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Spectrum of PTCH1 Mutations in French Patients with Gorlin Syndrome

à à Nathalie Boutet, Yves-Jean Bignon,w ValeŁ rie Drouin-Garraud,z Pierre Sarda,y Michel Longy, Didier Lacombe,z and Philippe GorryÃz à Laboratoire de GeŁ neŁ tique Oncologique, Institut BergonieŁ , Bordeaux, France; wUniteŁ d’OncogeŁ neŁ tique, Center Jean Perrin, Clermont-Ferrand, France; zUniteŁ de GeŁ neŁ tique Clinique, CHU Charles Nicolle, Rouen, France; yUniteŁ de GeŁ neŁ tique MeŁ dicale, CHU Arnaud de Villeneuve, Montpellier, France; zService de GeŁ neŁ tique MeŁ dicale, CHU Pellegrin-Enfants, Bordeaux, France

Gorlin syndrome or nevoid basal cell carcinoma syn- new polymorphisms were identi¢ed in this group of drome is an autosomal dominant disease characterized patients. One microdeletion without frameshift by developmental abnormalities and a predisposition to underlines the importance of one amino acid for Ptc cancers. The responsible for this syndrome is the function. Although no mutation hot-spot was des- PTCH encoding for the Sonic cribed, we identi¢ed a recurrent mutation. Key words: Hedgehog receptor. We screened for PTCH mutations basal cell/carcinoma/genetic/germline mutation/skin disease. in 65 French Gorlin syndrome families or sporadic cases J Invest Dermatol 121:478 ^481, 2003 for the ¢rst time. Nineteen novel mutations and ¢ve

orlin syndrome (Gorlin and Goltz, 1960) or nevoid In this study, we have assessed the spectrum of PTCH germ- basal cell carcinoma syndrome (NBCCS) (Mende- line mutation in the French population. lian Inheritance in Man, MIM no. 109400) is an autosomal dominant genetic disease with nearly complete penetrance and variable expressivity. MATERIALS AND METHODS GNBCCS is characterized by development abnormalities and tu- mor predisposition. The diagnosis is based on the occurrence of Patients Patients were evaluated by a geneticist for the presence of at three major criteria: basal cell carcinomas, palmar and plantar epi- least one of the three main features of NBCCS. Sixty-¢ve families were collected, 23 familial cases and 42 sporadic cases. Familial cases included dermal pits, jaw keratocysts. Patients might display other clinical 46 patients and 33 nona¡ected siblings. Nona¡ected unrelated individuals features (Lo Muzio et al, 1999, and references therein).The severity from the same area were used to de¢ne a control population. The local is due to the basal cell carcinomas and various di¡erent tumors ethics committee approved this study, and patient informed consent was (Lacombe et al, 1990; Evans et al,1993). obtained. The NBCCS gene was localized to 9q22^31 by genetic linkage studies (Farndon et al, 1992; Gailani et al, 1992). NBCCS tumors Mutation screening DNA was extracted with QIAmp Blood show loss of heterozygosity in this region, arguing for a tumor (Qiagen Courtaboeuf, France). We used 24 pairs of primers previously suppressor gene (Gailani et al, 1992; Levanat et al, 2000). The re- reported for the ampli¢cation of the 23 exons of the PTCH gene sponsible gene PTCH has been identi¢ed by positional cloning (sequence no. U59464; exons 1^8, 11, 14^20, 23, and intron 1 (Hahn et al, 1996); exons 9 and 10 (Wolter et al, 1997); exons 12, 13, and 21 (Pietsch et al, as the human homolog of the Drosophila gene (Hahn et al, 1997), and exon 22 (Chidambaram et al, 1996)) (detailed PCR procedures 1996; Johnson et al, 1996). PTCH has 23 exons, which span 34 kb. are available upon request). It encodes a transmembrane glycoprotein composed of 1447 ami- Single-strand conformation polymorphism or Heteroduplex Analysis no acids, with 12 transmembrane domains. (HA) analyses were performed as described previously (Orita et al, 1989; In 1996, two groups initially described germline mutations of White et al, 1992). HA was conducted at 100 V, for 14 h. Single-strand PTCH gene in Gorlin patients (Hahn et al, 1996; Johnson et al, conformation polymorphism was realized on a Genephore electrophoresis 1996). Since these two reports, many PTCH germline mutations apparatus with GeneGel Clean 15/24 at 41C, 201C, 600 V, during 1 h have been described; most were frameshift or nonsense mutations 20 min. Staining was done with a silver staining kit (Pharmacia). leading to the synthesis of a truncated Ptc (Chidambaram Sequencing of amplicons demonstrating band shifts was performed with a Big Dye Terminator Cycle Sequencing kit on an ABI Prism 377 et al, 1996; Unden et al, 1996; Lench et al, 1997; Wicking et al,1997; analyzer (Perkin-Elmer). Ampli¢cation product was sequenced on both Aszterbaum et al, 1998; Hasenpusch-Theil et al, 1998; Smyth et al, strands. Sequence analysis was conducted with a GCG sequence analysis 1998). software package (University of Wisconsin).

