Hum Genet (1998) 102 : 624–628
© Springer-Verlag 1998
ORIGINAL INVESTIGATION
Alessandro Stella · Nicoletta Resta · Angela Polizzi Mariapina Montera · Filomena Cariola Francesco Susca · Viviana Gismondi · Lucio Bertario Cristiana Marchese · Romano Tenconi Maria Grazia Tibiletti · Paola Izzo · Mattia Gentile Fernando Prete · Oronzo Pannarale Giovanni Di Matteo · Paola Sala · Liliana Varesco Cristina Mareni · Ginevra Guanti
The familial adenomatous polyposis region exhibits many different haplotypes Received: 5 November 1997 / Accepted: 3 February 1998
Abstract In the present study, we used five different polymorphic markers to construct the haplotype at the adenomatous polyposis coli (APC) locus in families with familial adenomatous polyposis (FAP) and in the normal Italian population. Non-ambiguous haplotypes were reconstructed from 246 normal chromosomes and 65 FAP chromosomes. In the control population, the four polymorphisms intragenic to APC gave rise to 16 haplotypes, the most common of which (II and XV) accounted for over 50% of all chromosomes. In FAP patients, 13 haplotypes were
A.S. and N.R. contributed equally to this work A. Stella · N. Resta · A. Polizzi · F. Susca · G. Guanti (✉) Sezione di Genetica Medica, Dipartimento di Medicina Interna e del Lavoro, Policlinico, Piazza Giulio Cesare, I-70124 Bari, Italy e-mail:
[email protected], Fax: +39-80-5478269 F. Cariola · M. Gentile · G. Di Matteo I.R.C.C.S. “De Bellis”, Castellana, Bari, Italy
found but their distribution was not statistically different from normal subjects. Eighty complete chromosomal haplotypes (many fewer than the theoretical maximum of 208) for the five polymorphic sites assayed were observed in the control population, 35 being found in the FAP patients. We compared the distribution of these haplotypes within the two groups; no statistically significant differences between normal and FAP chromosomes were found. The elevated heterogeneity of FAP chromosomes was clearly confirmed by the observation that 19 patients who carried one or other of the two most common APC mutations (nt 3183 and nt 3927) showed 18 different haplotypes. On the basis of these results, we were not able to identify a founder FAP chromosome. Various mechanisms are presented to explain this observation.
Introduction
M. Montera · C. Mareni Dipartimento di Medicina Interna,Università, Genoa, Italy V. Gismondi · L. Varesco Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy L. Bertario · P. Sala Istituto Nazionale Tumori, Milan, Italy C. Marchese Ospedale Mauriziano, Turin, Italy R. Tenconi Cattedra di Genetica Medica, Dipartimento di Pediatria, Università di Padova, Padua, Italy M. G. Tibiletti Dipartimento di Scienze Cliniche e Biologiche, Università di Pavia, Sede Varese, Italy P. Izzo Dipartimento di Biochimica e Biotecnologie Mediche, Università Federico II, Naples, Italy F. Prete V Divisione e Cattedra di Chirurgia Generale, Policlinico, Bari, Italy O. Pannarale IV Divisione e Cattedra di Chirurgia Generale, Policlinico, Bari, Italy
Familial adenomatous polyposis (FAP) is a precancerous syndrome transmitted as an autosomal dominant disorder primarily involving the colon and rectum. Penetrance is close to 100% and the syndrome is characterized by the appearance of hundreds to thousands of adenomatous polyps that inevitably lead to carcinoma if the large bowel is not removed. The family members of affected individuals (who are genetically heterozygous) have a 50% risk of inheriting the condition. Because the age of onset is variable, patients at risk are advised to enter an early detection program involving periodic endoscopic evaluation, the objective being the early detection of those who have inherited the gene so that they can be advised to undergo preventive colectomy. Indeed, the risk of colonic cancer in those individuals who manifest the polyposis is 100%, so that the large bowel must be removed before the development of colorectal malignancy. Presymptomatic diagnostic tests
