Accepted: 29 November 2001Published online: 2 February 2002© Springer-Verlag 2002

Abstract Background and aims:Familial adenomatous polyposis(FAP) is an autosomal dominantcondition characterized by multipleadenomatous polyps in the colon andrectum that inevitably develop intoadenocarcinomas if the patient’s co-lon is not removed in time. To datemore than 500 mutations related tothe disease have been identified inthe APC (adenomatous polyposis coli) gene. The molecular study ofFAP families was initially intro-du-ced in Cuba with the aim of iden-tifying the asymptomatic carriers ofAPC gene mutations in each family.Patients and methods: We studied 23individuals from 17 Cuban familieswho had been diagnosed clinicallywith FAP. Peripheral DNA was ex-tracted from the index case of eachfamily. Exon 15 of the APC genewas screened for germinal mutations using PCR and DNA heteroduplex.

Results: Three different germinalmutations were identified in the mu-tational clustering region of APCgene by sequencing analysis in fiveFAP unrelated families. Three fami-lies carry the most frequent mutationin APC in codon 1309, while the other two families carry mutations incodons 1061 and 1192, respectively.Two asymptomatic carriers of onefamily were detected, and later thedisease was confirmed by colonos-copy in a very early stage while sixmembers at risk were found to benegative. Conclusion: For the firsttime in Cuba molecular diagnosis ofFAP was performed and the develop-ment of colorectal cancer preventedin asymptomatic carriers.

Keywords Familial adenomatouspolyposis · APC gene · Mutation detection · At-risk relatives · Molecular diagnosis

Int J Colorectal Dis (2002) 17:344–347DOI 10.1007/s00384-001-0385-0 O R I G I N A L A RT I C L E

Diana Cruz-BustilloLuis VillasanaFrancisco LlorenteDamián CasadesúsElsa GarcíaPetros SyrrisNick CarterRosa GuerraRaimundo Llanio

Preliminary results of the molecular diagnosisof familial adenomatous polyposis in Cuban families

Introduction

Familial adenomatous polyposis (FAP) is an autosomaldominant syndrome with an almost complete penetrancethat occurs in the general population with a frequency of1/8,000–1/13,500 [1]. In a typical case of FAP the patientdevelops hundreds to thousands of adenomatous polypsin the colon and rectum generally after the first decade oflife [1, 2, 3, 4, 5, 6]. The disease is caused by germlinemutations in the tumour suppressor gene APC (adenoma-tous polyposis coli) localized in 5q21. The patients whoinherit a mutation in APC have a very high probability

(>90%) of developing adenomas in their colon. The ade-nomas appear when the normal allele of APC is lost ormutated, resulting in the inactivation of the gene [7]. Theage at which adenomas appear is variable. Only 10% ofthe mutated gene carriers developed adenomas by10 years of age; by age 20, the probability raises to 75%,and by age 30 some 90% of these individuals express thephenotype of the disease. If the colon is not eliminated ontime, the majority of FAP patients develop colorectal can-cer by the fourth decade of life [4, 5, 6].

The majority of germinal mutations identified in FAPpatients are in the 5′ half of the APC gene [8, 9, 10, 11,

D. Cruz-Bustillo (✉ ) · E. García · R. LlanioNational Institute of Gastroenterology,Calle 25 no. 503 entre H e I, Havana 10400, Cubae-mail: dcruzb@infomed.sld.cuTel.: +53-537-325067Fax: +53-537-333253

L. Villasana · D. CasadesúsDepartment of Coloproctology, General Calixto García Hospital, Havana, Cuba

F. LlorenteDepartment of Proctology, Manuel Fajardo Hospital, Havana, Cuba

P. Syrris · N. CarterMedicine School, St. George’s Hospital,London, UK

R. GuerraGenetics Laboratory, National Institute of Neurology and Neurosurgery, Havana, Cuba

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12]. Approximately 23% of the mutations are clusteredin 300 codons within exon 15 of the APC gene [13].

When a germinal mutation is detected in a patient, theaffected members at risk in the family carrying the muta-tion can be predicted with almost 100% precision be-cause all the carriers within a given family carry exactlythe same mutation [7]. In this case asymptomatic carriersare frequently screened by colonoscopy while noncarri-ers may be discharged from the severe screening sincetheir risk of developing colorectal cancer is the same asthat of the general population [14].

In Cuba FAP patients are diagnosed clinically, endo-scopically (colonoscopy or recto-sigmoidoscopy) andhistologically (biopsy). The aim of this study was to de-tect mutations in exon 15 of the APC gene in FAP pa-tients in order to screen family members and preventcancer development in these individuals. This contribu-tion shows the first results of molecular diagnosis of fa-milial adenomatous polyposis in Cuban families.

