Familial Cancer 1: 3–7, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Profuse familial adenomatous polyposis with an APC exon 3 mutation Steven Nasioulas1, Ian T. Jones2, D. James B. St. John3, Rodney J. Scott4, Susan M. Forrest1 and R. J. McKinlay Gardner1 1 Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Australia; 2 Department of Surgery and 3 Department of Gastroenterology, The Royal Melbourne Hospital, Melbourne, Australia; 4 Pathology Service, John Hunter Hospital, Newcastle, Australia Received 20 January 2000; accepted in revised form 14 May 2000
Key words: APC gene mutation, familial adenomatous polyposis
Abstract The attenuated form of familial adenomatous polyposis coli (AAPC) is associated with mutations in the adenomatous polyposis coli (APC) gene which cluster in the 5′ region of the gene. It has been proposed that a ‘genotype–phenotype boundary’ exists at codons 159–163, and mutations that are 5′ of this boundary will produce AAPC. Herein we document a three-generation family with an exon 3 mutation well to the 5′ side of the proposed boundary, in which two affected individuals have had, in their 40s, a profuse form of familial adenomatous polyposis coli. We conclude that the codon 159–163 ‘boundary’ is indicative rather than definitive. These two patients also had postoperative intra-abdominal adhesions, severely so in one.
Introduction In the attenuated form of familial adenomatous polyposis (AAPC) there is only a small number of colorectal polyps, generally fewer than one hundred, and there is a later onset of malignant change, compared with typical familial adenomatous polyposis (FAP). Lynch and Smyrk have reviewed the difficulties in the phenotypic subclassification of FAP, and suggest that although the proposition of a spectrum from very attenuated through attenuated, sparse, classic, and profuse polyposis may be somewhat arbitrary, such divisions may be relevant for clinical management [1]. Nagase et al. [2] made the initial observation that site of mutation correlated with the degree of polyposis. Subsequently, others have recorded an association of AAPC with mutations in exons 3–4 and in the splice sites of intron 3 in the extreme 5′ region of the gene [3–11], in exon 6 [12], at the splice site of [13] or within [9, 14] the alternatively spliced exon 9, in the 3′ end of the gene [15–20], and with a deletion of the whole gene, cytogenetically detectable [21]. With respect to the extreme 5′ APC mutations, localised within exons 3 and 4, it was originally proposed that there existed a specific ‘phenotypic boundary’ between AAPC and FAP, residing in codons 157–168 of exon 4 [3]. This was
further refined by Marshall et al. [7] to be between codons 159 and 168 and by Walon et al. [22] to codons 159–163. We report a family in which the observations may somewhat undermine the concept of this AAPC/FAP boundary. We have identified an exon 3 mutation, at codons 116–118, which is some 40 codons upstream of the boundary; and yet two affected individuals presented (admittedly in adult age) with a profuse FAP phenotype. Apparently, the AAPC/FAP boundary is not absolute, and other genetic or epigenetic factors can influence disease expression.