Manuscript received December 18, 2002; revised March 18, 2003; RESULTS accepted for publication April 3, 2003 We have screened the PTCH gene for mutations by PCR single- Reprint requests to: Dr Philippe Gorry, Laboratoire de GeŁ neŁ tique Oncolo- gique, Institut BergonieŁ , 33076 Bordeaux, France; Email: gorry@bergonie. strand conformation polymorphism or HA analysis in 65 family org or sporadic cases and identi¢ed 19 new mutations (Tab l e I ). Mu- Abbreviation: NBCCS, nevoid basal cell carcinoma syndrome. tation segregation with the disease in each family was tested.

0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc.

478 VOL. 121, NO. 3 SEPTEMBER 2003 PTCH1 MUTATIONS IN GORLIN SYNDROME 479

Table I. Mutations and polymorphisms detected in the PTCH 1 gene Mutations Exon Nucleotide change E¡ect on protein

1 2 c260del5TinsAA L87del, stop at 87 2 2 280insA 94 frameshift, stop at 138 3 3 c531del ACA Q177del 4 5 c672C4A Y224X 5 5 c742delCTinsGGAG 248 frameshift, stop at 249 6 intron 5 IVS5 þ3 del AA 7 8 c1194delCinsATATG 398 frameshift, stop at 436 8 8 c1208delAT 403 framshift, stop at 435 9 8 c1208delAT 403 frameshift, stop at 435 10 8 c1208delAT 403 frameshift, stop at 435 11 10 c1450G4A G484R 12 11 c1511C4TP504L 13 14 c1941C4AS647R 14 14 c2178insC 721 frameshift, stop at 736 15 15 c2465T4C L822P 16 16 c2619C4AY873X 17 18 c3042delC 1014 frameshift, stop at 1048 18 17 c2776T4C W926R 19 21 c3583A4TT1195S Polymorphisms 5c735A4GT245T Figure1. Identi¢cation of mutation Q177del in exon 3 of the PTCH intron 5 IVS5 þ12A4G gene. (A) DNA sequencing of the ACA deletion. (B) Amino acid sequence intron 7 IVS7 þ 74 A4G conservation of the mutated amino acid. Alignment of the amino acid se- intron 10 IVS10-52 G4C quences of human (Hs), mouse (Mm), zebra¢sh (Dr), chicken (Gg), and 12 c1665T4CN555N Xenopus (Xl) PTCH protein (GenBank Accession nos. U59464, U46155, 12 c1686C4TA562A AJ007742, U40074, and AF302765, respectively). The deleted amino acid is intron 15 IVS15 þ9C4G boxed. 17 c2787C4T N929N 21 c3471G4AA1157A 21 c3524T4GL1175W 23 c3944T4CL1315P localized at the beginning of the ¢rst extracellular loop of the Ptc receptor. This microdeletion was not detected in a population of 100 control individuals.

