Association of chromosome 19q13.2-3 haplotypes with basal cell carcinoma: tentative delineation of an involved region using data for single nucleotide polymorphisms in two cohorts

Eszter Rockenbauer1, Mette H. Bendixen1, Zuzanna Bukowy1,2, Jiaoyang Yin1,3, Nicklas R. Jacobsen4, Mohammad Hedayati5, Ulla Vogel4, Lawrence Grossman5, Lars Bolund1 and Bjørn A. Nexø1,6

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We have previously used single nucleotide polymorphisms to detect an association of basal cell carcinoma (BCC) in Caucasian Americans and Danes with the genome region 19q13.2-3, which contains several genes involved in the nucleotide excision repair of DNA. In this exploratory paper we have extended the data and used them in a chromosomal scan. The results indicate the presence of a gene variation modulating the risk of developing BSS in a submegabase region including and surrounding the gene RAI. Specifically, persons that are homozygous for the haplotype RAI intron 1A RAI exon 6A appear at increased risk for BCC. In addition, we have looked for possible synergisms between all pairs of markers. We find that a marker in GLTSCR1, presumably separated from RAI by several million bases, supplements the most significant marker in RAI in separating cases from controls, which may suggest the presence of an independent, risk-modulating variation in this second gene region.

Abbreviations: BCC, basal cell carcinoma; SNP, single nucleotide


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
One of the central questions in cancer biology is still largely unanswered: who gets cancer? Epidemiology has revealed a number of important risk factors in lifestyle and environment, but even with as potent a carcinogen as tobacco only ~10% of users will get lung cancer (1), and we do not know whom. Usually we excuse our ignorance by the fact that carcinogenesis is a stochastic process, but there could easily be major differences among persons with respect to intrinsic cancer risk. Indeed, it is our contention that genetic differences modulate cancer risk in the general population.

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 (7–9, 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Study groups
The groups of Caucasian Americans with and without BCC have been described previously (8,9). Briefly, the study was a clinic-based case-control study at the Johns Hopkins Hospital, which serves multiple participating dermatologists in Maryland. Cases were histo-pathologically confirmed primary BCCs and were diagnosed between 1987 and 1990. The controls were patients from the same physician practices and had a diagnosis of mild skin disorders. All participants were Caucasians living near Baltimore and were between 20 and 60 years of age. The controls were frequency matched to the cases by age and sex. Cases and controls with any other forms of cancer were excluded. In the questionnaire, the study subjects were asked if they had any blood relatives with skin cancer, and were asked to specify the type of cancer. Study subjects with relatives with BCC and squamous cell carcinoma and `skin cancer' were included in the group of subjects with a family of skin cancer. Subjects with relatives with melanoma were not included. At the clinic visit the subjects gave informed consent, were examined by dermatologists, completed a structured questionnaire and provided blood. Available frozen lymphocytes were genotyped.

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 1992–1995 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 IGo. 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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Table I. The markers used, their sources of information, and their currently estimated positions on chromosome 19, as well as their position in Figure 1Go
 
The XPD exon 10 polymorphism was typed using a LightcyclerTM (Roche, Basel, Switzerland) to amplify the genomic DNA in a real-time PCR reaction and then determine the temperature profile of the fluorescent energy transfer between an anchor and a sensor probe. The primers and probes were as follows: forward primer 5'-gatcaaagagacagacgagc-3'; reverse primer 5'-gaagcccaggaaatgc-3'; anchor probe 5'-ggacgcccacctggccaacc-fluorescein; sensor probe 5'-LCR ed640-cgtgctgcccaacgaagtg-p. Please note that the probe sequences given in ref. 8 are erroneous. The reaction mix contained 500 nM of each primer, 200 nM of anchor and sensor probes, 10% DNA Master Hybridization Probes Mix (Roche) which contains DNA polymerase, dNTP and buffer, 3.5 mM MgCl2 and 5% DMSO. After an initial denaturation of 2 min at 95°C, the regimen consisted of 45 cycles of 10 s at 95°C, 15 s at 53°C and 30 s at 72°C. The last cycle was extended by 2 min at 72°C for polishing. To read the temperature profile of the energy transfer the PCR products were denatured 10 s at 95°C, cooled to 50°C for 30 s and subjected to a temperature ramp from 50 to 95°C with a slope of 0.1°/s.

