PTCH2, a Novel Human Patched Gene, Undergoing Alternative Splicing and Up-Regulated in Basal Cell Carcinomas1

[CANCER RESEARCH 59, 787–792, February 15, 1999] Advances in Brief PTCH2, a Novel Human Patched Gene, Undergoing Alternative Splicing and Up-regulated in Basal Cell Carcinomas1 Peter G. Zaphiropoulos,2 Anne Birgitte Unde´n, Fahimeh Rahnama, Robert E. Hollingsworth,3 and Rune Toftgård2 Department of Bioscience, Center for Nutrition and Toxicology, Karolinska Institute, Novum 141 57, Huddinge, Sweden [P. G. Z., A. B. U., F. R., R. T.], and Cancer Research, Pharmacia and Upjohn, Inc., Kalamazoo, Michigan 49001 [R. E. H.] Abstract appears to be involved in the SHH/PTCH cell signaling, having similar but still distinct properties than PTCH1. By a combination of cDNA library screening, rapid amplification of cDNA ends analysis, and BAC sequencing, a novel human patched-like Materials and Methods gene (PTCH2) has been cloned and sequenced. The genomic organization is similar to PTCH1 with 22 exons and, by radiation hybrid mapping, cDNA and Gene Cloning and Radiation Hybrid Mapping. The RACE PTCH2 has been localized to chromosome 1p33-34, a region often lost in analysis was performed essentially as described before (10), using the Mara- a variety of tumors. Several alternatively spliced mRNA forms of PTCH2 thon kit (Promega). The primer sequences used for RACE are available on were identified, including transcripts lacking segments thought to be request. A BAC clone encompassing the PTCH2 gene was isolated using involved in sonic hedgehog binding and mRNAs with differentially de- primers corresponding to the 39 untranslated region of the cDNA. The genomic fined 3* terminal exons. In situ hybridization revealed high expression of organization was obtained by a combination of direct sequencing of the BAC PTCH2 transcripts in both familial and sporadic basal cell carcinomas in and by sequencing of PCR products generated with primers originating from similarity to what has been observed for PTCH1, suggesting a negative adjacent exons. The same 39 end primers used to isolate the BAC clone were regulation of PTCH2 by PTCH1. This finding tightly links PTCH2 with also used to screen the GENEBRIDGE radiation hybrid panel. the sonic hedgehog/PTCH signaling pathway, implying that PTCH2 has Tumor Samples. A total of 11 formalin-fixed paraffin-embedded BCCs related, but yet distinct, functions than PTCH1. derived from seven patients were obtained from the Department of Dermatol- ogy, Karolinska Hospital (Stockholm, Sweden). Five tumors were from Introduction NBCCS patients, two tumors were from a young patient with multiple BCCs (not classified as NBCCS), and four tumors were sporadic BCCs. All histo- PTCH 4 was recently identified as the gene responsible for the logical subtypes of BCCs were represented. In addition, samples from two TEs Gorlin’s syndrome, an autosomal dominant disorder characterized by were included. Both were sporadic tumors. The histological diagnoses were confirmed by an experienced dermatopathologist. multiple BCCs, medulloblastomas, and ovarian fibromas, as well as In Situ Hybridization. Preparation of PTCH2-mRNA probes and in situ numerous developmental anomalies (1, 2). PTCH codes for a mem- hybridization were performed as described previously (9). Briefly, two human brane receptor of the autocatalytically cleaved (protein-spliced), ami- PTCH2 cDNA fragments corresponding to positions 218–437 and 838–920, no-terminal domain of SHH (3, 4). In the nonsignaling state, PTCH is cloned into pGEM5, were used. Tumor sections were treated with proteinase thought to inhibit the constitutive signaling of another membrane K and washed in 0.1 M triethanolamine buffer containing 0.25% acetic anhy- protein, SMO, however, binding of SHH to PTCH relieves this dride. Sections were hybridized with 2.5 3 106 cpm of each of the two labeled inhibition (5). This cascade of signaling events, best characterized in antisense or sense probes at 55°C. Autoradiography was performed for 2 Drosophila, also involves a number of intracellular components in- weeks. Immunohistochemistry. A monoclonal mouse antibody directed against cluding fused (a serine threonine kinase), suppressor of fused, costal human Ki-67 was used (Immunotech). Sections from each tumor sample were 2, and cubitus interruptus (6). The latter is a transcription factor that incubated with the antibody for 1 h and processed using a standard avidin- positively regulates the expression of target genes that also include biotin immunoperioxidase/diaminobenzidine detection system. PTCH itself. Mutations in the PTCH gene have been identified in both sporadic and familial BCCs (7, 8). The lack of the normal PTCH Results and Discussion protein in these cells allows the constitutive signaling of SMO to To identify additional components of the PTCH/SHH cascade of occur, resulting in the accumulation of mutant PTCH mRNAs (9). In signaling events, the Incyte LifeSeqTM database was searched using this