1 Yorkshire Regional DNA Laboratory, Department of Clinical Genetics, St James’s University Teaching Hospital , Leeds, England

Keywords: familial adenomatous polyposis, adenomatous polyposis coli, quantitative PCR, dosage quotients

Familial adenomatous polyposis (FAP) is an autosomal dominant colon cancer predisposition syndrome in which patients develop hundreds to thousands of precancerous colonic polyps that have a high risk of becoming malignant.

Despite the fact that deletions of the APC locus were originally used to map [1][2], and identify [3][4] the APC gene most studies of mutations in APC have used techniques which would not detect deletions [5][6][7]. Molecular analysis of the APC gene in FAP patients has found the pathogenic mutation in 70% of cases [8], however a substantial minority have no mutation identified.

Interstitial deletions of chromosome 5q, which include the APC locus, have been reported in patients with polyposis coli and mental retardation [9]. Submicroscopic deletions have been reported in only a few cases [10][11]. In one family [10] linkage analysis with flanking and intragenic markers followed by in situ hybridisation with intragenic cosmid clones showed the deletion was approximately 200 kb and included more than the 3’ half of the APC gene and the 3’ adjacent D5S346 microsatellite. A recent report [11] described a quantitative PCR assay to detect submicroscopic deletions which included the entire APC gene and the adjacent D5S346 microsatellite in three unrelated Italian FAP families. These submicroscopic deletions do not appear to be associated with mental retardation.

Microsatellite analysis of families with FAP in our region showed one family with apparent non-maternity at D5S346 in one of the affected offspring of an affected mother, suggesting the possibility of an APC deletion. A quantitative PCR assay was therefore developed to detect submicroscopic deletions of the APC gene.

Previous analysis of 68 FAP families from the Yorkshire region has found the pathogenic mutation in 46 cases (67%). These cases were referred for testing (with informed consent) from the Yorkshire Regional Clinical Genetics Service after clinical examination and counselling. All families with an unidentified mutation have been analysed by quantitative PCR; deletions have been identified in 4 families which is about 5% of the Yorkshire FAP families. In family 1 we studied 3 individuals affected with FAP. Cytogenetic analysis on one of the patients showed a normal female karyotype. In family 2 we studied 4 individuals affected with FAP. One of these patients, the father of the other 3, did not develop the disease until he was 62 years old. In family 3 we studied 2 affected individuals. Only one sample was available from family 4. A patient with a cytogenetic deletion 46,XX,del(5),(q14.2q22.3) was also analysed by the quantitative PCR method.

Linkage analysis was performed using several flanking microsatellite markers: D5S299 [12], D5S82 [13], DS5122 [14], D5S346 [15], MCC [16] and D5S318 [17]. Products were analysed by GeneScan analysis on an ABI 373A DNA sequencer with 672 software.

Dosage quotient and standard deviation results are shown in table 3. The values were close to expected values with standard deviations approximately 10% of the mean dosage quotient. Two clear groups of results emerged with no overlap seen between values close to unity or values close to 0.5. Results indicating deletions (dosage quotients close to 0.5) were seen in each case with more than one amplicon, excluding allelic dropout as a cause of reduced amplification.

Three of the four deletions identified were distinct. Family 1 showed apparent non-maternity with marker D5S346 between the affected mother and one of her affected daughters. Quantitative PCR showed the affected individuals to have only one copy of exon 8, exon 15A, exon 15D and exon 15F of the APC gene and we concluded that the mutation in this family was a deletion which extended from at least exon 8 of APC to the 3’ D5S346 microsatellite. A similar deletion was identified in family 2. In this family all affected individuals were homozygous for the same MCC allele which suggests the deletion could include this locus. In family 3 the deletion extended from at least exon 8 to exon 15F of APC. Microsatellite analysis showed the proband to be heterozygous at D5S346 and D5S82. In family 4 dosage quotients for exon 8 of APC showed that there were two copies of this exon present but only one copy of the regions of exon 15 examined. The proband was heterozygous at D5S346. We concluded that the mutation in this family was a deletion which included exons 15A, 15D and 15F but did not extend as far as D5S346. A summary of the microsatellite data and quantitative PCR is shown in table 2.

Using a quantitative PCR assay we found submicroscopic gene deletions of APC to be the pathogenic mutation in four unrelated FAP families in Yorkshire, accounting for about 5% of our FAP families. Taken together with the report of [11] who found submicroscopic deletions in three Italian families with FAP (17% of their FAP pedigrees) these data suggest that submicroscopic deletions of the APC gene may be more common than is evident from the literature, perhaps because they would be undetected by conventional mutation scanning methods. For example, a recent study [7] found APC mutations in 106/190 FAP families but did not search for gene deletions. Our evidence suggests that gene deletions may be found in a substantial minority of FAP patients. That at least three of the four submicroscopic deletions were distinct, differing in the loss of D5S346 and exon 8, eliminates an undue bias in our sample due to a common founder mutation. As the assay is relatively quick and easy assay to perform it will now form part of our initial diagnostic mutation screen for a new FAP patient.

A number of quantitative methods have been described to measure gene dosage, including Southern blotting [20][21][22], oligonucleotide hybridisation , competitive PCR [23][24][25] and more recently Taqman assays [26] and comparative genomic hybridisation (CGH) either using metaphase spreads [27] or arrayed targets [28]. Any useful dosage assay should meet criteria of specificity (absence of false positives), sensitivity (absence of false negatives), reproducibility and be reasonably economical in time and material. Hybridization assays using genomic DNA digests are somewhat more time consuming than PCR-based methods and usually involve the use of radiolabelled probes. CGH is a promising alternative, but limitations in the size of deletions or duplications detectable by metaphase spreads must be overcome by the availability of arrayed DNA before the technique can be generally applicable. Since automated fragment analysis is already widely used in genetic diagnostic laboratories, the use of dosage quotient analysis offers a robust and accessible means to test for microdeletions and duplications.

Pathogenic duplications or deletions have long been reported in the literature, from alpha globin [23] and dystrophin [29] to tumour suppressor genes including BRCA1 [30], BRCA2 [31] and hMSH2 [32]. It is therefore important that genetic testing includes the detection of altered gene dosage where appropriate as conventional mutation scanning methods may miss submicroscopic deletions.

Acknowledgements

We thank Michael Yau, NE Thames Regional DNA Laboratory, Guy's hospital, London for providing primer sequences for STSs from the MPZ gene.

References

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Legends

Figure legend

Key: Hom - homozygous at locus, het - heterozygous at locus.

The order of the markers and exons is 5’ of APC to 3’ of APC.