pp.64-71 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.

Copyright 1992, Eubios Ethics Institute All commercial rights reserved. The copyrights for the employees of the US Government, are subject to other copyright arrangements. This publication may be reproduced for limited educational or academic use, however please enquire with Eubios Ethics Institute.

Characterization of germ-line mutations of the APC gene: application to presymptomatic diagnosis

Isamu Nishisho,
Dept. of Medical Genetics, Osaka University Medical School, JAPAN

Thank you, I am honoured to be giving this presentation here today. There may be a mixture of people in the audience, therefore I wasn't sure what subject was most suitable for today. But since this is a scientific seminar I will focus on issues using reverse genetics in the analysis of the APC gene. I want to talk an outline of identification of the APC gene, structure of the gene, nature of mutations found in the patients, and presymptomatic diagnosis.

Familial adenomatous polyposis (FAP), which is an autosomal dominant inherited disease, is characterized by the development of hundreds to thousands of adenomatous polyps in the colon and rectum, one or more of which can progress to cancer if left without surgical treatment. Without surgery, the polyps will develop into cancer with 100% certainty. About 50% of the children will have onset of this disease.

The gene responsible for FAP has been assigned to the long arm of chromosome 5(5q21) by linkage analysis in 1987. We isolated about 1,000 cosmid markers from the chromosomal region in which the APC gene is supposed to reside. About 40 clones out of 400 RFLP clones were mapped physically and by linkage analysis close to the responsible gene (Figure 1). No recombination was found with the markers located between L5.62 and KK5.97, therefore these markers were considered to be very close to the responsible gene. In reverse genetics, we don't know what protein is involved in the disease or pathogenic mechanisms, the only clue is the gene location.

Next, we examined the DNA from leucocytes of FAP patients on pulsed field gels by using these cosmid markers as a probe. L5.71, one of the cosmid markers, detected abnormal band in one FAP kindred. An approximately 200kb germ-line deletion was confirmed in this kindred by several restriction enzyme digestions. We considered that the gene responsible for the disease might be included in the deleted region. To isolate expressed genes from the deleted region, several YAC (Yeast Artificial Chromosome) clones that encompassed the deletion were isolated. Potential exon sequences were identified by a cross-species hybridization approach and used as probes to screen cDNA libraries. Eventually, several genes were isolated from the deleted region, two of which were MCC (Mutated in Colorectal Cancers) and APC (Adenomatous Poliposis Coli) gene (Figure 2). The MCC gene was found to be not included in the deleted region of the patient. Subsequent analysis revealed that APC gene is inactivated by point mutation in germ-line of FAP patients, and this is the candidate gene responsible for FAP.

Figure 1: Cosmid markers that detect restriction fragment length polymorphisms around APC locus. The responsible gene is supposed to locate approximately 5,000kb segment indicated by bold line.

Figure 2: Schematic representation of locations of MCC and APC genes. Some part of the APC gene is located in the deletion detected in one patient.

As shown in Figure 3, the APC gene encodes 2,843 amino acids and is expressed in a variety of tissue types including a normal colonic mucosa. Table 1 shows the intron-exon structure of APC gene. The gene is divided into 15 exons, and the final exon is about 6.5kb which is very large. When genes are isolated the functions should be determined. Searches of an amino acid database with the putative APC protein sequence did not reveal any extensive similarities. However, the APC gene, like the MCC gene, contained local similarity to the region of m3 muscarinic acetylcholine receptor (mAChR) known to regulate the specificity of G-protein coupling (Figure 4). This similarity is in the area of the proteins involved with signal transmission. We therefore estimate that it has some significant role in signal transmission pathway of cells.
Table 1: Intron-exon structure of the APC gene

Figure 3: The predicted amino acid sequence of the APC gene

Figure 4: Local similarity among the APC and MCC genes and the m3 mAChR. The connecting lines indicate identities dots indicate related amino acid residues.

Table 2: Germ-line mutations of the APC gene in FAP patients and somatic mutations in sporadic colorectal cancers. The mutated nucleotide are underlined.

Germline Mutations of the APC Gene

Somatic Mutations of the APC Gene

These results made it clear that there was one 200kb deletion, which destroyed the APC genes, in one FAP kindred. However, what about other familial groups? We conducted Southern blotting analysis probed with the APC cDNA, but we could not identify any clear abnormality. This still left the possibility of inactivation by point mutations. In order to search for subtle alterations of the APC gene, ribonuclease (RNase) protection analysis coupled with PCR amplification of genomic DNA was carried out in 80 unrelated patients with FAP, representing each familial group. The coding region of the APC gene was divided into 31 segments, and each segment was separately amplified using PCR. The amplified fragments were hybridized to radio-labeled RNA transcripts corresponding to normal APC sequences. If any mismatching would occur, the RNA probe would be divided by RNase A treatment. By electrophoresis and subsequent autoradiography, we can see if the original length of the probe is divided into two bands, which means that the labelled probe was cut, because there was a mutation causing mismatching.

