Contribution of APC and MUTYH mutations to familial adenomatous polyposis susceptibility in Hungary

Abstract

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome with considerable genetic and phenotypic heterogeneity, defined by the development of multiple adenomas throughout the colorectum. FAP is caused either by monoallelic mutations in the adenomatous polyposis coli gene APC, or by biallelic germline mutations of MUTYH, this latter usually presenting with milder phenotype. The aim of the present study was to characterize the genotype and phenotype of Hungarian FAP patients. Mutation screening of 87 unrelated probands from FAP families (21 of them presented as the attenuated variant of the disease, showing <100 polyps) was performed using DNA sequencing and multiplex ligation-dependent probe amplification. Twenty-four different pathogenic mutations in APC were identified in 65 patients (75 %), including nine cases (37.5 %) with large genomic alterations. Twelve of the point mutations were novel. In addition, APC-negative samples were also tested for MUTYH mutations and we were able to identify biallelic pathogenic mutations in 23 % of these cases (5/22). Correlations between the localization of APC mutations and the clinical manifestations of the disease were observed, cases with a mutation in the codon 1200-1400 region showing earlier age of disease onset (p < 0.003). There were only a few, but definitive dissimilarities between APC- and MUTYH-associated FAP in our cohort: the age at onset of polyposis was significantly delayed for biallelic MUTYH mutation carriers as compared to patients with an APC mutation. Our data represent the first comprehensive study delineating the mutation spectra of both APC and MUTYH in Hungarian FAP families, and underscore the overlap between the clinical characteristics of APC- and MUTYH-associated phenotypes, necessitating a more appropriate clinical characterization of FAP families.

Keywords: APC; Colorectal cancer; Familial adenomatous polyposis; Genotype–phenotype correlations; Germline mutations; MUTYH.

Fig. 1

Identification and characterization of the large deletion (c.1627-185_1958+651del7146insGATCCT) in case HFC220. a MLPA analysis results of a heterozygous deletion removing exon 13 and 14 of the APC gene. The electropherogram of the patient (red) is superimposed on that of a control sample (blue). The peaks showing 50 % reduction of intensity are marked by red diamonds. The agarose gel image of the amplification product with primers located in intron 12 and intron 14 is shown on b. PCR was performed using the Multiplex PCR Kit (Qiagen) with short extension time (3.5 min), so the 9866 bp long normal fragment cannot be amplified (empty control lane). Case HFC220 shows a PCR product of approximately 3000 bp. The DRIgest III (GE Healthcare) molecular weight marker (MW) was used for this experiment, the 4.36, 2.32 and 2.03 kb fragments are indicated by arrows on the left side of the gel image. c The sequencing results of the above PCR product with a nested primer, showing the nucleotide sequence around the breakpoints. Red nucleotides are non-templated insertions, light grey letters are applied to mark deleted nucleotides. Grey arrows on top and below the sequences show the orientation of the Alu elements involved in the deletion. (Color figure online)

Fig. 2

Approximate localization of large genomic deletions by robust dosage PCR (RD-PCR). a Examples of copy number determination at several regions in duplex and triplex RD-PCR assays. The electropherograms of the patient (case HFC208, red) were superimposed on those of a control sample (blue), and shifted slightly to the right for better visibility. The heights of the cntrol peaks (C) were adjusted to the same level. The deletion for a given amplicon (names given under each peak, reflecting the position of the given marker) is seen as a ~50 % reduction of peak intensity (red diamonds). b Diagram showing the approximate size of two deletions (case HFC106: upper; and HFC208: lower). The distance of the markers from the APC gene is given on the X axis (in kilobases, using negative numbers for upstream and positive numbers for downstream markers), while Y axis indicates the copy numbers normalized for the average of three control samples. A 40–60 % reduction for a given marker indicates the presence of the heterozygous deletion (red bars). Not all samples were tested for all positions. (Color figure online)

Fig. 3

Genotype–phenotype correlations for substitution and small indels of the APC gene. The age of disease onset is shown in relation to the position of the mutated codon. For splice site mutations resulting in exon skipping, the last codon of the previous exon was used. To demonstrate genotype–phenotype correlations, mutations found in AFAP cases are depicted as empty symbols (clustering near the 5′ part of the gene), while the mutations of patients diagnosed with Gardner syndrome are shown as triangles (mostly after codon 1400). Red symbols are used to highlight the mutations within the codon 1200–1400 region, their carriers showing a significantly reduced age at disease onset as compared to the carriers of mutations located elsewhere in the gene. (Color figure online)

Fig. 4

Germline large deletions extending over the boundaries of the APC gene. The localization of the known RefSeq genes of the chromosome 5 region 104,000,000–114,000,000 (coordinates are given according to the GRCh37/gh19 chromosome assembly) are shown schematically. The minimal and maximal sizes of the large genomic deletions are indicated for our six samples as red and pink bars, respectively. The loci where copy number analyses were done (RD-PCR markers) are shown on the left, their names reflecting their localization with respect to the coding portion of the APC gene. APC*: the 5′–30 kb RD-PCR marker is located in the first non-coding exon of APC. Markers without gene names are in intergenic regions. (Color figure online)