RB1 gene inactivation by chromothripsis in human retinoblastoma

Abstract

Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [1]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identified recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.

Figure 1. Copy Number Changes and LOH in Human Retinoblastoma

Inferred log2 ratio (A) and LOH (B) for DNA isolated from 94 retinoblastomas and matched normal germline tissue. Red is gain and blue is loss in (A). LOH is indicated by blue and yellow indicates no change in genotype in (B). (C) Heatmap of chromosomal, regional and focal lesions for the retinoblastoma cohort. The highest rate of copy number variations (CNVs) was found in 10 tumors (>20 lesions per tumor). An intermediate rate (10–20 lesions per tumor) was found in 18 retinoblastomas and the remaining had low rate of CNV (1–10 lesions per tumor) or none.

Figure 2. Changes in Gene Expression Associated with 6p Gain

(A) Scatterplot of the number of tumors with large regional gains (>3Mb) spanning chromosome 6p. Plot is overlaid with a median spline (red). Below the scatterplot is the corresponding heat map of the log2 ratio of tumor to normal copy number signal across chromosome 6 for all 94 retinoblastomas. (B) Scatterplot of mean gene expression signal for chromosome 6p genes in tumor samples with a gain of chromosome 6p and tumors that are wild type for 6p (diploid). The red line is the unity line where x=y. The genes highlighted with red circles are those that are significantly increased in their expression and have at least a 2-fold upregulation in the tumors with 6p gain. Genes previously identified with increased expression correlated with 6p gain are highlighted with blue circles nuclear ribonucleoprotein (U11/U12). Similar analysis was performed for 1q, 16q, and 2p (Fig. S1). Only 1 gene (COX4I1) was differentially expressed in tumors with a 16q loss and no genes were identified on 1q or 2p. The COX4I1 gene encodes a subunit of cytochrome c oxidase and there is some evidence that this gene is downregulated in skin cancer[20].

Figure 3. Amplification of OTX2 in Retinoblastomas

(A) Heat map and (B) Manhattan plot of inferred log2 ratio of copy number for germline (G) and diagnostic (D) DNA samples of 3 out of 94 samples that carry an amplification in OTX2 detected by SNP6.0 array analysis. (C) OTX2 amplification was validated by quantitative real-time PCR. All data was normalized to CTNNA3 with normal diploid copy number. The dashed line is the cutoff for amplification ≥ 10 copies relative to matched normal DNA for that sample.

Figure 4. RB1 Gene Analysis in Retinoblastomas with MYCN Amplification

(A) Inferred log2 ratio normalized signal for germline and diagnostic DNA samples for samples with MYCN gene amplification. (B) One of the samples indicated by an (*) was previously validated to have a MYCN amplification (SJRB001). Validation of MYCN amplification in the remaining 7 samples using quantitative real-time PCR with a cutoff of 10 copies (dashed line). (C) Inferred log2 ratio for tumor and normal samples and loss of heterozygosity (LOH) for the RB1 gene as measured by SNP 6.0 analysis. (D) Table summarizing single nucleotide variations (SNV), loss of heterozygosity (LOH), promoter hypermethylation and insertions/deletions (INDEL) at the RB1 locus. (E) Immunohistochemistry for RB1 in normal retina, SJRB051 with biallelic RB1 loss and SJRB011 with wild type RB1. Arrows indicate RB1 immunopositive cells in the normal retina and vascular endothelial cells. (F) PCA plot of gene expression array analysis for the RB1 wild type sample (SJRBO11, yellow) and RB1 deficient retinoblastomas (blue). Scale bars in (E): 10μm.

Figure 5. Whole Genome Sequence Analysis of Retinoblastoma

(A) Copy number alterations on chromosome 13 spanning the RB1 gene (box). Blue indicates a loss and red indicates a gain. (B) LOH analysis on chromosome 13 spanning the RB1 locus (box). Blue indicates LOH and white indicates normal diploid copy number. (C) CIRCOS plot of SJRB031 with chromothripsis at the RB1 gene (arrow). (D) Half-oval chromothripsis plot of copy number gain (red) and loss (blue) and deletions (gray lines), intrachromosomal translocations (red lines) and insertions (yellow lines). (E) Position of the 5′ and 3′ FISH probes used for 2-color FISH of the RB1 gene. Blue arrowheads indicate the breakpoints in SJRB031 in the RB1 gene. (F) FISH for the 5′ (red) and 3′ (green) probes for the RB1 gene on one sample with chromothripsis (SJRB031) and a normal control (tonsil). (G) Summary of results for immunohistochemistry for RB1 (IHC), deletion analysis (DEL), chromothripsis (CHRP), single nucleotide variation (SNV), loss of heterozygosity (LOH), small insertions/deletions (INDEL) in the RB1 gene in each of the 10 samples. The red box indicates the one tumor among 94 with wild type RB1 and expression of RB1 protein. The gray boxes indicate those with one intacti RB1 gene but no RB1 protein by IHC. (H) Immunohistochemistry for RB1 in SJRB031. Scale bars in F,H: 10¼m.