Point mutations identified in our initial analysis are listed in Table 2. For example, Patient 93 has a C to G transition at codon 280 that results in a change from serine (TCA) to a stop codon (TGA). Patient 24 and 34, they are from separate kindreds, have an identical C to T transition at codon 302 that results in a change from arginine (CGA) to a stop codon (TGA). As this point mutation results in the predicted loss of the recognition site for the enzyme TaqI, appropriate PCR products can be digested with TaqI to detect the mutation.

The examples are shown in Figure 5. This allowed us to determine that the stop codon precisely co-segregate with disease phenotype in 21 members with FAP. In this kindred, presymptomatic DNA diagnosis can now be made with virtually 100% accuracy simply by testing for the relevant mutation, that is, digesting the PCR products with restriction enzyme TaqI. Up to date, approximately 70% of the FAP patients tested have mutations in the APC gene. The mutations are point mutations, small deletions, and 1-2 bp insertions. Moreover, over 90% of the mutations are predicted to result in truncations of APC protein.

Figure 5: TaqI digestion of PCR products of the APC gene.

PCR products before (lane 1) or after (lane 2) digestion with TaqI were separated by electrophoresis on 10% polyacrylamide gel. TaqI cleaves the normal 215-bp PCR product into 134- and 81-bp subfragments. A mutation at codon 302 results in loss of the TaqI site. PCR products were derived from the DNA of: (N) normal individual (P24, P32, and P71) DNA from three affected members of a single FAP kindred (P34) DNA from an affected member of a second FAP kindred.

Figure 6: Segregation of APC mutation in a FAP kindred. A C to A transition newly generates MseI recognition site, and PCR products originated from mutated APC sequences can be cleaved with MseI. All affected members has the base substitution.

Figure 7: Example of presymptomatic diagnosis by linkage analysis. Diagnosis could not be made by these data. We could not conclude whether the mutated gene is linked to allele A of B.

In the case of FAP patients, like all people they have about 3 billion base pairs of DNA, but if there is only one base change, polyposis was created. It is quite a tragic and unfortunate case I think. Figure 6 is an example of presymptomatic diagnosis in a FAP kindred. As shown in the figure, when the mutation was characterized once in FAP kindred, presymptomatic DNA diagnosis can be made by testing whether the member of the kindred inherits the mutation characteristic to the kindred.

A recent case of diagnosis is shown in Figure 7. We could not identify any mutations of APC gene in this kindred. Then we tried to diagnose by linkage analysis. Allelotype of the members at polymorphic site located in the APC gene itself were studied. The father is a FAP patient, and there are two children. We were requested to diagnose whether the children inherited the mutant gene or not. The father has A and B, and one of the children inherited A from his father and the other inherited B from the father. But we could not conclude from the data obtained whether the pathogenic gene is linked to A or B in this kindred. That is, in other words, whether the mutated gene is contained in chromosome A or B. Therefore, we could not predict which is the potential patient. If the elder son develops it, then the second son will not, or vice verse. We may get some answer by testing DNA samples from another patients of the kindred to determine whether the mutated APC gene is linked to A or B in this kindred.

In addition to these germ-line mutations, we identified several somatic mutations of APC in sporadic colorectal carcinomas. These finding strongly suggest that the mutation of the APC gene may contribute to tumour development in patients with noninherited forms of colorectal cancer.

Finally, the demonstration that APC is mutated in the germ-line of FAP patients has obvious clinical implications. Members of kindreds with FAP can now be directly tested for mutations of this gene, pre- or postnatally. Those individuals who have not inherited the gene will spared the discomfort of repeated medical evaluations and colonoscopies as well as the anxiety associated with disease expectation. Because adenomas do not generally develop until the second of third decade of life in FAP patients, attempts to prevent this development in presymptomatic individuals with suitable drugs may prove worthwhile.
This study was done with Dr. Yusuke Nakamura of Cancer Institute and Dr. Bert Vogelstein of Johns Hopkins Oncology Center. I would like to express my thanks to all of the researchers involved in the APC project.

1. Kinzler, K.W., Nilbert, M.C., Vogelstein, B., Bryan, T.M., Levy, D.B., Smith, K.J., Preisinger, A.C., Hamilton, S.R., Hedge, P., Markham, A., Carlson, M., Joslyn, G., Groden, J., White, R., Miki, Y., Miyoshi, Y., Nishisho, I.,and Nakamura, Y. (1991) "Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers", Science 251,1366-1370.

2. Kinzler, K.W., Nilbert, M.C., Su, L.K., Vogelstein, B., Bryan, T.M., Levy, D.B., Smith, K.J., Preisinger, A.C., Hedge, P., Mckechnie, D., Finniear, R., Markham, A., Groffen, J., Bogouski, M.S., Altschul, S.F., Horii, A., Ando, H., Miyoshi, Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) "Identification of FAP locus genes from chromosome 5q21", Science 253: 661-665.

3. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., Hedge, P., Markham, A., Krush, A.J., Petersen,G., Hamilton, S.R., Nilbert, M.C., Levy, D.B., Bryan, T.M., Preisinger, A.C., Smith, K.J., Su, L.K., Kinzler, K.W., and Vogelstein, B (1991) "Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients", Science 253: 665-669.

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