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may enable the screening program to focus on those relatives who have inherited the mutated gene. Cloning (Groden et al. 1991; Joslyn et al. 1991; Kinzler et al. 1991; Nishisho et al. 1991) of the APC (adenomatous polyposis coli) gene has led to the molecular characterization of specific mutations responsible for the disease. So far, a myriad of mutations, including deletions, insertions and nonsense mutations, have been described (Cottrell et al. 1992; Fodde et al. 1992; Groden et al. 1993; Mandl et al. 1994; Nagase and Nakamura 1993; Béroud and Soussi 1996). Because most of the germline APC mutations are different from each other, the entire open reading frame (8535 nucleotides) must be scanned to identify the specific mutation in any particular sporadic patient, even though a mutation is not found in about 20%–25% of cases. As the detection of individual mutations remains a difficult task, the most realistic approach for identifying carriers relies on cosegregation analysis of the disease allele with nearby polymorphic markers. The information provided by polymorphic marker analysis can be markedly improved by constructing haplotypes consisting of a defined pattern of alleles at polymorphic loci, linked together on a chromosome. Besides its use in linkage studies, haplotype analysis affords the opportunity to characterize the history (origin and migration) of a particular allele and to study allele-specific expression. Furthermore, if an association between a specific mutation and a particular haplotype is found, the typing of the APC gene mutations can be facilitated. In the current study, we have used polymorphic markers located within the APC locus and a microsatellite localized in a region flanking the APC gene and tightly linked to it to reconstruct and analyse the haplotypes from individuals of normal families and FAP kindreds.
Table 1 Comparison of FAP and normal haplotypes
aExon 11, 15 and 3´ UTR polymorphisms: + restriction site present, – restriction site absent; exon 13 polymorphism: + fast allele, – slow allele
Haplotypesa
Materials and methods Patients High molecular weight DNA was extracted from peripheral blood or lymphoblastoid cell lines according to standard methods (Sambrook et al. 1989). Sixty control families originating, where possible, from the same regions as the FAP families were sampled. In several instances, the families of the in-laws of the FAP patients and some unaffected family members of the patients were typed as controls. From the 468 individuals who were fully genotyped, 246 different haplotypes were reconstructed unambiguously. From a pool of Italian FAP families, 65 were selected for the study. Unfortunately, we were only able to study five cases of fresh mutations, it being difficult to find families in which the fresh APC mutated carrier had unaffected living parents and at least one affected offspring. In 57 cases, the APC gene mutation had previously been fully characterized (Stella et al. 1992, 1994; Varesco et al. 1993; Scarano et al. 1997; Gismondi et al.1997). In 2 cases, it had been localized (by using Protein Truncation Test PTT) but not yet identified and, in the remaining 6 cases, it is being sought but has not yet been found. In all cases, the family members of the patients were available for linkage study. Haplotype analysis Haplotypes were derived by using five different polymorphic markers, the following two allele systems being intragenic to the APC gene: (1) a T/C polymorphism at nucleotide (nt) 1458 (Groden et al.1991) detected by RsaI digestion, (2) an A/G polymorphism at nt 1635 detected by single-strand conformation polymorphism analysis, (3) an A/G polymorphism at nt 5037 detected by HgiAI digestion (Kraus and Ballausen 1992), (4) an SspI polymorphism with two alleles of 270 bp and 135 bp in an 850-bp fragment encoding part of the 3´ untranslated region of the gene (Heighway et al. 1991), detected by agarose gel electrophoresis, and (5) a microsatellite marker, D5S346 (Spirio et al. 1991), which is a highly polymorphic dinucleotide (CA) repeat locus located 70 kb from the APC gene (Spirio et
FAP chromosomes
Normal chromosomes
11 13 15 3´
Number
Frequency
Number
Frequency
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI
+ + + + + + + + – – – – – – – –
4 24 / 2 3 4 / 1 3 / 4 1 2 1 14 2
