed on the Affymetrix 10K platform. Copy-number changes were calculated with the analysis software DChipSNP. The amplification was later verified at the GLI3 locus by qPCR. (A) Copy-number analysis indicated that chromosome 7p had a copy number change to 2.5 copies for the tissue and not the blood, whereas all other samples showed no significant stretches of copy-number change. The assay was repeated for verification that the finding was not assay specific. (B) The percent of SNPs receiving a No Call across a 50 SNP window is shown for sample 2020. It would be expected that the No Call rate would increase in a region containing an amplification because SNPs would not easily bin into AA, AB, and BB, as would be the case if the copy number were two or one. Within the 7p region is the GLI3 gene, previously shown to harbor germline mutations causing Pallister Hall syndrome, which is highly associated with HH.

Figure 2

Observed Loss of Heterozygosity within GLI3 Gene for HH Blood and Tissue Paired Samples LOH was detected either by resequencing, genotyping on the Affymetrix platform, or custom SNP-genotyping with Tm-Shift assays. LOH events were defined as pairs in which the blood sample contained a heterozygous polymorphism (e.g., a heterozygote SNP) and the tissue sample was homozygous. SNPs that were genotyped homozygous in blood were uninformative (shown as gray bars). Samples where a heterozygote was observed in both the tissue and blood samples indicate the absence of LOH. (A) A total of eight samples contained LOH events observed by either genotype data from the Affymetrix 10K GeneChip mapping array (yellow bar), sequencing of a heterozygous SNP within exons (green bar), or SNP genotyping (red bar). Blue bars indicate the nearest flanking SNP to an LOH even (green, yellow, and red bars) where LOH was definitively not observed. Uninformative SNPs that were not within a region of LOH or SNPs that were heterozygous in blood and tissue but did not neighbor an LOH event are not shown, for clarity. (B) Schematic of GLI3 gene aligned to above LOH graphs. (C) Mutations within the GLI3 gene have previously been found to lead to Pallister-Hall Syndrome (PHS).