study, we describe the cloning and characterization, including PTCH sequences. In addition to clones representing the PTCH cDNA, differentially spliced mRNA forms, of a related gene, PTCH2,5 that two nearly identical cDNAs were identified from the parotid gland and the colon, which contained sequences similar to, but distinct from, Received 11/30/98; accepted 12/31/98. the 39 end of PTCH. By 59 RACE analysis using fetal brain cDNAs, 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 additional sequence information from these transcripts (termed 18 U.S.C. Section 1734 solely to indicate this fact. PTCH2) and, corresponding to a full-length cDNA, was obtained (Fig. 1 Supported by the Swedish Cancer Fund, the Welander-Finsen Foundation, the Swedish Radiation Protection Institute, the Swedish Children Cancer Fund, and the 1A). PTCH2 is 57% identical to PTCH1, with a significantly variable Karolinska Institute. region present between the transmembrane domains 6 and 7, and 91% 2 To whom requests for reprints should be addressed, at Department of Bioscience, identical to the recently published mouse Ptch2 sequence (11). In Center for Nutrition and Toxicology, Karolinska Institute, Novum 141 57, Huddinge, Sweden. Phone: 46-8-6089151 (P. G. Z.) or 46-8-6089152 (R. T.); Fax: 46-8-6081501; similarity with the mouse gene, PTCH2 lacks the COOH-terminal E-mail: [email protected] or [email protected]. extension present in human, mouse, and chicken PTCH1 (12, 13). 3 Present address: Genomic Sciences, Glaxo Wellcome Inc., Research Triangle Park, However, the human PTCH2 cDNA terminates 36 amino acids earlier NC 27709. 4 The abbreviations used are: PTCH, human patched; BCC, basal cell carcinoma; SHH, than the mouse Ptch2 sequence. Characterization of the PTCH2 gene sonic hedgehog; SMO, smoothened; TE, trichoepithelioma; RACE, rapid amplification of revealed 22 coding exons and conservation of the intron-exon junc- cDNA ends; NBCCS, nevoid basal cell carcinoma syndrome. 5 The PTCH2 sequence has been submitted to the GenBank (accession number tions relative to PTCH1, except for the last intron (Fig. 1B). More- AF119569). over, when 39 RACE was performed from fetal brain, an alternate 787 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. A NOVEL PTCH GENE Fig. 1. A, amino acid sequence comparison of the human PTCH2 (top lines) and PTCH1 sequences. Vertical lines, identical amino acids; dots, similar amino acids. The PTCH2 sequence presented is composed of the original cDNA clones and of the products of the 59 RACE analysis. B, intron-exon organization of the PTCH2 gene. The intron-exon borders are shown, with small letters representing intronic sequences and capital letters representing exonic sequences. C, schematic representation of the alternative splicing events that result in different COOH termini. In the parotid gland and the colon, the penultimate and the last exon are canonically joined together. In fetal brain, however, the penultimate exon with part of the 39 intron functions as the terminal exon. The intronic sequence is shown by small letters, with the flanking exonic shown by capital letters. Above the nucleotide sequence, the deduced amino acid sequence is shown, and below is the corresponding sequence of mouse Ptch2. The conserved intronic dinucleotides are shown by bold letters, and the termination signals are indicated by *. Note the absence of conservation of the position of the termination codons between the mouse and human PTCH2 sequences. The putative polyadenylation signals are also shown in this diagram. The genomic organization was obtained by analyzing a BAC clone encompassing the PTCH2 gene. D, schematic representation of the different variations of spliced transcripts encompassing exon 1 and exon 2 sequences. The canonical exons 1 and 2 are shown by boxes, and the intron between them is shown by a solid line. The GT and AG dinucleotides spanning the sequences that are used as introns in individual transcripts are indicated by small letters. G, genomic structure, derived from sequencing segments of a BAC clone encompassing the PTCH2 gene; C, canonical transcript; A, transcript A (the skipped exons 9 and 10 of this product are not shown in the diagram); B, transcript B. 788 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. A NOVEL PTCH GENE Fig. 1. C, D, Continued. 789 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. A NOVEL PTCH GENE Fig. 2. A, a dark-field photomicrograph of a BCC tumor hybridized with 35S-labeled antisense probe showing abundant signal for PTCH1 mRNA (light grains) in all BCC tumor cells. B, PTCH2 mRNA overexpression in BCC is in contrast mainly expressed in the basaloid cells in the periphery of the tumor nests (arrow). C, another BCC showing a strong PTCH2 mRNA signal in the periphery of the tumor nest (Tu), whereas no signal is detected in epidermis (Ep). D, sections of the same tumor (C) hybridized with the PTCH2 sense probe showed no signal.

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