GREM1 and POLE variants in hereditary colorectal cancer syndromes

Abstract

Hereditary factors are thought to play a role in at least one third of patients with colorectal cancer (CRC) but only a limited proportion of these have mutations in known high-penetrant genes. In a relatively large part of patients with a few or multiple colorectal polyps the underlying genetic cause of the disease is still unknown. Using exome sequencing in combination with linkage analyses together with detection of copy-number variations (CNV), we have identified a duplication in the regulatory region of the GREM1 gene in a family with an attenuated/atypical polyposis syndrome. In addition, 107 patients with colorectal cancer and/or polyposis were analyzed for mutations in the candidate genes identified. We also performed screening of the exonuclease domain of the POLE gene in a subset of these patients. The duplication of 16 kb in the regulatory region of GREM1 was found to be disease-causing in the family. Functional analyses revealed a higher expression of the GREM1 gene in colorectal tissue in duplication carriers. Screening of the exonuclease domain of POLE in additional CRC patients identified a probable causative novel variant c.1274A>G, p.Lys425Arg. In conclusion a high penetrant duplication in the regulatory region of GREM1, predisposing to CRC, was identified in a family with attenuated/atypical polyposis. A POLE variant was identified in a patient with early onset CRC and a microsatellite stable (MSS) tumor. Mutations leading to increased expression of genes can constitute disease-causing mutations in hereditary CRC syndromes.

Figure 1

Pedigree of the AFAP/atypical polyposis family. Individuals with clinical diagnosis indicated, patients marked with * were sequenced by whole exome sequencing and linkage analysis was also performed on these patients. The duplication was present in heterozygote form in patients indicated with a (+) and family members which did not carry the mutation are indicated with a (−). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Schematic view of the duplicated region on chromosome 15. The tandem repeat of 16,229 bp (chr15:32,986,220‐33,002,449 (GRCh37/hg19) including the 3′part of the SCG5 gene. The rs4779584 C allele in the duplicated region is indicated with an arrow as is the c.−76 C/G SNP in the CpG island upstream of the GREM1 gene. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

CpG‐methylation level boxplot showing the overall methylation levels of each sample compared side by side. The methylation level on the Y‐axis is the fraction of methylated reads/total number of reads. Sample IV:2, V:2, IV:6, and V:8 are from affected members and Q1, Q2, Q3 are blood controls. The overall degree of methylation detected is low and there is no significant difference between the patients and the control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

The absolute expression analysis of the GREM1 gene in patients and controls. The number of copies/µl is shown in this absolute expression analysis of GREM1. Six controls of normal colon mucosa and normal colon mucosa from two samples each from patient IV:2 and V:2 were analyzed. The GREM1 gene is significantly higher expressed in samples from colon mucosa in the affected family members compared with controls (P = 0.013). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Sanger sequencing over rs4779584. (a) Family member V:2 (affected), presents with three alleles C,C,T (where C is blue and T is red). The duplication of C on the affected allele is clearly seen compared with the normal control in b. (b) Normal control (alleles C,T). (c) Family member IV:2 (affected) with three C alleles, C,C,C. The duplicated C allele cannot be distinguished from the other normal C allele in this case. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

Superposition of yeast DNA polymerase and ssDNA substrate from Escherichia coli DNA polymerase Klenow fragment. Yeast DNA polymerase epsilon (yellow) and bound dsDNA (magenta) with ssDNA substrate (red, green, blue, orange) from E. coli DNA polymerase Klenow fragment superimposed onto the exonuclease domain of the yeast DNA polymerase. The magnification shows a slice through the polE structure (4M8O.pdb) superimposed with the ssDNA (red, green, blue, orange) in the exo‐site from the structure of the Klenow fragment (18Y.pdb). The side chains of the Lys425 and the Leu424 residues are shown in red and green, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]