Case reports Patient 1 is a male now 69 years old, who had presented with abdominal symptoms at age 45 years. At surgery at that time, two synchronous cancers had been identified, both well-differentiated Dukes stage A adenocarcinomas, one 20 cm from the ileocaecal valve, and the other in the sigmoid colon. There were innumerable sessile and pedunculated polyps, from 0.2 to 4.5 cm in diameter. The maximum concentration was in the ascending and proximal transverse colon, and in the sigmoid colon and proximal rectum, where almost the entire bowel surface was recorded as being covered
Correspondence to: R.J.M. Gardner, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia 3052; Tel: +61-3-8341-6293; Fax: +61-3-8341-6390; E-mail:
[email protected]
4 with polyps. There were no polyps in the terminal ileum. Some 5 months thereafter, he presented with a small bowel obstruction, and required surgery for the division of adhesions. At age 48, a leiomyoma of the scrotum was removed. Latterly, he provided a blood sample to enable a mutation search of the APC gene. He himself had been adopted, but had recently made contact with his elderly biological mother, and she was not aware of any history of bowel cancer in her own family. Patient 2, the son of patient 1, presented to our service for predictive gene testing at the age of 42 years, and received a mutation-positive result. Colonoscopy at this time showed dense polyposis throughout the colon, with a moderate number of small polyps in the rectum. After discussing the options available to him, he elected to have total colectomy with ileorectal anastomosis. The operative specimen comprised 1100 mm of the colon, from caecum to sigmoid, and 30 mm of the terminal ileum. The entire colonic mucosa was covered with sessile and pedunculated polyps, from 1 to 25 mm in size, numbering greater than 900. The polyps were particularly dense in the transverse and sigmoid colon. There was no macroscopic invasion by tumour, and on microscopy, no stromal invasion. The polyps showed mild to moderate and occasionally severe dysplastic change. Five days postoperatively he developed a small bowel obstruction, and came to laparotomy on the 16th post-operative day, which revealed an obliterative peritonitis due to dense intra-abdominal adhesions. The greater omentum and small bowel were completely adherent en masse. After prolonged dissection, the parietal peritoneum could be separated from this mass, but the operation was then stopped due to concern for a risk of small bowel injury. After several days, the obstruction spontaneously resolved. He has remained well at follow-up: some rectal polyps were destroyed 11/2 years following surgery, but none was seen at 3 years. Patient 3 is aged 15 years, and is the second of the three daughters of patient 2. These three children had undergone predictive testing at the ages of 15, 13 and 11 respectively, following parental consent and with individual counselling of the girls, and patient 3 had the mutation. Limited study by sigmoidoscopy at age 14 was normal, but a colonoscopy at age 15 showed about 50 diminutive (less than 5 mm diameter) polyps throughout the colon, although relatively fewer in the rectum.
Materials and methods Blood samples were drawn from family members and lymphoblastoid cell lines established, in order to isolate RNA for the synthesis of cDNA. The protein truncation test (PTT) required the amplification by PCR of the hexamer-primed cDNA. 5 µl of the reaction mix was used with a final concentration of 1.5 mM MgCl2, 2 mM dNTPs (deoxynucleoside triphosphates), and 1 unit of
Taq polymerase in a total volume of 50 µl, with standard cycling conditions, for amplification of codons 1–811 (exons 1 to 14 and the first part of exon 15) [23]. The PTT was based on the method of Roest et al. [24] using the TnT T7 coupled reticulocyte system essentially according to the manufacturer’s conditions (Promega, Sydney). For sequencing of the mutation, exon 3 (250 bp) was amplified under standard PCR conditions, using published primers [25]. The PCR conditions were as follows: 3.5 mM MgCl2, 2 mM dNTPs, 1 unit Taq polymerase, 120 ng DNA. The total PCR product was excised from the gel and purified with Qiagen (Adelaide) spin columns. Sequencing was done using a Sequenase version 2.0 T7 DNA polymerase kit (Amersham, Sydney).
Results The translated products derived from the first 14 exons of the APC gene in the sample from patient 1 comprised the expected product, along with a small and faint prematurely terminated product of approximately 16 kiloDaltons (data not shown), which indicated a premature truncation in the region of exon 3. Sequencing of exon 3 demonstrated a 5 bp deletion. The mutation comprised the deletion of the sequence TTCAT at nucleotides 348–352 inclusive, encompassing codons 116–118. A stop codon is created seven amino acids downstream, which causes the premature termination of protein synthesis. The deletion is demonstrable upon electrophoresis (10% polyacrylamide) of the MboII digested exon 3 PCR product: the heterozygote shows 143 bp and 148 bp fragments, as well as a distinct heteroduplex band (Figure 1).
Discussion The clinical categorization of the degree of polyposis in FAP, attenuated versus typical, may seem simple if a boundary is established at the number of 100 polyps: less than 100 defines AAPC, while in typical FAP there are more than 100. The issue becomes more complex if age and site within the colon are taken into account. In our own material, might the two affected men, first assessed by us in their 40s, have had an attenuated phenotype in youth, which could never have proceeded to the profuse picture of their later age if they had had timely prophylactic surgery? Does the relative rectal sparing disqualify them from a label of ‘typical’ FAP? – at any rate, with innumerable polyps more proximally, a designation of ‘profuse’ colonic polyposis is not unjustified. Patient 3, at her present young age of 15 years, can be described as having AAPC; we expect that prophylactic surgery will deny much opportunity further to observe the natural history. Her case is to be contrasted with, for example, the codon 1309 mutation, which is particularly associated with a profuse pancolic
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Figure 1. Family tree with patients 1, 2 and 3 indicated above their own lanes on a DNA gel. The bands in the gel are due to DNA segments from part of exon 3 of the APC gene. The normal APC gene gives a segment size of 148 bp, and the family mutation, with a deletion of 5 bp within this part of exon 3, gives a segment size of 143 bp. Those who carry the abnormal gene also display an extra band due to a heteroduplex (HD) formed from one strand of mutant and one strand of normal DNA.