Materials and methods

We studied 23 patients from 17 unrelated Cuban families clinical-ly diagnosed with FAP. After genetic counselling was providedand consent to be studied was obtained, the pedigree of the familywas determined, and 5 ml peripheral blood was extracted from thepatients. Genomic DNA was isolated from venous blood [15], anda single-step method based on heteroduplex formation was used todetect mutations in codons 1027–1384 (nucleotides 3082–4152) ofexon 15 of the APC gene [16]. The region amplified by theseprimers is the mutational cluster region (MCR) described for theAPC gene [17].

Whenever variants of the heteroduplex pattern were detectedin 8% nondenaturing polyacrylamide minigels for a FAP patient,the members at risk for that family were screened. A completescreening of exon 15 of the APC gene was made in noninforma-tive patients for the above primers. In this case previously de-scribed primers and methods [18] were used. The bands were visualized with silver nitrate or ethidium bromide staining. The se-quencing analysis used fluorescent BigDye chemistry according tothe manufacturer’s protocol in an ABI PRISM 377 DNA se-quencer from PE Applied Biosystems–Perkin Elmers [19].

Results

Of the 17 families 5 had unclear family histories, eitherbecause they did not know their parents’ age and causeof death, because they are still alive and healthy, or be-cause they died young before expressing the disease.Therefore the possibility of a de novo mutation cannot

be rejected. Two index cases had desmoid tumours; onewas diagnosed with congenital hypertrophy of the retinalpigment epithelium (CHRPE), and three members of an-other three families had benign brain tumours, which oc-curred without polyposis in two cases.

In 65% of the patients the first symptoms of the dis-ease appeared or the disease was diagnosed before theage of 30 years. Four of the six patients with onset be-fore the age of 20 years were diagnosed by colonoscopybecause they had ancestors who had died from colorectalcancer or FAP; one started with rectal bleeding, and thepresentation form of the sixth was unknown. The two pa-tients with the oldest age at onset (46 and 68 years, re-spectively) had not had surgery because they presented afew polyps that were eliminated by polypectomy. One ofthe parents of each of these individuals died of colorectalcancer at an elderly age (>75 years).

Exon 15 of the APC gene was completely screened in23 unrelated FAP patients. Germinal mutations were de-tected and identified in 5 of the 17 FAP families studied(29.4%). All the mutations were detected in the MCR ofthe APC gene. The sequencing analysis results areshown in Table 1 below. The identified mutations pro-duced truncated APC proteins in all cases. All patientswith mutations expressed the typical FAP phenotypewith severe polyposis, and the five had had colectomy.

In the five informative families the germinal mutationwas confirmed in several family members at risk. Theseindividuals had not previously been analysed and wereall asymptomatic. In family 1 the oldest son (22 yearsold) of the index case did not carry his father’s mutation,and he will therefore not express the disease as a resultof that mutation. A healthy aunt (50 years old) and twofirst cousins (32 and 23 years old, respectively) of the in-dex case in family 2 were studied; the oldest cousin hadrectal bleeding. The three were negative to the genetictest and the coloproctologist analysis of the bleeding in-dividual resulted in haemorrhoids and no FAP. Two ofthe four children of the index case in family 4 werefound to be FAP carriers and two were noncarriers. Thetwo carriers are still asymptomatic, and colonoscopy re-vealed the disease in its early stages in the youngest.

Discussion

The average age at onset was within the range often re-ported for classical FAP [1, 2, 3, 4, 5, 6]. All patients aged

Table 1 Germinal mutations identified in Cuban FAP families

Family Mutation Consequence

1 3615–3616 del CA; deletion 1192 2-bp deletion with frameshift and stop codon TAA 39 nucleotides downstream [20]2 3183–3187del ACAAA; deletion 1061 5-bp deletion with frameshift and stop codon TGA immediately downstream [8]3, 4, 5 3921–3925 del AAAAG; deletion 1309 5-bp deletion with frameshift and stop codon TAG 8 or 13 nucleotides downstream [8]

under 40 years expressed the typical phenotype of the dis-ease. Two index cases had later age at onset than the rest,and their phenotype was more benign. There is an attenu-ated form of the disease which has been related both to alater onset and to mutations in APC nearer the 5′ end ofthe gene [7, 21] than could be scanned in this study.