A total of 29 Gorlin patients bearing a PTCH mutation were Missense mutations In this study, we report six missense identi¢ed. mutations (Tab l e I ) occurring in exons 10, 11, 14, 15, 17, and 21. G484R and P504L occur in the tenth transmembrane domain. Nonsense and frameshift mutations Two nonsense mutations S647R is in the intracellular loop between the two groups of were identi¢ed: Y224X, localized after the ¢rst transmembrane transmembrane domains. L822P and W926R are localized in an domain, and Y873X, after the ¢rst group of transmembrane extracellular loop. Finally, T1195S occurs at the C-terminal domains. intracellular end. These sequence variations have not been found We observed seven frameshift mutations giving rise to various in a population of 200 control . They segregate with truncated Ptc . They are deleted of at least the last four the disease in familial cases of NBCCS. transmembrane domains (cytosine deletion at 3042 in exon 18) or deleted of all transmembrane domains at the most (complex Polymorphisms DNA variants were classi¢ed as mutation in exon 2). polymorphisms either because they do not lead to amino acid A two-nucleotide microdeletion at 1208^1209 in exon 8 was change, or because there is no segregation with the disease in the identi¢ed in three di¡erent cases of NBCCS (one sporadic and family, or because they were found in a cohort of 200 normal two familial cases), coming from di¡erent parts of France chromosomes (Ta b l e I ). Eleven polymorphisms were identi¢ed, without any evidence of familial links. Research for a common of which ¢ve have not been described before (N929N, A1157A, allele was done with microsatellite markers D9S196, D9S287, L1175W, IVS5 þ12 A4G, and IVS7 þ 74 A4G). They appear in D9S1786, D9S180, D9S1783, and D9S109. No common morbid the general population at the following frequencies: T245T haplotype is shared between these three cases (data not shown). (10%), intron 5 (2%), intron 7 (o1%), intron 10 (14.5%), Finally, we identi¢ed, in a typical sporadic case of Gorlin N555N (10%), A562A (13%), intron 15 (6%), N929N (o1%), syndrome, a deletion of two nucleotides in intron 5 with A to A11571 (2%), L1175W (2%), and L1315P (6%). G substitution that occurs at the exonintron junction and changes the nature of the nucleotide after the consensus sequence (GT). It has not been found in a population of 100 DISCUSSION control individuals and may alter the synthesis of the normal mRNA. We screened French NBCCS patients and identi¢ed 19 novel mu- tations with 11 polymorphisms (Tab l e I ). In agreement with the Inframe deletion In a sporadic case, a three-nucleotide literature, we found mainly frameshift mutations; most of them microdeletion was identi¢ed in exon 3 deleting a conserved are characterized by a truncated protein lacking the last group of glutamine among vertebrate species (Fig 1). This glutamine is six transmembrane domains. The high prevalence of typical 480 BOUTET ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table II. Clinical ¢ndings in French Gorlin patients Dysmorphology Tumors Mutation Case identi¢er Familial/Sporadic Sexe Age Pits Cysts Other BCC Other identi¢er

A.1 Familial F 18 þþ þ þ 1 A.2 Familial F 40 þþþ 1 B Sporadic F 38 þþ2 C Sporadic M 20 þþþ 3 D Sporadic M 38 þþ 4 E Familial F 41 þþþ 5 F Sporadic M 16 þþ þ þ 6 G Familial M 7 þ 7 G.1 Familial M 64 þþ þ þ 8 G.2 Familial M 33 þþ 8 G.3 Familial M 33 þþ þ þ 8 H Sporadic F 13 þþ þ 9 I.1 Familial F 15 þþ þ þ 10 I.1 Familial F 18 þþ þ 10 I.1 Familial F 39 þ 10 J Familial G 39 þþ þ þ 11 K Familial F 33 þþ þ 12 L Familial F 37 þþ13 M.1 Familial F 15 þ 14 M.2 Familial M 41 þ 14 N.1 Familial M 55 þþ þ þþ 15 N.2 Familial F þþ þþ 15 O Sporadic M 30 þþ16 P Sporadic M 33 þþþ 17 Q.1 Familial F 19 þþ þ þ 18 Q.2 Familial F 9 þþ 18 Q.3 Familial F 16 þþ þ 18 Q.4 Familial F 36 þþþ 18 R Sporadic M 50 þþ19 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.

