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The Sebaceous Nevus

A Nevus with Deletions of the PTCH Gene

Department of Dermatology, University Hospital, 8091 Zürich, Switzerland

	ABSTRACT

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Sebaceous nevi (SN) are congenital malformations of the skin with the potential to develop into basal cell carcinoma (BCC). To date, the molecular basis for their carcinogenic potential remains unknown. The genetic defect in BCC is known and involves the human homologue of Drosophilapatched (PTCH) on chromosome 9q22.3. The objective of this study was to test whether allelic deletion of the PTCH gene could already be detected in SN. Twenty-one paraffin-embedded SN were investigated in this study. Basaloid cells in conjunction with mature sebaceous glands as well as epidermal layer apart from SN were microdissected and subjected to single-step DNA extraction. We performed the analysis with polymorphic markers at 9q22.3 (D9S15, D9S252, D9S287, and D9S303). Of the 20 informative SN, 8 (40%) exhibited loss of heterozygosity at least at one locus. Here, we provide the first evidence of the involvement of the tumor suppressor gene PTCH in SN. Whether PTCH deletion in SN is associated with progression to BCC and/or other appendageal tumors should be addressed in future studies.

	Introduction

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SN² are congenital malformations of the skin that appear as slightly raised, yellowish, smooth or velvety, hairless plaques on the head or neck. The growth of the lesion is accelerated at puberty, showing a verrucous and nodular appearance. During adult life, it may be complicated by secondary tumor development, which is regarded as third stage in the natural history of the lesion (1) . Quite commonly, a large variety of appendage tumors may develop within SN, e.g., syringocystadenoma papilliferum, primitive follicular germs, trichoblastomas, and, most often, BCCs (1, 2, 3) . For this reason, SN are usually excised. Despite the fact that substantial data have proven the potential of SN to develop mainly into BCC, the molecular basis for the carcinogenesis of SN remains unknown.

During the past few years, the genetic changes in BCCs have been described in detail. For the nevoid BCC syndrome, an autosomal dominant disorder characterized by multiple BCCs, one particular locus, the human homologue of Drosophilapatched gene (PTCH gene) at 9q22.3 has been shown to be involved and to act as a tumor suppressor gene (4, 5, 6) . The inactivation of a tumor suppressor gene frequently occurs through point mutation of one homologue and loss of the other homologue by nondisjunction, deletion, or mitotic recombination. The deletion of genetic material results in LOH when alleles are examined using polymorphic markers in the chromosomal region surrounding the tumor suppressor gene, making it possible to screen chromosomal areas containing putative tumor suppressor genes with LOH studies (7) .

This study was undertaken to investigate the possible LOH in the 9q22.3 region (PTCH gene) in a set of randomly selected SN. We aimed to compare the pattern of LOH with that found in BCCs and expound the close association between SN and BCC with cytogenetic evidence.

	Materials and Methods

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Histological Specimens.
Twenty-one paraffin-embedded histological specimen of SN (from 17 males and 4 females; mean age, 29 ± 15 years), obtained from the archives of the Department of Dermatology, University Hospital of Zurich, were investigated in this study. For each specimen, one section was used for H&E staining and microdissection (Fig. 1) . To assure correct diagnosis, we reviewed all histological slides.

View larger version (110K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, clinical picture of a sebaceous nevus. B, histology showing microdissected basaloid cells in conjunction with sebaceous glands (arrows). C, sequence gel analysis results obtained after subjection of microdissected cells to single-step DNA extraction and amplification using the primers D9S15, D9S252, and D9S303. Loss of an allele was scored as >50% reduction of the band intensity (arrow). Lanes N, normal epidermal layer apart from SN; Lanes T, basaloid cells in conjunction with sebaceous glands within SN.

DNA Extraction.
Procured cells were immediately suspended in a 20-µl solution containing 0.05 M Tris-HCl, 1 mM EDTA, 1% Tween 20, and 2.5 mg/ml proteinase K (pH 8.0) and incubated for 2 days at 37°C. The mixture was boiled for 10 min at 94°C to inactivate proteinase K, and 1.5 µl of this solution were used as template DNA in PCR amplification.