polyposis, and which can present even in early childhood [26]. It may be that in some cases of AAPC there is an evolution over time, and more so in the proximal than the distal colon, into a profuse polyposis. This proposition would be consistent with the data presented in [11], a large kindred having an intron 3 mutation (vide infra). In the presently reported family, the mutation is well upstream of the codon 159–163 AAPC/FAP phenotypic boundary, and in fact is the fourth-most 5′ mutation ever recorded (Table 1 and Figure 2). A small number of other examples are on record of a profuse FAP phenotype in association with a mutation 5′ of the boundary. We set out in Table 1 the reported extreme 5′ APC mutations (exons 3 and 4), noting whether the phenotype is an ‘atypical’ polyposis (AAPC) or ‘typical’ polyposis (FAP), and depict the positions of the different mutations in Figure 2. As a rule, the atypical phenotypes have had mutations at or preceding codon 159, and mutations at codon 163 and beyond in the remainder of exon 4 have been associated with typical phenotypes. It was from these observations that the original proposition arose of a functional boundary in this vicinity. Exceptions exist in the following cases, along with the family we report here. One of the mutations reported in Dobbie et al. [4] originally described at codon 151 and associated with an atypical phenotype, is in fact at codon 173, and this is some 10 codons 3′ of the codon 159–163 phenotypic boundary. Similarly, the codon 168 mutation in [10] is beyond the boundary, by 5 codons, and the phenotype is that of AAPC. The codon 163
Table 1. Those 5′ APC mutations from the literature in which the associated FAP phenotype is recorded. The dividing line identifies the codon 159–163 AAPC/FAP boundary that has been proposed. The ‘exceptions’ to the genotype–phenotype rule are annotated a–f, as they also are in Figure 2. Phenotypea
Codon
Exon
Mutation
Stop codonb
Reference
AAPC AAPC AAPC FAP AAPC AAPC/FAP AAPC AAPC AAPC FAP AAPC AAPC
78–79 99 101 116–118 121 141 141 141–177c 142–143 157 157 159
3 3 3 3 3 intron 3 intron 3 4 4 4 4 4
232–235 delGATA 295 C → T (missense) 301 G → T 348–352 delTTCAT 361 A → T g → a splice acceptor t → a splice donor del exon 4 426–427 delAT 469 insT 470 G → A 477 C → G
85–86 – 101 137–138 121 184–185 184–185 184–185 147–148 167 157 159
[3] [4, 5] [10] Present report (a) [8] [10, 11] (b) [3] [9] [3, 6] [22] (c) [3] [7]
AAPC AAPC FAP FAP FAP AAPC
163 168 168–169 169–170 172 173d
4 4 4 4 4 4
487 C → T 502 A → T 504–507 delAATA 507–510 delATAG 515 insT (517–518 delC)
163 168 174–175 174–175 175–176 174–175
[9] (d) [10] (e) [33] [4, 5] [8] [4] (f )
Codon/nucleotide numbering according to Joslyn et al., 1991 [34]. AAPC = atypical FAP, FAP = typical FAP. b As deduced from the cDNA sequence given in Joslyn et al., 1991 [34]. c Three kindreds with this mutation. d Originally published as the deletion of a cytosine in codon 151. a
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Figure 2. Positions of recorded 5′ mutations in the APC gene according to phenotype, atypical polyposis (AAPC) or typical FAP (detail in Table 1), one symbol per kindred. The half-filled symbol at codon 141 (b) is for the kindred in [11], in which both attenuated and typical FAP was observed. The segment of the APC gene from codons 78–180 is shown. The putative AAPC/FAP boundary at codons 159–163 is indicated as the dashed line. The 3′ extent of the homodimerisation domain at codon 171 is marked x. The cases which do not follow the ‘rule’ of mutation location according to phenotype are indicated a (present family), b (kindred of [11]), c (families 22, 32 in [22]), d (kindreds 1–3 in [9]), e (case 3 of [10]), and f (case B of [4], and as per corrigendum, R.J.S.).
