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Spectrum of PTCH1 Mutations in French Patients with Gorlin Syndrome

2003; Elsevier BV; Volume: 121; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2003.12423.x

1523-1747

Nathalie Boutet, Yves‐Jean Bignon, Valérie Drouin‐Garraud, Pierre Sarda, Michel Longy, Didier Lacombe, Philippe Gorry,

Oral and Maxillofacial Pathology

Gorlin syndrome or nevoid basal cell carcinoma syndrome is an autosomal dominant disease characterized by developmental abnormalities and a predisposition to cancers. The responsible gene for this syndrome is the PTCH tumor suppressor gene encoding for the Sonic Hedgehog receptor. We screened for PTCH mutations in 65 French Gorlin syndrome families or sporadic cases for the first time. Nineteen novel mutations and five new polymorphisms were identified in this group of patients. One microdeletion without frameshift underlines the importance of one amino acid for Ptc receptor function. Although no mutation hot-spot was described, we identified a recurrent mutation. Gorlin syndrome or nevoid basal cell carcinoma syndrome is an autosomal dominant disease characterized by developmental abnormalities and a predisposition to cancers. The responsible gene for this syndrome is the PTCH tumor suppressor gene encoding for the Sonic Hedgehog receptor. We screened for PTCH mutations in 65 French Gorlin syndrome families or sporadic cases for the first time. Nineteen novel mutations and five new polymorphisms were identified in this group of patients. One microdeletion without frameshift underlines the importance of one amino acid for Ptc receptor function. Although no mutation hot-spot was described, we identified a recurrent mutation. nevoid basal cell carcinoma syndrome Gorlin syndrome (Gorlin and Goltz, 1960Gorlin R.J. Goltz R.W. Multiple nevoid basal-cell epithelioma, jaw cysts and bifid rib: A syndrome.N Engl J Med. 1960; 262: 908-912Crossref PubMed Scopus (644) Google Scholar) or nevoid basal cell carcinoma syndrome (NBCCS) (Mendelian Inheritance in Man, MIM no. 109400) is an autosomal dominant genetic disease with nearly complete penetrance and variable expressivity. NBCCS is characterized by development abnormalities and tumor predisposition. The diagnosis is based on the occurrence of three major criteria: basal cell carcinomas, palmar and plantar epidermal pits, jaw keratocysts. Patients might display other clinical features (LoMuzio et al., 1999LoMuzio L. Nocini P.F. Savoia A. et al.Nevoid basal cell carcinoma syndrome. Clinical findings in 37 Italian affected individuals.Clin Genet. 1999; 55: 34-40Crossref PubMed Scopus (125) Google Scholar, and references therein). The severity is due to the basal cell carcinomas and various different tumors (Lacombe et al., 1990Lacombe D. Chateil J.F. Fontan D. Battin J. Medulloblastoma in the nevoid basal-cell carcinoma syndrome: Case reports and review of the literature.Genet Couns. 1990; 1: 273-277PubMed Google Scholar;Evans et al., 1993Evans D.G. Ladusans E.J. Rimmer S. Burnell L.D. Thakker N. Farndon P.A. Complications of the naevoid basal cell carcinoma syndrome: Results of a population based study.J Med Genet. 1993; 30: 460-464Crossref PubMed Scopus (392) Google Scholar). The NBCCS gene was localized to 9q22–31 by genetic linkage studies (Farndon et al., 1992Farndon P.A. Del Mastro R.G. Evans D.G. Kilpatrick M.W. Location of gene for Gorlin syndrome.Lancet. 1992; 339: 581-582Abstract PubMed Scopus (317) Google Scholar;Gailani et al., 1992Gailani M.R. Bale S.J. Leffell D.J. et al.Developmental defects in Gorlin syndrome related to a putative tumor suppressor gene on chromosome 9.Cell. 1992; 69: 111-117Abstract Full Text PDF PubMed Scopus (359) Google Scholar). NBCCS tumors show loss of heterozygosity in this region, arguing for a tumor suppressor gene (Gailani et al., 1992Gailani M.R. Bale S.J. Leffell D.J. et al.Developmental defects in Gorlin syndrome related to a putative tumor suppressor gene on chromosome 9.Cell. 1992; 69: 111-117Abstract Full Text PDF PubMed Scopus (359) Google Scholar;Levanat et al., 2000Levanat S. Pavelic B. Crnic I. Oreskovic S. Manojlovic S. Involvement of PTCH gene in various noninflammatory cysts.J Mol Med. 2000; 78: 140-146Crossref PubMed Scopus (45) Google Scholar). The responsible gene PTCH has been identified by positional cloning as the human homolog of the Drosophila patched gene (Hahn et al., 1996Hahn H. Wicking C. Zaphiropoulous P.G. et al.Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.Cell. 1996; 85: 841-851Abstract Full Text Full Text PDF PubMed Scopus (1611) Google Scholar;Johnson et al., 1996Johnson R.L. Rothman A.L. Xie J. et al.Human homolog of patched, a candidate gene for the basal cell nevus syndrome.Science. 1996; 272: 1668-1671Crossref PubMed Scopus (1571) Google Scholar). PTCH has 23 exons, which span 34 kb. It encodes a transmembrane glycoprotein composed of 1447 amino acids, with 12 transmembrane domains. In 1996, two groups initially described germline mutations of PTCH gene in Gorlin patients (Hahn et al., 1996Hahn H. Wicking C. Zaphiropoulous P.G. et al.Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.Cell. 1996; 85: 841-851Abstract Full Text Full Text PDF PubMed Scopus (1611) Google Scholar;Johnson et al., 1996Johnson R.L. Rothman A.L. Xie J. et al.Human homolog of patched, a candidate gene for the basal cell nevus syndrome.Science. 1996; 272: 1668-1671Crossref PubMed Scopus (1571) Google Scholar). Since these two reports, many PTCH germline mutations have been described; most were frameshift or nonsense mutations leading to the synthesis of a truncated Ptc protein (Chidambaram et al., 1996Chidambaram A. Goldstein A.M. Gailani M.R. et al.Mutations in the human homologue of the Drosophila patched gene in Caucasian and African-American nevoid basal cell carcinoma syndrome patients.Cancer Res. 1996; 56: 4599-4601PubMed Google Scholar;Unden et al., 1996Unden A.B. Holmberg E. Lundh-Rozell B. Stahle-Backdahl M. Zaphiropoulos P.G. Toftgard R. Vorechovsky I. Mutations in the human homologue of Drosophila patched (PTCH) in basal cell carcinomas and the Gorlin syndrome: Different in vivo mechanisms of PTCH inactivation.Cancer Res. 1996; 56: 4562-4565PubMed Google Scholar;Lench et al., 1997Lench N.J. Telford E.A. High A.S. Markham A.F. Wicking C. Wainwright B.J. Characterisation of human patched germ line mutations in naevoid basal cell carcinoma syndrome.Hum Genet. 1997; 100: 497-502Crossref PubMed Scopus (73) Google Scholar;Wicking et al., 1997Wicking C. Gillies S. Smyth I. et al.De novo mutations of the Patched gene in nevoid basal cell carcinoma syndrome help to define the clinical phenotype.Am J Med Genet. 1997; 73: 304-307Crossref PubMed Scopus (37) Google Scholar;Aszterbaum et al., 1998Aszterbaum M. Rothman A. Johnson R.L. et al.Identification of mutations in the human PATCHED gene in sporadic basal cell carcinomas and in patients with the basal cell nevus syndrome.J Invest Dermatol. 1998; 110: 885-888Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar;Hasenpusch-Theil et al., 1998Hasenpusch-Theil K. Bataille V. Laehdetie J. Obermayr F. Sampson J.R. Frischauf A.M. Gorlin syndrome: Identification of 4 novel germ-line mutations of the human patched (PTCH) gene. Mutations in brief no. 137. Online.Hum Mutat. 1998; 11: 480Crossref PubMed Google Scholar;Smyth et al., 1998Smyth I. Wicking C. Wainwright B. Chenevix-Trench G. The effects of splice site mutations in patients with naevoid basal cell carcinoma syndrome.Hum Genet. 1998; 102: 598-601Crossref PubMed Scopus (19) Google Scholar). In this study, we have assessed the spectrum of PTCH germline mutation in the French population. Patients were evaluated by a geneticist for the presence of at least one of the three main features of NBCCS. Sixty-five families were collected, 23 familial cases and 42 sporadic cases. Familial cases included 46 patients and 33 nonaffected siblings. Nonaffected unrelated individuals from the same area were used to define a control population. The local ethics committee approved this study, and patient informed consent was obtained. DNA was extracted with QIAmp Blood kit (Qiagen Courtaboeuf, France). We used 24 pairs of primers previously reported for the amplification of the 23 exons of the PTCH gene (sequence no. U59464; exons 1–8, 11, 14–20, 23, and intron 1 (Hahn et al., 1996Hahn H. Wicking C. Zaphiropoulous P.G. et al.Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.Cell. 1996; 85: 841-851Abstract Full Text Full Text PDF PubMed Scopus (1611) Google Scholar); exons 9 and 10 (Wolter et al., 1997Wolter M. Reifenberger J. Sommer C. Ruzicka T. Reifenberger G. Mutations in the human homologue of the Drosophila segment polarity gene patched (PTCH) in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system.Cancer Res. 1997; 57: 2581-2585PubMed Google Scholar); exons 12, 13, and 21 (Pietsch et al., 1997Pietsch T. Waha A. Koch A. et al.Medulloblastomas of the desmoplastic variant carry mutations of the human homologue of Drosophila patched.Cancer Res. 1997; 57: 2085-2088PubMed Google Scholar), and exon 22 (Chidambaram et al., 1996Chidambaram A. Goldstein A.M. Gailani M.R. et al.Mutations in the human homologue of the Drosophila patched gene in Caucasian and African-American nevoid basal cell carcinoma syndrome patients.Cancer Res. 1996; 56: 4599-4601PubMed Google Scholar)) (detailed PCR procedures are available upon request). Single-strand conformation polymorphism or Heteroduplex Analysis (HA) analyses were performed as described previously (Orita et al., 1989Orita M. Iwahana H. Kanazawa H. Hayashi K. Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.Proc Natl Acad Sci USA. 1989; 86: 2766-2770Crossref PubMed Scopus (3327) Google Scholar;White et al., 1992White M.B. Carvalho M. Derse D. O'Brien S.J. Dean M. Detecting single base substitutions as heteroduplex polymorphisms.Genomics. 1992; 12: 301-306Crossref PubMed Scopus (340) Google Scholar). HA was conducted at 100 V, for 14 h. Single-strand conformation polymorphism was realized on a Genephore electrophoresis apparatus with GeneGel Clean 15/24 at 4°C, 20°C, 600 V, during 1 h 20 min. Staining was done with a silver staining kit (Pharmacia). Sequencing of amplicons demonstrating band shifts was performed with a Big Dye Terminator Cycle Sequencing kit on an ABI Prism 377 analyzer (Perkin-Elmer). Amplification product was sequenced on both strands. Sequence analysis was conducted with a GCG sequence analysis software package (University of Wisconsin). We have screened the PTCH gene for mutations by PCR single-strand conformation polymorphism or HA analysis in 65 family or sporadic cases and identified 19 new mutations (Table I). Mutation segregation with the disease in each family was tested. A total of 29 Gorlin patients bearing a PTCH mutation were identified.Table IMutations and polymorphisms detected in the PTCH 1 geneMutationsExonNucleotide changeEffect on protein12c260del5TinsAAL87del, stop at 8722280insA94 frameshift, stop at 13833c531del ACAQ177del45c672C>AY224X55c742delCTinsGGAG248 frameshift, stop at 2496intron 5IVS5+3 del AA78c1194delCinsATATG398 frameshift, stop at 43688c1208delAT403 framshift, stop at 43598c1208delAT403 frameshift, stop at 435108c1208delAT403 frameshift, stop at 4351110c1450G>AG484R1211c1511C>TP504L1314c1941C>AS647R1414c2178insC721 frameshift, stop at 7361515c2465T>CL822P1616c2619C>AY873X1718c3042delC1014 frameshift, stop at 10481817c2776T>CW926R1921c3583A>TT1195SPolymorphisms5c735A>GT245Tintron 5IVS5+12A>Gintron 7IVS7+74 A>Gintron 10IVS10-52 G>C12c1665T>CN555N12c1686C>TA562Aintron 15IVS15+9 C>G17c2787C>TN929N21c3471G>AA1157A21c3524T>GL1175W23c3944T>CL1315P Open table in a new tab Two nonsense mutations were identified: Y224X, localized after the first transmembrane domain, and Y873X, after the first group of transmembrane domains. We observed seven frameshift mutations giving rise to various truncated Ptc proteins. They are deleted of at least the last four transmembrane domains (cytosine deletion at 3042 in exon 18) or deleted of all transmembrane domains at the most (complex mutation in exon 2). A two-nucleotide microdeletion at 1208–1209 in exon 8 was identified in three different cases of NBCCS (one sporadic and two familial cases), coming from different parts of France without any evidence of familial links. Research for a common allele was done with microsatellite markers D9S196, D9S287, D9S1786, D9S180, D9S1783, and D9S109. No common morbid haplotype is shared between these three cases (data not shown). Finally, we identified, in a typical sporadic case of Gorlin syndrome, a deletion of two nucleotides in intron 5 with A to G substitution that occurs at the exon-intron junction and changes the nature of the nucleotide after the consensus sequence (GT). It has not been found in a population of 100 control individuals and may alter the synthesis of the normal mRNA. In a sporadic case, a three-nucleotide microdeletion was identified in exon 3 deleting a conserved glutamine among vertebrate species (Figure 1). This glutamine is localized at the beginning of the first extracellular loop of the Ptc receptor. This microdeletion was not detected in a population of 100 control individuals. In this study, we report six missense mutations (Table I) occurring in exons 10, 11, 14, 15, 17, and 21. G484R and P504L occur in the tenth transmembrane domain. S647R is in the intracellular loop between the two groups of transmembrane domains. L822P and W926R are localized in an extracellular loop. Finally, T1195S occurs at the C-terminal intracellular end. These sequence variations have not been found in a population of 200 control chromosomes. They segregate with the disease in familial cases of NBCCS. DNA variants were classified as polymorphisms either because they do not lead to amino acid change, or because there is no segregation with the disease in the family, or because they were found in a cohort of 200 normal chromosomes (Table I). Eleven polymorphisms were identified, of which five have not been described before (N929N, A1157A, L1175W, IVS5+12 A>G, and IVS7+74 A>G). They appear in the general population at the following frequencies: T245T (10%), intron 5 (2%), intron 7 (<1%), intron 10 (14.5%), N555N (10%), A562A (13%), intron 15 (6%), N929N (<1%), A11571 (2%), L1175W (2%), and L1315P (6%). We screened French NBCCS patients and identified 19 novel mutations with 11 polymorphisms (Table I). In agreement with the literature, we found mainly frameshift mutations; most of them are characterized by a truncated protein lacking the last group of six transmembrane domains. The high prevalence of typical sporadic cases in our study, however, underlines the high frequency of PTCH spontaneous mutations (Aszterbaum et al., 1998Aszterbaum M. Rothman A. Johnson R.L. et al.Identification of mutations in the human PATCHED gene in sporadic basal cell carcinomas and in patients with the basal cell nevus syndrome.J Invest Dermatol. 1998; 110: 885-888Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar;Hasenpusch-Theil et al., 1998Hasenpusch-Theil K. Bataille V. Laehdetie J. Obermayr F. Sampson J.R. Frischauf A.M. Gorlin syndrome: Identification of 4 novel germ-line mutations of the human patched (PTCH) gene. Mutations in brief no. 137. Online.Hum Mutat. 1998; 11: 480Crossref PubMed Google Scholar;Smyth et al., 1998Smyth I. Wicking C. Wainwright B. Chenevix-Trench G. The effects of splice site mutations in patients with naevoid basal cell carcinoma syndrome.Hum Genet. 1998; 102: 598-601Crossref PubMed Scopus (19) Google Scholar; and references therein). PTCH mutations are irregularly distributed along the coding sequence and no hot-spot mutations have been reported yet. The frameshift mutation in exon 8 is found in three different families, however (G, H, I in Table II). So far, no founder effect of PTCH gene mutations has been described in the literature. We did not find any arguments to support a founder effect. Indeed, some mutations in different exons have been reported twice in the literature without any knowledge of recurrence (Hahn et al., 1996Hahn H. Wicking C. Zaphiropoulous P.G. et al.Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.Cell. 1996; 85: 841-851Abstract Full Text Full Text PDF PubMed Scopus (1611) Google Scholar;Wicking et al., 1997Wicking C. Gillies S. Smyth I. et al.De novo mutations of the Patched gene in nevoid basal cell carcinoma syndrome help to define the clinical phenotype.Am J Med Genet. 1997; 73: 304-307Crossref PubMed Scopus (37) Google Scholar). We propose that mutation c1208–1209delAT is the first truly recurrent mutation identified in PTCH gene. Patients bearing this mutation have a different clinical outcome, which pinpoints the possible involvement of modifier genes as identified for instance in neurofibromatosis type 1 (Bahuau et al., 2001Bahuau M. Pelet A. Vidaud D. et al.GDNF as a candidate modifier in a type 1 neurofibromatosis (NF1) enteric phenotype.J Med Genet. 2001; 38: 638-643Crossref PubMed Google Scholar). The identification of this mutation in monozygotic twins displaying different clinical features, however (family G, probands G2 and G3, Table II), supports also the role of the environment (Nelson et al., 2002Nelson H.H. Kelsey K.T. Mott L.A. Karagas M.R. The XRCC1 Arg399Gln polymorphism, sunburn, and non-melanoma skin cancer: Evidence of gene–environnement interaction.Cancer Res. 2002; 62: 152-155PubMed Google Scholar).Table IIClinical findings in French Gorlin patientsDysmorphologyTumorsCase identifierFamilial/SporadicSexeAgePitsCystsOtherBCCOtherMutation