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 {chi}2-tests of the allele frequencies. {chi}2-tests (including Tables II, IV and VGoGoGo) 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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Table II. Genotype occurrences for RAI intron 1 and RAI exon 6 in American cases occurring before 50 years of age and in controls
 

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Table IV. Genotype occurrences for RAI intron 1 and GLTSCR1 exon 1 in American cases occurring before 50 years of age and in controls
 

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Table V. Genotype occurrences for RAI intron 1 and SLC1A5 exon 8 in American cases occurring before 50 years of age and in controls
 

    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In this study we have added more SNPs to our previous data on the relation of BCC with chromosome 19q13.2-3 and combined all data. Our intention was to locate the chromosomal subregion with the highest association to disease. Thus, the polymorphisms were selected on the basis of their chromosome localization, and the majority are unlikely to play a functional role in BCC. Figure 1Go is a representation of the results showing the logarithm of the P-values of the associations as a function of the approximate positions of the polymorphisms on the chromosome. The map positions are derived from current NCBI maps of this region of chromosome 19 (http://www.ncbi.nlm.nih.gov/). All markers were located in a 6 Mb region with 10 of the markers within 250 kb. The identifications and current chromosome positions of the various markers are listed in Table IGo.



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Fig. 1. The association of occurrence of BCC with various markers on chromosome region 19q13.2-3. ({blacklozenge}) All cases of BCC in the cohort of Caucasian Americans. ({blacksquare}) Cases of BCC occurring before 50 years of age in the cohort of Caucasian Americans. (Triangle) Cases of BCC occurring in the cohort of psoriatic Danes. The numbers next to the curve refer to the listing of markers in Table IGo. The absolute chromosome positions are from a particular build of NCBI's map of chromosome 19 and are expected to change with time.

 
The results for the American cohort showed a very sharp drop in P-values with minimum in the gene RAI (P = 0.004). As cancers with a known genetic component tend to occur earlier than sporadic cancer, we also limited the dataset to cancers occurring before 50 years of age. This further reduced the P-value, and thus strengthened the association (P = 0.00014). Bonferroni adjustment shows that with 14 tests and a mean correlation of 0.15 between the variables any P-value below 0.0054 is unlikely to be a random event at a level of significance of 0.05. In other words, the P-values of the minima are so low that they are unlikely to be due to chance in a large number of tests.

Figure 1Go 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 IIGo, odds ratio = 12.8; P({chi}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 IIIGo 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 III. Pair-wise linkage disequilibria between markers in controls and pair-wise association of markers to BCC
 
The association of the two markers RAI intron 1 and RAI exon 6 with BCC was not significant as part of a global test for dual interactions, i.e. after Bonferroni adjustment, and thus disregarding the very obvious nature of this particular combination, However, two combinations of markers encompassing RAI intron 1 were significantly associated with the occurrence of BCC, thus confirming the involvement of RAI. These were RAI intron 1 combined with SLC1A5 exon 8 (P = 0.00007) and RAI intron 1 combined with GLTSCR1 exon 1 (P = 0.00013). The two sets of markers also satisfy an additional criterion: the P-values of the combinations are lower than the lowest obtained with the markers individually. This is not automatically the case, as the introduction of an additional marker not only increases the discriminative power of the variables, but also increases the degrees of freedom in the statistical test.

Table IVGo 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({chi}2) = 0.000002].

Table VGo shows similar results for RAI intron 1 and SLC1A5 exon 8. Analysis of Table VGo shows that while SLC1A5 exon 8T in combination with RAI intron 1AA may be associated with BCC [P({chi}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 VGoGoGo 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 (15–17). 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.


    Notes
 
6 To whom correspondence should be addressed Email: nexo{at}humgen.au.dk Back


    Acknowledgments
 
We thank Thrine Schneidermann for expert technical assistance. The Karen Elise Jensen Foundation, the Danish Cancer Society (J. no. 9810028), the Danish Medical Research Council (J. no. 9600259) and the Novo Nordisk Foundation supported this paper.


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received January 7, 2002; revised February 5, 2002; accepted March 22, 2002.