Numerous correlations have been made between thesite of the APC mutation and the phenotype of the dis-ease, and it has been confirmed that several extracolicforms of FAP are correlated with specific germinal muta-tions. The presence of benign tumours in the central ner-vous system is a well known variant of the disease calledTurcot syndrome. These patients express the polyp clas-sical phenotype together with meduloblastomas when themutations are mainly in the APC gene and with glio-blastomas when the mutations occur in the mismatch er-ror repair genes [7]. Until now we have not found muta-tions associated with benign central nervous system tu-mours in our patients or their at-risk relatives.

In Gardner syndrome the presence of desmoid tu-mours in patients with polyposis is associated with muta-tions in APC [22]. Mutations downstream from exon 9 ofAPC gene have been associated with CHRPE [21], a re-gion of the gene which we have not been able to scan yet.

All the identified mutations have already been reported.Two of them (1061 and 1309) are associated with the se-vere phenotype of FAP, and this was confirmed in our sam-ple. The two most frequent mutational sites in FAP patientshave been found in codons 1061 and 1309 [6] (T. Soussi“APC,” available at: http://perso.curie.fr/Thierry.Soussi/APC.html, updated November 1999). Mutation 1309 is themost frequent in the gene and represents 18.01% of thegerminal mutations found in APC, followed by 1061 witha frequency of 11.86%. Mutation 1192 is rarer, with a fre-quency of 0.60% [22]. All three mutations result in prema-ture stop codons downstream which give rise to truncatedAPC proteins [6, 13, 21, 23].

The unrelated individuals from the 12 noninformativefamilies and their relatives will continue under colonosco-pic surveillance. We could only scan exon 15 of the APCgene using only one type of molecular analysis. There areother ways of detecting FAP genetically; for example, theprotein truncation test can detect up to 80% of the casesand gives an idea of the site of the mutation by the size ofthe truncated product [24]. Until the present, however, inour country it has not been possible to use it.

The carrier or noncarrier status of FAP at-risk rela-tives was predicted in three of the families carrying ger-minal mutations. The noncarrier individuals have thesame risk of developing polyps or colorectal cancer asthe general population, and therefore they do not need tobe so strictly surveyed [25]. The two relatives who carrythe same germinal mutation in APC as their mother willhave colectomy and will continue under strict surveil-lance to prevent bowel cancer from developing. At pres-ent in Cuba the type of surgery a FAP patient receives

depends on the extension and localization of the polypsand/or the tumour. Nevertheless, the possibility has beenreported that in the future the knowledge of the site andnature of the germinal mutation may modify the surgicalstrategy for each individual [25].

The active APC protein is a dimer. The APC producttruncated in the MCR forms a stable heterodimer withthe wild-type protein which presents its tumour suppres-sor activity reduced or missing [13, 26]. It has been sug-gested that this happens because the mutated proteins arecapable of inactivating the normal allele of APC [26].

APC is a hypermutable gene which presents a highlyheterogeneous mutational pattern [27] (Human GeneMutation Database, Cardiff, UK). It has been shown [28]that the different mutations provide cells with differentselective advantages, with mutations close to codon 1300providing the greatest advantage. When the function ofthe APC protein is totally lost, the cell acquires a grow-ing advantage, resulting in clonal expansion [25]. Itseems that when APC interacts with the complex formedby β-catenin and E-cadherin, it mediates the transmis-sion of growth regulating signals [13].

APC has three binding domains for β-catenin: twowould be present in the truncated protein codified byAPC mutated in 1309 [13]. It has been shown that the1309-mutated protein can exert a dominant negative ef-fect in the wnt signalling pathway that could represent astep preceding colorectal tumorigenesis [26].

The clustering of mutations in the MCR of APC andthe correlation of FAP’s more severe phenotypes withmutations in that region suggest that either this part ofthe gene is specifically susceptible to mutations, or thatthe induction of a tumour is caused, at least partially, byselection due to a mutation in the MCR [26].

It is possible that the rest of the mutations in the MCRhave a similar mechanism of tumorigenic action, an is-sue yet to be solved, but in our sample we observed themore severe form of the disease to be associated withmutations in this region.

Conclusion

For the first time in Cuba the molecular diagnosis ofFAP was made, and germinal mutations were identifiedin the mutational clustering region of the APC gene inasymptomatic carriers of the disease. Similarly, the pres-ence of the more severe phenotype of the disease wasconfirmed in patients carrying mutations in this region.

Acknowledgements We are grateful to the National Institute ofNeurology and Neurosurgery for allowing us to perform the ana-lytical work in their Genetics Laboratory. Some of the primersused to scan exon 15 of the APC gene were generously donated byDr. Ian Frayling and the Imperial Cancer Research Fund, Colorec-tal Cancer Unit of St. Mark’s Hospital in the United Kingdom.The authors thank all the workers of the National Institute of Gas-troenterology who collaborated in carrying out this work.

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