identifierA.1FamilialF18++++1A.2FamilialF40+++1BSporadicF38++2CSporadicM20+++3DSporadicM38++4EFamilialF41+++5FSporadicM16++++6GFamilialM7+7G.1FamilialM64++++8G.2FamilialM33++8G.3FamilialM33++++8HSporadicF13+++9I.1FamilialF15++++10I.1FamilialF18+++10I.1FamilialF39+10JFamilialG39++++11KFamilialF33+++12LFamilialF37++13M.1FamilialF15+14M.2FamilialM41+14N.1FamilialM55+++++15N.2FamilialF++++15OSporadicM30++16PSporadicM33+++17Q.1FamilialF19++++18Q.2FamilialF9++18Q.3FamilialF16+++18Q.4FamilialF36+++18RSporadicM50++19BCC, Basal Cell Carcinoma; M, male; F, female; Pits, palmar and plantar pits; Cysts, keratocysts; Dysmorphology other, any other dysmorphic symptom; Tumors other, any type of tumor. Open table in a new tab BCC, Basal Cell Carcinoma; M, male; F, female; Pits, palmar and plantar pits; Cysts, keratocysts; Dysmorphology other, any other dysmorphic symptom; Tumors other, any type of tumor. Interestingly, we report a unique deletion of a single amino acid conserved in vertebrate species localized at the beginning of the first extracellular loop of the receptor (Figure 1). It would be interesting to know if this amino acid is important for the interaction of Hedgehog with its Ptc receptor. Unfortunately, no mutagenesis data or binding studies are available to document the effect of this mutation. Finally, we describe several sequence variations not found in the general population segregating with the disease in the family, considered as missense mutations. According to the literature, missense mutations do not occur as frequently as frameshift or nonsense mutations (15% vs 85%). They are spread along the PTCH gene (Aszterbaum et al., 1998Aszterbaum M. Rothman A. Johnson R.L. et al.Identification of mutations in the human PATCHED gene in sporadic basal cell carcinomas and in patients with the basal cell nevus syndrome.J Invest Dermatol. 1998; 110: 885-888Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar;Smyth et al., 1998Smyth I. Wicking C. Wainwright B. Chenevix-Trench G. The effects of splice site mutations in patients with naevoid basal cell carcinoma syndrome.Hum Genet. 1998; 102: 598-601Crossref PubMed Scopus (19) Google Scholar; and references therein). In our study, missense mutations span the second group of six transmembrane domains of the protein. Molecular and cellular studies on Ptc mutant proteins in Drosophila would argue for disruption of the normal topology and/or sorting of the receptor (Strutt et al., 2001Strutt H. Thomas C. Nakano Y. Stark D. Neave B. Taylor A.M. Ingham P.W. Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation.Curr Biol. 2001; 11: 608-613Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Unfortunately, such data are not available to interpret the pathologic value of the missense mutations described in humans. Improvement of the PTCH gene screening is necessary but the nondetection of PTCH mutations in half of the typical familial cases might also underline the nonexploration of other genetic events. Many studies have shown splice mutations (Aszterbaum et al., 1998Aszterbaum M. Rothman A. Johnson R.L. et al.Identification of mutations in the human PATCHED gene in sporadic basal cell carcinomas and in patients with the basal cell nevus syndrome.J Invest Dermatol. 1998; 110: 885-888Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar;Smyth et al., 1998Smyth I. Wicking C. Wainwright B. Chenevix-Trench G. The effects of splice site mutations in patients with naevoid basal cell carcinoma syndrome.Hum Genet. 1998; 102: 598-601Crossref PubMed Scopus (19) Google Scholar; and references therein). Small deletions encompassing several exons might also take place as has already been demonstrated in other cancer-prone genetic diseases (Dorschner et al., 2000Dorschner M.O. Sybert V.P. Weaver M. Pletcher B.A. Stephens K. NF1 microdeletion breakpoints are clustered at flanking repetitive sequences.Hum Mol Genet. 2000; 9: 35-46Crossref PubMed Scopus (149) Google Scholar). We are grateful to the patients and the physicians, especially Drs D. Augias, P. Bitoun, Devauchelle, H. Journel, G. Lecanelle, M. Le Merrer, S. Lyonnet, G. Matthijs, N. Philip, P. Saiag, B. Sassolas, and A. Zankl. We thank Dr F. Bonnet-Dorion for advice and D. Lafon for technical help. This work was supported by grants from the Association pour la Recherche sur le Cancer, Fondation pour la Recherche Médicale, Ligue National Contre le Cancer, and Plan Hospitalier pour la Recherche Clinique to P.G. N.B. was a recipient of a Fédération des Maladies Orphelines fellowship.