mutation seen in [9], with AAPC, actually abuts the 3′ extent of the boundary. The recently reported kindred of [11] with an intron 3 splice acceptor mutation has the particular merits of being large (90 individuals studied), and including more than one endoscopic observation over a period of time in some persons. From this vantage point, they were able to record polyp numbers from single figures through to hundreds, and in just three individuals there were greater than a thousand. This variability in clinical expression led them to comment on the ‘need for defining AAPC in the context of a familial phenotype rather than on an individual basis’. While some doubt therefore arises with respect to a clear distinction of molecular-phenotypic correlation, it remains true that most exon 3 and 4 mutations which are at or 5′ of codon 159 lead to an attenuated phenotype, as shown in Figure 2, and that most exon 4 mutations which are 3′ of codon 163 produce a typical phenotype. The ‘rule’ that extreme 5′ mutations necessarily determine a milder phenotype does not hold absolutely, and the ‘boundary’ in the region of codons 159–163 is indicative rather than definitive. The age at which the phenotype is documented is also to be considered. The underlying biological basis for these phenotypic differences within the spectrum of recorded 5′ APC mutations remains to be defined. A most remarkable hypothesis has recently been put forward, in which it is proposed that a particular quality of an AAPCassociated abnormal allele actually enables it to influence the nature of the somatic mutation on the other chromosome, or within itself [27, 28]. In AAPC (in contrast to the generality of polypogenesis) it is the germline abnormal allele which is typically lost, with
retention of the normal chromosome, and with a somatic mutation having occurred in this (initially normal) APC gene. Only a limited repertoire of somatic mutations would, as a rule, be ‘permitted’, and those which might have a more powerful dominant-negative polypogenic property are not generated. More conventional explanations for the ‘attenuating’ effects of 5′ mutations include the following. Firstly, it may be a common property of 5′ mutations that they exert their effect via a mechanism of haplo-insufficiency, and this is a mechanism that a 5′ mutation would promote [29]. Secondly, the alternative splicing of exons in the 5′ part of the gene may, in some, moderate the effect of the mutation, by deleting that part of the genetic message which contains it [13, 14, 30]. Thirdly, other loci may influence the expression of the APC locus, and, for example, Dobbie et al. have identified a candidate modifying locus at 1p35–36 [31]. Fourthly, the gene product of some mutations may be prone to the ‘rescuing’ influence of nonsense-mediated mRNA decay, a physiological process that removes mRNA which contains premature stop codons. Finally, any mutation is entitled to have its own very specific effect (the desmoid-inducing codon 1924 mutation being a striking example [32]), for which no clear explanation can presently be offered. Concerning the history of post-operative adhesions in patients 1 and 2, it is the experience of surgeons familiar with operating upon patients with FAP that dense intra-abdominal adhesions are not uncommonly observed when repeat surgery is undertaken (V. Fazio, pers. comm.). The question arises whether the pathogenesis of this readily provoked adhesion process might be related to that which determines susceptibility to desmoid disease, albeit this complication is typically a 3′ APC mutation association.
Acknowledgements We thank MaryAnne Young, Genetic Counsellor, and Dorothy Weigall, Registrar of the Esso Victorian FAP Register, for their contributions to this study. Dr Kendall Francis provided historical clinical information concerning patient 1. The study of this family was presented at the 2nd joint meeting of the Leeds Castle Polyposis Group and the International Collaborative Group for Hereditary Non-Polyposis Colorectal Cancer at Lorne, Victoria, Australia in March 1999.
Addendum A recently published Japanese series [35] documents three FAP patients having a germline mutation within the exon 3–4 region of present interest, at codons 157, 169–171, and 175 respectively. The latter two mutations are 3′ of the codon 159–163 ‘boundary’. All three patients showed an AAPC phenotype.
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