Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant

Abstract

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Figure 1

GAPPS Pedigrees Abbreviations are as follows: POSITIVE/neg, APC promoter 1B mutation status by Sanger sequencing; WES, whole-exome sequencing; WGS, whole-genome sequencing by HiSeq or Complete Genomics; WGS-X Ten, whole-genome sequencing by X Ten. Only affected individuals and carriers are shown. (A) Family 1. Individuals and clinical presentation are as follows: I-4, unconfirmed gastric cancer; II-2, normal endoscopy aged 72 years; II-4, normal endoscopy aged 77 years; II-6, no endoscopy; II-11, normal endoscopy aged 68 years; III-33, normal endoscopy aged 60 years; IV-24, normal endoscopy aged 42 years. Thirty or fewer fundic gland polyps were observed in these non-carriers: III-22, 17 FGPs; III-23, 15 FGPs; IV-13, 30 FGPs; III-21, 7 FGPs; III-25, 25 FGPs. In two polyps from individuals III-7 and IV-4 where parental origin could be evaluated, the wild-type haplotype was lost. Asterisk (^∗) indicates sample was included in the linkage analysis. (B) Family 2. I-4, non-melanoma skin cancer, unconfirmed colon or gastric cancer; III-21, thyroid cancer (and fundic gland polyposis). (C) Family 3. I-2, leukemia; I-3, brain cancer; I-5, antral gastric cancer, no gastric polyps; I-6, prostate cancer; I-7, lung cancer. (D) Family 4. (E) Family 5. (F) Family 6. II-1, prostate cancer; II-2, ovarian cancer and unconfirmed colon cancer; II-3, unconfirmed colon cancer; III-1, unconfirmed colon cancer; IV-21, bone cancer, gastro-intestinal cancer.

Figure 2

Genomic Copy-Number Changes of Six Gastric Fundic Gland Polyps Whole-genome copy-number plots from copy-number analysis of each polyp represented by log R ratio and B allele frequency. Deviation of B allele frequency suggests allelic imbalances and copy-number variation at 5q. Genomic SNP data of the remaining eight polyps showing no LOH are presented in Figure S2.

Figure 3

Schematic of the APC 1A and 1B Promoters Showing the Location of Deletions Described in FAP and Point Mutations in GAPPS Black bars denote large deletions in FAP-affected families reported by Pavicic et al., Rohlin et al., Snow et al., Kadiyska et al., and Lin et al. “RefSeq Genes” track shows APC 1A transcripts (GenBank: NM_000038 and NM_001127510) and APC 1B transcript (GenBank: NM_001127511). Bottom part depicts the sizes of two promoters, the distance in between, and point mutations in the families.

Figure 4

GAPPS and FAP Mutations Alter YY1 Binding to the APC Promoter 1B (A) Position weight matrix (PWM) of YY1 binding. c.−195A>C (g.112043220C), c.−192A>G (g.112043223G) and c.−192A>T (g.112043223T), c.−191T>C (g.112043224C), and c.−190G>A (g.112043225A) in the APC promoter 1B are predicted to disrupt YY1 binding at positions 1, 4, 5, and 6, respectively. (B) ChIP-qPCR on the APC promoter 1B region in AGS and MKN74 cells. ChIP assays were performed with a YY1 antibody or IgG control antibody on the region of 1B containing the predicted YY1 binding site. A negative control region was used to represent nonspecific binding. One of two biological replicates is shown. Error bars denote SD. (C and D) EMSA of promoter 1B using with a biotinylated DNA duplex representing the predicted YY1 binding region with and without (WT) GAPPS mutations c.−195A>C, c.−192A>G, and c.−191T>C (C) and FAP mutations c.−190G>A and c.−192A>T (D). (E) EMSA-supershift using the WT DNA duplex and a polyclonal antibody against YY1 with AGS nuclear extracts. Rabbit IgG was used as a negative control. The black arrowhead denotes the YY1 supershifted complex.

Figure 5

Mutations in APC Promoter 1B Show Decreased Transcriptional Activity in Gastric Carcinoma Cell Lines AGS and MKN74 and Colorectal Cancer Cell Lines HCT116 and RKO Error bars denote 95% confidence intervals from three independent experiments performed in triplicate. p values were determined by two-way ANOVA followed by Dunnett’s multiple comparisons test (^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 compared to wild-type pGL3-APC 1B construct) on log transformed data; for ease of interpretation, back-transformed data have been graphed.

Figure 6

Sanger Sequencing Showing Allelic Imbalance in Mutation Carriers from Families 1 and 3 (A) Allelic imbalance in blood from affected subject (IV-29) and unaffected carrier (II-11) from family 1 using SNP rs448475 as a marker. (B) Allelic imbalance in saliva from affected carrier (II-3) from family 3 using rs2229992 as a marker.