European Journal of Human Genetics (2002) 10, 505 – 510 ª 2002 Nature Publishing Group All rights reserved 1018 – 4813/02 $25.00 www.nature.com/ejhg ARTICLE Pathogenic mutations and rare variants of the APC gene identified in 75 Belgian patients with familial adenomatous polyposis by fluorescent enzymatic mutation detection (EMD) Genevie`ve Michils1, Sabine Tejpar1,2, Jean-Pierre Fryns1, Eric Legius1, Eric Van Cutsem2, Jean-Jacques Cassiman1 and Gert Matthijs*,1 1Center for Human Genetics, University of Leuven, Leuven, Belgium; 2Department of Gastro-enterology, University of Leuven, Leuven, Belgium Familial adenomatous polyposis (FAP) is a dominant inherited colorectal cancer syndrome which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Enzymatic mutation detection (EMD) has potential advantages over the standard protein truncation test (PTT) that is currently used in screening the APC gene for mutations. First we wanted to validate the EMD technique in comparison to PTT. Secondly, we wanted to develop an efficient working protocol for EMD screening of APC. Seventy-five unrelated patients were screened for mutations. All mutations that had previously been detected by PTT were also identified by EMD; the sizes of the cleavage fragments were as expected according to the position of the mutations within the amplicons. A new screening strategy based on EMD allows the analysis of the APC gene in 31 overlapping PCR fragments. In total, EMD efficiently detected the 26 truncating mutations in this series. In addition, two rare variants were also detected: the first is the typical Ashkenazi missense mutation I1307K while the second variant, E1317Q, has been identifed in Belgian patients and controls, and should no longer be considered as a pathogenic mutation, but rather classified as a polymorphism. European Journal of Human Genetics (2002) 10, 505 – 510. doi:10.1038/sj.ejhg.5200825 Keywords: enzymatic mutation detection; EMD; variants; hypermutable; FAP; adenomatous polyposis coli (APC); protein truncation test Introduction Familial adenomatous polyposis (FAP) is an autosomal 99, appearing at a later age and being still associated with dominant disorder characterised by the development of at an increased risk of cancer.1 The profuse phenotype is asso- least 100 colorectal adenomas at a young age (before 20 ciated with the appearance of more than five thousand years), some of which will progress to colorectal cancer polyps, whereas patients with the classical sparse phenotype one or two decades later. In addition to the classical form eventually develop one to two thousand polyps. The adeno- of FAP, an attenuated form was described: attenuated FAP matous polyposis coli (APC) gene was discovered in 1991,2,3 (AFAP) or attenuated adenomatous polyposis coli (AAPC), and the germline mutation spectrum is generally spread in which the number of polyps ranges between 10 and over its entire coding region. Hot spot regions for somatic mutations in colorectal tumors (mutation cluster region (MCR) in exon 15),4 and in desmoids (middle part of exon *Correspondence: G Matthijs, Center for Human Genetics, UZ Gasthuis- 15)5 are well characterised. The positions of the germline berg, Herestraat 49, B-3000 Leuven, Belgium. Tel: +32 16 346070; Fax: +32 16 346060; E-mail: [email protected] mutations correlate with the severity of the disease. For Received 23 July 2001; revised 18 April 2002; accepted 23 April 2002 example, in classical FAP, mutations are located between Enzymatic mutation detection for APC screening GMichilset al 506 codons 213 – 1249, whereas in the profuse phenotype muta- to the samples and incubation for 30 min at 378C followed. tions are located between codons 1250 – 1464.6 Mutations All the incubation steps were performed in a standard ther- in exon 9 and at the 5’ and the 3’ end of APC are also mocycler. EMD products were loaded on a 6% Long Ranger responsible for AFAP.7–9 Known germline and somatic Urea gel (SanverTech) and detected with an ALF DNA mutations can be found at the internet site address: sequencer (Amersham Pharmacia Biotech). PTT analysis of http://perso.curie.fr/Thierry.Soussi/APC.html#Ancrage3. exon 15 has been performed as described.11 Due to the extended mutation spectrum of the APC gene, Exon 6 has been screened for all patients using Denatur- and the size of the gene itself, there is a need for an effi- ing Gradient Gel Electrophoresis (DGGE) according to cient and sensitive mutation detection method. Currently Olschwang et al.18 the protein truncation test (PTT) is the technique most widely used to screen for mutations in the APC gene.10,11 Results However, PTT only allows the detection of truncating muta- PTT was initially used for the APC exon 15 screening