Germline mutations and somatic inactivation of TRIM28 in Wilms tumour

Abstract

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.

Fig 1. DNA sequence and methylation of TRIM28.

(A) Family 1. 2-bp deletion (c.525_526del) in the blood of case 39 (39B), the kidney of case 37 (37K) and the blood of their mother (37M). The father (37F) was unaffected. The tumours from cases 37 and 39 (37T and 39T) showed loss of heterozygosity. (B) Family 2. Germline deletion/insertion (c.1746_1747delinsC) in blood DNA from case 399 (399N) with loss of heterozygosity in tumours 399T and 249T. (C) Somatic deletion/insertion mutation (c.1935delinsGA) in W117 tumour (W117T) and reference sequence in the adjacent kidney (W117K). (D) The proportion of methylated CpGs in exon 1 of TRIM28 in W117T as measured by targeted bisulfite PCR. For each CpG site the black portion of the bar shows the proportion of methylated reads.

Fig 2. TRIM28 immunohistochemistry.

(A) Monomorphic epithelial Wilms tumours showing absence of TRIM28 expression in 37T and W117. (B) Absence of TRIM28 protein expression in tumour (T) but not in adjacent kidney (K) in case 39. (C) Positive control showing TRIM28 expression in two representative Wilms tumours. Black line = 50 μM.

Fig 3. Somatic genetic changes in Wilms tumours.

The left side shows the number of somatic non-synonymous and truncating mutations for each tumour detected by MuTect2. The single somatic variant in W117 is the TRIM28 mutation. The right side shows the fractional length of aberrant copy number segments as determined by ADTEx.

Fig 4. Dendrogram from unsupervised hierarchical clustering of gene expression of 17 Wilms tumours.

IGF2, refers to IGF2 status where blue = loss of imprinting, and red = loss of heterozygosity at IGF2. Rests refers to the presence of nephrogenic rests (NR) were blue = intralobar NR, red perilobar NR and purple NR of unknown type. For each gene, red boxes indicate the presence of mutation, whereas the grey box denotes gene deletion.

Fig 5. Comparison of gene expression between S1 and other Wilms tumours.

The upper panels show the five most down-regulated and five most up-regulated genes in the S1 subgroup (n = 11) compared to S2-S5 tumours (n = 213) in the study of Gadd et al. [30]. The lower panels show expression of these genes in TRIM28-mutated tumours and 13 other tumours from this study. Red circles, S1 or TRIM28-mutated tumours. Blue circles, favourable histology tumours. Note that two tumours with anaplastic histology, both of which had TP53 mutations, are not included to maintain comparability with the favourable histology tumours reported by Gadd et al. [30].