1 Institute of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark,
2 Department of Biology and Environmental Protection, University of Silesia, Katowice, Poland,
3 Department of Medical Genetics, Shenyang Medical College, Shenyang 110034, Liaoning Province, P. R. China,
4 Institute of Occupational Health, DK-2100 Copenhagen O, Denmark and
5 Department of Biochemistry, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA
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Abstract |
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Abbreviations: BCC, basal cell carcinoma; SNP, single nucleotide
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Introduction |
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Traditionally, localization of genes for specific traits on the chromosomes has involved the study of the co-segregation of the phenotype with specific markers in affected families. This technique has been highly successful, particularly in the study of rare diseases caused by one or a few genes (2,3). Recently, it has become realistic to use molecular epidemiology performed directly on samples of the underlying population for the localization of genes (3). These studies use linkage disequilibrium, presumably arising from a distant founder effect, to study the association of markers with the phenotypic trait. It has been speculated that this approach may be superior for the study of common complex traits (3,4), but results are still limited.
Rather than to start a random search in the genome for cancer-disposing or cancer-preventing genes we have focused on genes of the DNA repair system. The argument for this is simple: a number of rare genetic syndromes with cancer predisposition are already known. Many of these syndromes have turned out to be caused by mutations in DNA repair genes (5,6). In other words, we know that frank mutation of these genes will increase cancer risk dramatically. Thus, it is an obvious hypothesis that other variations in genes for DNA repair may modulate cancer risk.
We have worked with basal cell carcinoma (BCC) of the skin, the most common cancer among Caucasians, and have reported an association of this cancer with the genome region 19q13.2-3, which contains several genes involved in nucleotide excision repair of DNA (79, J.Yin, E.Rockenbauer, M.Hedayati, N.R.Jacobsen, V.Vogel, L.Grossman, L.Bolund and B.A.Nexø, submitted for publication. J.Vin, V.Vogel, L.V.Gerdes, M.Dybdahd, L.Bolund and B.A.Nexø in press). In this paper we have performed a search along the chromosome for association with disease using a number of single nucleotide polymorphisms (SNP) in the relevant region from two cohorts consisting of Caucasian Americans and Danes, respectively. Both sets of data indicate the presence of a gene variation modulating risk of BCC. We also suggest tentative limits for the location of this variation. Furthermore, we have investigated possible synergisms between the markers studied. Remarkably, we have found that markers on chromosome 19 presumably separated by several million bases supplemented each other in separating cases from controls, which suggests the presence of a risk-modulating variation in a second gene region.
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Materials and methods |
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Initially, 71 cases and 118 controls were included in this study. However, the number of persons varied between analyses, as the supply of DNA gradually was depleted. In the case of SNP RAI intron 1 only 133 persons could genotyped reliably.
The groups of 20 psoriatic Danes with and 20 psoriatic Danes without BCC have been described previously (7). Briefly, BCC subjects were identified from a population-based cohort of persons treated by Danish dermatologists in the year 1995, and fulfilled the following criteria: (i) age in 1995 <50 years and (ii) clinically verified diagnosis of psoriasis. The diagnosis of BCC was clinically and histologically confirmed. The controls consisting of psoriasis cases without BCC were selected from among patients treated in the year 19921995 for psoriasis by dermatologists who participated in the national cohort study 1995. The controls were matched by age and sex to the cases. The patients with psoriasis and BCC differed from the national cohort of BCC in that the average age of first BCC was 38 years against 56 years in the cohort. A number of cases had had multiple BCCs. There was a tendency that cases had been treated for a longer time than the controls, and also that the treatments were more intense. This was to be expected as treatment of psoriasis involves a number of carcinogenic treatment modalities.
Genotyping
The total list of markers analysed in the present study is given in Table I. Determination of the genetic polymorphisms in the American cohort in CKM exon 8, ERCC1 exon 4, FOSB exon 4, GLTSCR1 exon 1, LIG1 exon 6, RAI intron 1, RAI exon 6, SLC1A5 exon 8, and XPD intron 4, XPD exon 6, XPD exon 23 and XRCC1 exon 10 has been described previously (8, J.Lin, E.Rockenbauer, M.Hedayati, N.R.Jacobsen, V.Vogel, L.Grossman, L.Bolund and B.A.Nexø submitted for publication).
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Typing of the genetic polymorphisms in the Danish psoriatics was performed as described (7, J.Yin, U.Vogel, L.U.Gerdes, M.Dybdahl, L.Bolund and B.A.Nexø, submitted for publication). In addition, FOSB exon 4 and SLC1A5 exon 8 were examined as for the American cohort.
Finally, we determined the polymorphism ASE1 exon 1 by Lightcycler in both cohorts using the above program and the following primers and probes: forward 5'-ggttttctgctctgcacacg; reverse 5'-cctttctccttccaccaacg; sensor 5'-LCRed640-cgggctacagggttacctgag-p; anchor 5'-tctgcaacctggtgcgagcagc-fluorescein. The primers were obtained from DNA Technology (Aarhus, Denmark) and the probes from TIB-MOLBIOL (Berlin, Germany).
Statistical tests
Tests for association of individual markers with BCC were performed by 2-tests of the allele frequencies.
2-tests (including Tables II, IV and V
) were performed in Excel (Microsoft, Redmond, WA). Tests for linkage disequilibria and for association of two markers with BCC were performed using the program Arlequin (11, obtainable at http://lgb.unige.ch/arlequin/). Bonferroni adjustment was performed online (http://home.clara.net/sisa/bonfer.htm).