of a tions. While over 90% of pathogenic mutations detected in series of 40 patients. It has been performed in four overlap- classic FAP are truncating mutations, missense mutations, ping fragments. In fifteen out of the 40 patients, a polymorphisms and variants of unknown significance that truncated peptide was detected; sequencing revealed ten have been reported are spread over the whole gene. The different alterations that were all localized in the first half majority of these would not have been detected by PTT of exon 15. (e.g. E1317Q, S1971R, A2119V).12,13 For comparisons, these samples were submitted to EMD We wanted to evaluate the feasibility and efficiency of in amplicons of several sizes, ranging from 277 to 910 bp. enzymatic mutation detection (EMD) screening of the The size of the cleavage peaks could be calculated according APC gene. This is potentially an easy, rapid and accurate to the position of the mutation within the tested amplicon. method that has the advantage of detecting all types of As both primers were labelled, two peaks were expected for mutations.14 EMD is based on the properties of the T4 detection after cleavage. All peaks were positively detected endonuclease VII that has the ability to detect mismatches and some mutations were observed in overlapping ampli- in a double-stranded DNA molecule and to cleave the DNA cons (Table 1). strand at this locus.15 The cleavage products can easily be The samples showing a negative PTT result have been detected after electrophoresis. EMD has been originally further analysed by EMD of the exons 1 – 14. A second developed from the enzymatic mismatch cleavage method series of 35 patients, which had not been previously tested (EMC),16 and has been improved to allow extended screen- ing. In this study, we aimed to validate EMD by comparing it Table 1 Germline mutations of the APC gene identified in to PTT. In addition, we established an efficacious screening Belgian FAP patients protocol which allows rapid scanning of all exons of APC. Exon Detection Mutation Codon (fragment) method Patient Subjects and methods DNA from clinically affected FAP or AFAP patients R216X 216 6 DGGE 187832 submitted for molecular diagnostic analysis was used for R232X 232 6 DGGE 191930 R302X 302 8 EMD 186794 this study. Lymphocyte DNA from patients and controls 1087 ins A 357 9 EMD 202032 was prepared using standard salt precipitations. S457X 457 10 EMD 179129 For EMD analysis of exon 15, amplicons of different sizes 1494 del C 491 11 EMD 207230 R499X 499 11 EMD 151913; 178620 were generated using several combinations of described 2128 del G 704 15-ac EMD 110175 2 primers. In addition, a few new primers were designed: c3 Y935X 935 15-ac/c3d PTT – EMD 38378 (forward) 5’-CACAAGCAAAGTCTCTATGGTG, h2 (reverse) 2911 del AATA 966 15-c3d/de PTT – EMD 30913 5’-TTTCTGCCTCTTTCTCTTGG, i3 (forward) 5’-ACCAAGA- 3104 del TT 1028 15-c3d/de PTT – EMD 175491 3201 del ACAAA 1061 15-de EMD 153316 GAAAGAGGCAGAA. The amplicons were : a-c, c3-d, d-e, f- Q1175X 1175 15-ff EMD 228789 f, g-h, h-h2, i3-i, j-k, k-l, m-n, o-p, q-r, s-t, u-v, and v-w. S1194X 1194 15-ff PTT – EMD 180798 The remaining 16 segments of APC (1 – 9, 10, 11 – 14, and 3614 ins A 1199 15-ff PTT – EMD 35963 3945 del AAAGA 1309 15-gh PTT – EMD 167202; 167877; the alternative spliced exons 9A and 10A) were amplified 2,17 163927; 185236; as described. For each amplicon, both forward and 185266; 216937 reverse primers were labelled with fluorescein isothiocya- 4057 GC?A 1347 15-gh PTT – EMD 193859 nate (FITC). Fluorescently labelled PCR fragments were 4411 del AG 1465 15-gh/hh2 PTT – EMD 187805 4439 del C 1474 15-gh/hh2 PTT – EMD 177751 submitted to the EMD reaction with the Passport kit for 4630 del GA 1538 15-hh2 PTT – EMD 101206 ABI 377 detection (Amersham Pharmacia Biotech). Dena- Numbering of nucleotides (nt) and codons is related to the turation (958C, 4 min) was followed by a cooling step GenBank file M74088 (nt 1 in position 1 of cDNA; codon 1 is first from 958Cto378C at 0.028C/s. Enzyme was then added ATG at nucleotide 19 of cDNA). European Journal of Human Genetics Enzymatic mutation detection for APC screening G Michils et al 507 by PTT, was also submitted to EMD according to the presented protocol (see Subjects and methods). In total, 26 pathogenic APC mutations were identified in 75 FAP or AFAP patients (35%). They represent twenty different DNA alterations (nine deletions, two insertions, eight nonsense mutations and one GC?A mutation) (Table 1). The mutations were located in exons 6, 8, 9, 10, 11, and in fragments a to i of exon 15. Six patients carried the same 5 bp deletion at codon 1309. This particular mutation represents nearly a quarter of all positive samples in this study.