Primers and PCR Conditions.
Four polymorphic DNA markers (obtained from Research Genetics, Huntsville, AL) were used: D9S252 (9q22), D9S15 (9q21), D9S287 (9q22), and D9S303 (9q21–22).

The PCR was performed in 10-µl volumes by using 1.5 µl of template DNA, as described above; 50 pmol of each primer; 20 nM each dATP, dCTP, dGTP, and dTTP; 0.2 µl of [³²P]dCTP (6000 Ci/mmol); 0.1 unit of Taq polymerase; and 1 µl of 10+ buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂, and 0.1% w/v gelatin]. Each sample was overlaid with mineral oil and was amplified in a thermal cycler (Gene Amp PCR System 9600; Perkin-Elmer, Zurich, Switzerland) for 35 cycles of denaturation at 94°C for 1 min, annealing for 1 min at the temperature described above for each primer, and extension for 1 min at 72°C, with a final 10-min extension at 72°C.

LOH Analysis.
Labeled amplified DNA was mixed with an equal volume of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). Samples were then denatured for 5 min at 94°C, followed by rapid cooling, and loaded on to a commercial 8% acrylamide gel (Gel-Mix8; Life Technologies, Inc.). The gel was run at 1600 V for 3 h. After electrophoresis, the gel was transferred to 3-mm Whatman paper and dried. Autoradiography was performed with Typon X-Ray DX-41 film (Typon, Burgdorf, Switzerland). A case was considered informative for a polymorphic marker if normal tissue DNA showed two different alleles. LOH of the informative polymorphic loci was compared with heterozygosity at the analyzed locus by visually evaluating allele band intensity of normal epidermal layer apart from SN basaloid cells and basaloid cells in conjunction with sebaceous glands from the same patient. Complete loss or >50% reduction in a single band intensity in an informative locus was scored as LOH by two independent observers.

	Results

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LOH analysis was performed on 21 SN using four polymorphic markers at 9q22.3 (Table 1) . Twenty SN were informative at one or more loci. The DNA from one sample (case 13) failed to be amplified by any of the polymorphic markers, probably because of the extreme fragmentation of DNA from this sample preserved in paraffin.

	Discussion

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BCC and SN share 9q22.3 deletions and are, therefore, likely to be pathogenetically related. Deletions at the PTCH locus in basaloid cells of SN suggest the presence of a tumor suppressor gene, which could be responsible for the transformation of SN to BCC and, possibly, into other appendageal tumors.

Tumor suppressor genes play an important role in the carcinogenesis of many tissue types. These genes normally exert a negative control on cell growth and inactivation of both homologues ("two hits") is required for a growth-promoting effect. One hit is almost invariably a point mutation. The other homologue is frequently lost through a gross chromosomal rearrangement such as deletion or nondisjunction, which is manifested as LOH for polymorphisms surrounding the gene (8) .

The frequency of LOH in SN as found in our study is strikingly similar to the frequency found in a series of BCC (9) . The possible reasons for why LOH was not detected in 50% of SN include: mutation without deletion; intragenic microdeletion, or deletions that did not expand to adjacent microsatellite markers; methylation of CpG islands; and other undetected genetic factors that might contribute to SN/BCC development (10) . The genetic defect at 9q22.3 might possibly also contribute to the development of other appendageal tumors in SN, e.g., syringocystadenoma papilliferum, primitive follicular germs, and trichoblastomas. To date, however, these tumors have not been genetically analyzed.

Here, we provide the first evidence of the involvement of the tumor suppressor gene PTCH in SN. Our study may support, on a molecular basis, the clinical observation of the development of a subset of SN into BCC.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at University Hospital, Department of Dermatology, Gloriastrasse 31, 8091 Zürich, Switzerland. Phone: 411 255 28 11; Fax: 411 255 44 03.

² The abbreviations used are: SN, sebaceous nevi; BCC, basal cell carcinoma; LOH, loss of heterozygosity.

Received 11/23/98. Accepted 3/ 2/99.

	REFERENCES

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