sporadic cases in our study, however, underlines the high fre- considered as missense mutations. According to the literature, quency of PTCH spontaneous mutations (Aszterbaum et al,1998; missense mutations do not occur as frequently as frameshift or Hasenpusch-Theil et al, 1998; Smyth et al, 1998; and references nonsense mutations (15% vs 85%). They are spread along the therein). PTCH gene (Aszterbaum et al, 1998; Smyth et al, 1998; and refer- PTCH mutations are irregularly distributed along the coding ences therein). In our study, missense mutations span the second sequence and no hot-spot mutations have been reported yet. The group of six transmembrane domains of the protein. Molecular frameshift mutation in exon 8 is found in three di¡erent families, and cellular studies on Ptc mutant proteins in Drosophila would however (G, H, I in Tab l e I I ). So far, no founder e¡ect of PTCH argue for disruption of the normal topology and/or sorting of gene mutations has been described in the literature. We did not the receptor (Strutt et al, 2001). Unfortunately, such data are not ¢nd any arguments to support a founder e¡ect. Indeed, some available to interpret the pathologic value of the missense muta- mutations in di¡erent exons have been reported twice in the lit- tions described in humans. erature without any knowledge of recurrence (Hahn et al,1996; Improvement of the PTCH gene screening is necessary but the Wicking et al, 1997).We propose that mutation c1208^1209delAT nondetection of PTCH mutations in half of the typical familial is the ¢rst truly recurrent mutation identi¢ed in PTCH gene. Pa- cases might also underline the nonexploration of other genetic tients bearing this mutation have a di¡erent clinical outcome, events. Many studies have shown splice mutations (Aszterbaum which pinpoints the possible involvement of modi¢er as et al, 1998; Smyth et al, 1998; and references therein). Small dele- identi¢ed for instance in neuro¢bromatosis type 1 (Bahuau et al, tions encompassing several exons might also take place as has al- 2001). The identi¢cation of this mutation in monozygotic twins ready been demonstrated in other cancer-prone genetic diseases displaying di¡erent clinical features, however (family G, pro- (Dorschner et al,2000). bands G2 and G3,Ta b l e I I ), supports also the role of the environ- ment (Nelson et al,2002). Interestingly, we report a unique deletion of a single amino We are grateful to the patients and the physicians, especially Drs D. Augias, acid conserved in vertebrate species localized at the beginning of P. Bitoun, Devauchelle, H. Journel, G. Lecanelle, M. Le Merrer, S. Lyonnet, the ¢rst extracellular loop of the receptor (Fig 1). It would be G. Matthijs, N. Philip, P. Saiag, B. Sassolas, and A. Zankl.We thank Dr F. Bon- interesting to know if this amino acid is important for the inter- net-Dorion for advice and D. Lafon for technical help.This work was supported by action of Hedgehog with its Ptc receptor. Unfortunately, no mu- grants from the Association pour la Recherche sur le Cancer, Fondation pour la Re- tagenesis data or binding studies are available to document the cherche MeŁdicale, Ligue National Contre le Cancer, and Plan Hospitalier pour la Re- e¡ect of this mutation. cherche Clinique to P.G. N.B. was a recipient of a FeŁdeŁration des Maladies Finally, we describe several sequence variations not found in Orphelines fellowship. the general population segregating with the disease in the family, VOL. 121, NO. 3 SEPTEMBER 2003 PTCH1 MUTATIONS IN GORLIN SYNDROME 481

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