0.060 0.369 / 0.030 0.045 0.060 / 0.015 0.045 / 0.060 0.015 0.030 0.015 0.215 0.030
14 83 4 3 12 17 6 1 7 4 24 7 7 3 48 6
0.056 0.338 0.016 0.012 0.048 0.069 0.024 0.004 0.028 0.016 0.098 0.028 0.028 0.013 0.196 0.024
Total
65
+ + + – + – – – + + + – + – – –
+ + – + – – + – + – + + – + – –
+ – + + – + – – + + – + – – + –
246
626 Table 2 D5S346 allele distribution and frequencies on FAP and normal chromosomes
Allele
FAP chromosomes Number Frequency
Normal chromosomes Number Frequency
1 2 3 4 5 6 7 8 9 10 11 12 13
– 2 7 9 3 2 7 16 15 4 – – –
2 3 25 31 9 12 30 59 58 13 1 – 3
Total
65
– 0.031 0.108 0.138 0.046 0.031 0.108 0.246 0.231 0.062 – – –
0.008 0.012 0.102 0.126 0.037 0.049 0.122 0.240 0.236 0.052 0.004 – 0.012
246
al 1993). All the markers were assayed by the polymerase chain reaction, the conditions for which have been previously described (Stella et al 1993). The difference in allele distribution between normal and FAP chromosomes was evaluated by the χ2 test. Yates´ correction was used when appropriate. Reconstruction of the haplotypes was based on the assumption of the minimum number of recombinations in each family.
Table 3 Comparison of the most frequent APC-D5S346 haplotypes Haplotype
FAP chromosomes Number Frequency
Normal chromosomes Number Frequency
4-XV 7-II 8-II 9-II
5 4 5 11
16 12 27 30
0.077 0.062 0.077 0.169
0.065 0.049 0.110 0.120
Table 4 Haplotypes of the two most common mutations detected in FAP patients Patient
Mutation
APC haplotype
D5S346 allele
2324 1864 173 N E830 T2 T8 2078 T28 2291 1512 1637 F725 2314 1223 2301 1666 T45 F168 T22
3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3926 del 5 bp 3183 del 5 bp 3183 del 5 bp 3183 del 5 bp 3183 del 5 bp 3183 del 5 bp 3183 del 5 bp 3183 del 5 bp
XV XII II V XI VI XV IX XV II II XV II XV II II II I V
2 8 7 4 8 3 4 8 4 8 9 7 9 5 4 3 9 8 3
Results A panel of 60 normal families comprising over 468 individuals was typed to reconstruct the haplotype of the APC locus. Haplotypes for each individual were constructed by analysing the cosegregation of the marker alleles in all the members of each pedigree. Non-ambiguous haplotypes were determined for 246 chromosomes. The same procedure allowed the determination of the marker alleles present on the chromosome bearing the disease mutation in all of the FAP families studied. The haplotypes derived from the characterization of the four polymorphisms intragenic to the APC gene in control families and FAP patients are reported in Table 1. In the normal population, 16 haplotypes were observed; the two most common, viz. haplotypes II and XV, accounted for over 50% of all chromosomes. In FAP patients, 13 haplotypes were found but their frequency did not show statistically significant differences from the controls. The haplotypes most often associated with FAP chromosomes, viz. II and XV, accounted for 57.8% of the disease chromosomes. Analysis of microsatellite D5S346 in the control population revealed 12 of the 13 alleles described in the literature; in FAP patients, three alleles (1, 11 and 12) were not detected. The distribution and frequencies of D5S346 alleles, reported in Table 2, did not show statistically signifi-
cant differences between the two groups of subjects analysed. Taken together, the five polymorphisms that we have characterized can give rise to 208 theoretical haplotypes; of these, a total of only 80 have been observed in the normal chromosomes and 35 in the FAP chromosomes analysed (data not shown). The most common APCD5S346 haplotypes are shown in Table 3. The frequencies of FAP and control haplotypes were not significantly different. As reported in the literature (Nagase and Nakamura 1993 ), a few APC mutations are recurrent, the most frequent being the 5 bp deletion at nt 3926 and the 5 bp deletion at nt 3183. In our study, 19 patients carried one of these two mutations (see Table 4). Characterization of the five markers revealed that all but two chromosomes presenting the same mutation showed a different haplotype. Moreover, in the few cases in which we observed fresh mutations identical to the inherited ones, the chromosomes with fresh mutations showed different haplotypes from those of chromosomes of carriers of the inherited mutations.