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Results and discussion |
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Figure 1 also shows comparable results obtained with the set of psoriatic Danes of which some had acquired BCC (5, Yin et al., in press). The sizes of the groups were considerably smaller in this study and the P-values consequently not so low. This curve had a minimum in the gene ERCC1 33 kb from the previously described minimum in RAI (P = 0.01). Viewed in isolation the results of this curve could be due to chance in a large number of tests, but the minima in the two cohorts are located remarkably close to each other. Considering the stochastic nature of linkage disequilibrium (12) and the slightly different ethnicity of the two samples we view the coincidence as agreement and suggest that the latter curve corroborates the previous data.
Combining the neighbouring markers in RAI led to highly significant results in the American cohort. Thus, virtually all American cases occurring before the age of 50 years were homozygote for RAI intron 1A RAI exon 6A, while only 50% of the controls were so [Table II
, odds ratio = 12.8; P(
2) = 0.00006]. The logical conclusion is that the region must contain a sequence of major importance for the occurrence of BCC. The haplotype RAI intron 1A RAI exon 6A is presumably located close to this sequence and associated with the recessive version.
Delineation of the region, which contains the causative gene sequence, can only be tentative as linkage disequilibrium mapping is still in its infancy. A conservative interpretation would suggest the 125 kb interval between XPD exon 23 and FOSB exon 4. All significant P-values were observed well inside this interval. An optimistic interpretation would be that the causative gene variation lies in the 55 kb region from XPD exon 6 to ERCC1 exon 4. This region contains four genes: XPD, RAI, ASE1 and ERCC1 and contains both minima in P-values. RAI, which is located at the minimum of the curves from the American cohort, was discovered as an inhibitor of RelA and is thus presumably involved in the control of transcription (13), but its function is poorly understood. RAI could conceivably play a role in the carcinogenic process, for instance by regulating some element of DNA repair or by influencing the cellular balance between repair and apoptosis. Alternatively, the gene variation could directly influence the function of the nearby genes for DNA repair, ERCC1 and XPD. This remains largely speculation. Thus, the chromosomal search neither supported nor excluded the original premise that these genes for DNA repair modulate cancer risk. However, it reduced the size of the region of interest considerably and provided a superior combination of markers.
In an attempt to further elucidate the significance of chromosome 19q13.2-3 for the occurrence of BCC we investigated the effect of combining markers in general. We chose to focus on the dataset, which presumably contained least statistical noise: the Caucasian Americans that had aquired BCC before the age of 50 years. We investigated two parameters: P-values for linkage disequilibria between pairs of markers among the controls, and P-values for differences in occurrence of genotypes of marker pairs between cases and controls.
Table III summarizes the results. The P-values for pair-wise linkage disequilibria are shown above the diagonal while P-values for association of pairs of markers with BCC are shown below. Values that were significant after Bonferroni adjustment (72 tests, no correlation) are shown in boldface letters. In general, data for linkage disequilibria above the diagonal confirmed the order of the markers, i.e. neighbouring markers tended to be in strong disequilibrium.
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Table IV shows the ocurrence of combined genotypes of RAI intron 1 and GLTSCR1 exon 1 in relation to BCC. It is obvious to define `risk-genotypes' as having two As in RAI and at least one C in GLTSCR1. This corresponds to the assumptions that RAI intron 1A is recessive, and GLTSCR1 exon 1C is dominant. If one does so, one finds that 25 out of 25 cases have a `risk-genotype', while only 28 out of 62 controls have one [P(
2) = 0.000002].
Table V shows similar results for RAI intron 1 and SLC1A5 exon 8. Analysis of Table V
shows that while SLC1A5 exon 8T in combination with RAI intron 1AA may be associated with BCC [P(
2) = 0.002], most discriminative power arises from RAI intron 1. One should also remember that the subdivisions of the haplotypes were performed a posteriori. Thus, the P-values derived from Tables II, IV and V
must be interpreted with caution.
It is obvious from the positions of RAI and GLTSCR1 (assuming that the present map of chromosome 19 is correct) that the result of combining the two markers is unlikely to arise from their association to a common contiguous risk-sequence. The markers are several mega bases apart and linkage disequilibrium over such distances is rare in humans (12). Also, it is difficult to reconcile the suggetion that the `risk-allele' of one marker is dominant while the other is recessive with the hypothesis that they associate to the same causative gene variation. Probably, a second independent `risk-sequence' near GLTSCR1 exon 1 either works in synergy or additively with the one near RAI. We note with interest that GLTSCR1 is a candidate tumour suppressor gene for gliomas (14). It could conceivably be involved in skin tumourigenesis too.
No efforts were made to exclude patients suffering from Gorlin's syndrome (nevoid BCC), but these patients are rare and not likely to contribute to the data. Moreover, the Gorlin gene PTCH is located on chromosome 9, and cannot account for our findings.
The present case-control studies are fairly small in terms of the number of persons studied. Both because the experimental design and because of the limited number of persons one must be wary of sampling bias. We think that the differences between cases and controls in the markers in RAI are too dramatic to be caused by differential sampling, but are in the process of verifying the central results on larger population-based cohorts.
Recent studies have found markers in XPD located near RAI to be associated with the development of melanoma, glioma and lung cancer (1517). No attempts have yet been made to pinpoint the gene(s) responsible, but it is possibly the same one(s) as for BCC. Thus, this region of chromosome 19, which clearly plays a role in skin tumourigenesis, may be important for other cancers as well.
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Notes |
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Acknowledgments |
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References |
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