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Discussion In this study, we have reconstructed the haplotype of the APC locus in the Italian population and defined the respective haplotype association in 65 patients affected by FAP. The typing of the four polymorphisms intragenic to APC in the normal population has revealed 16 different haplotypes, of which two (II and XV) are very common, accounting for 33.8% and 19.6%, and four relatively frequent, accounting for 9.8%, 6.9%, 5.6% and 4.8% of the population. We have also characterized the polymorphic marker D5S346, a microsatellite lying approximately 70 kb telomeric to the APC gene. The 12 different alleles that we have been able to identify in combination with intragenic markers give rise to 80 haplotypes. Of the 16 APC haplotypes observed in the controls, 13 have been found in FAP patients . These, in combination with the nine different D5S346 alleles, generate 35 haplotypes with very similar frequencies to those observed in the controls. Our results exclude the existence of a particular haplotype being recurrently associated with a specific germline mutation, in contrast to the hypothesis proposed by Almeida et al. (1996). However, on their own admission, the small number of cases analysed made it difficult to extrapolate statistically significant conclusions (Almeida et al. 1996). The simplest explanation of the variety of haplotypes seen in FAP chromosomes is that multiple different germline mutations give rise to the disease. Notoriously, about 350 different germline APC mutations have been described so far and very few of them have been found in more than one independent FAP kindred. Among these, the mutations located at codon 1061 (the 5-bp deletion at nt 3183) and at codon 1309 (the 5-bp deletion at nt 3926) are the most common, occurring in about 20% of FAP patients. However, the analysis of the chromosomes of 19 families who carry one or other of these two mutations has shown the presence of 16 different haplotype backgrounds. The existence of a high mutation rate and/or a selective disadvantage of the mutated chromosomes could be responsible for spreading the APC mutations over many different haplotype backgrounds. A mutation rate of nine mutations per million gametes per generation and a proportion of 25% of cases of fresh mutations have been estimated (Bisgaard et al. 1994), whereas there is virtually a 100% risk of colonic cancer in people who carry an APC mutation and manifest the adenomatous polyps. However, although the age of onset of the first symptoms is variable even within families, the mean age at diagnosis is 35.8 years (Bussey 1975), which would not compromise the chance of reproduction of an individual. Data in the literature (Bisgaard et al. 1994) indicate a reproductive fitness of 0.87 with a growing trend as the consequence of the efficiency of the surgical treatment and, more recently, genetic analyses. Therefore, the elevated heterogeneity of FAP chromosomes, demonstrated by the observation that the most
common APC mutations are found associated with many different haplotypes, remains to be clarified. The FAP chromosomes might be more interrelated than they seem but they could have been altered by other mechanisms, such as gene conversion or recombination. Thus, the different FAP haplotypes could nevertheless have descended from the same founder chromosome. In conclusion, although there is some presumptive evidence that the APC region is a hot spot of mutation and recombination, all these hypotheses are essentially untestable given the available data. Studies with polymorphic markers located at a large genetic distance from the APC locus and the typing of normal and FAP chromosomes from different geographical regions could provide additional insights into the origin and age of FAP chromosomes. Acknowledgements We are very grateful for the cooperation of the Italian FAP families.We also wish to thank Paola Fiorente for her excellent technical assistance and Mary V. C. Pragnell (B.A.) for careful linguistic text revision. This work was financially supported by grants from Associazione Italiana Ricerca sul Cancro, C.N.R. Progetto Finalizzato A.C.R.O. 94.1142.O39, MURST 40% and 60%, and Byk Gulden (to C. Mareni). The experiments complied with the current laws of Italy.
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