A Recurrent Mosaic Mutation in SMO , Encoding the Hedgehog Signal Transducer Smoothened, Is the Major Cause of Curry-Jones Syndrome
2016; Elsevier BV; Volume: 98; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2016.04.007
ISSN1537-6605
AutoresStephen R.F. Twigg, Robert B. Hufnagel, Kerry A. Miller, Yan Zhou, Simon J. McGowan, John Taylor, Jude Craft, Jenny C. Taylor, Stephanie L. Santoro, Taosheng Huang, Robert J. Hopkin, Angela F. Brady, Jill Clayton‐Smith, Carol L. Clericuzio, Dorothy K. Grange, Leopold Groesser, Christian Hafner, Denise Horn, I. Karen Temple, William B. Dobyns, Cynthia J. Curry, Marilyn C. Jones, Andrew O.M. Wilkie,
Tópico(s)Genetic and rare skin diseases.
ResumoCurry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T [p.Leu412Phe]), encoding smoothened (SMO), a G-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism. Curry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T [p.Leu412Phe]), encoding smoothened (SMO), a G-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism. The multiple-congenital-anomalies disorder Curry-Jones syndrome (MIM: 601707) was first presented (in abstract form) by Cynthia Curry and Marilyn Jones at the David W. Smith Workshop on Malformations and Morphogenesis in 1987. These authors described two unrelated individuals with the shared features of unilateral coronal craniosynostosis, cutaneous syndactyly, bilateral preaxial polydactyly of the feet, and unusual streaky skin lesions. Subsequently, the term Curry-Jones syndrome (CJS) was applied to this condition.1Cohen Jr., M.M. Craniosynostosis update 1987.Am. J. Med. Genet. Suppl. 1988; 4: 99-148Crossref PubMed Google Scholar, 2Gorlin R.J. Cohen Jr., M.M. Levin L.S. Syndromes with craniosynostosis: general aspects and well-known syndromes.in: Syndromes of the Head and Neck. Oxford University Press, 1990: 519-539Google Scholar The first formal publication on CJS was by Temple et al.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar and included detailed clinical descriptions of the two original subjects and three further unrelated individuals. Four more simplex cases have since been added to the literature; each affected individual had abnormal skin patches and preaxial polydactyly of the feet.4Mingarelli R. Mokini V. Castriota Scanderbeg A. Dallapiccola B. Brachycephalosyndactyly with ptosis, cataract, colobomas, and linear areas of skin depigmentation.Clin. Dysmorphol. 1999; 8: 73-75Crossref PubMed Scopus (5) Google Scholar, 5Thomas E.R. Wakeling E.L. Goodman F.R. Dickinson J.C. Hall C.M. Brady A.F. Mild case of Curry-Jones syndrome.Clin. Dysmorphol. 2006; 15: 115-117Crossref PubMed Scopus (5) Google Scholar, 6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar Additional features found in most individuals have included ectopic hair growth, abnormalities of brain development, coloboma and/or microphthalmia, coronal suture synostosis, cutaneous syndactyly, and intestinal malrotation and/or obstruction (reviewed by Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar). Figures 1A–1D show the major craniofacial, limb, and dermatological features of CJS in a previously unreported individual (subject 8 in our series); MRI scans of three individuals (Figures 1E–1G) illustrate the diversity of cerebral malformations (described in more detail below). Mild intellectual disability has been present in some individuals. In a single individual, a desmoplastic medulloblastoma (World Health Organization grade IV) of the right cerebellum, found incidentally on a follow-up brain MRI scan at 17 months, was treated successfully by surgical resection, chemotherapy, and radiotherapy.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar In two individuals, biopsies of active skin lesions were reported to show features of either trichoblastoma6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar or nevus sebaceus.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar In addition, odontogenic keratocysts have been found in one individual, and lesions in the bowel, identified histologically as hamartomas or myofibromas, were reported in three instances.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar, 6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar The etiology of CJS was previously unknown, but three clinical observations are relevant to hypotheses for causation. First, the nine previously reported individuals comprised seven males and two females with similar disease severity, making an X-linked mutation unlikely. Second, the sporadic origin of all individuals in association with patchy skin lesions and asymmetric cranial findings led Temple et al.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar to propose an underlying mosaic mutation; however, Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar favored a germline constitutional mutation in view of the consistent presentation and bilaterality of some of the other features. Third, the occurrence of medulloblastoma in basal cell nevus syndrome (BCNS [MIM: 109400])—caused by mutations in PTCH1 (MIM: 601309), which encodes the hedgehog (Hh) receptor—as well as the overlapping cranial and limb abnormalities found in disorders caused by mutations in GLI3 (MIM: 165240), a downstream effector of the Hh pathway, led Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar to propose that perturbation of Hh signaling could underlie CJS. However, sequencing of PTCH1 and GLI3 in DNA obtained from the blood of two individuals with CJS was normal.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar The objective of the present work was to identify the causative mutation(s) underlying CJS through whole-exome sequencing (WES) by initially using an overlap strategy in four individuals to pinpoint disease-causing variants in the same gene. The study was approved by Oxfordshire Research Ethics Committee B (reference C02.143) and the Riverside Research Ethics Committee (reference 09/H0706/20). Participants or their parents provided informed, written consent for genetic studies. Four of five samples initially chosen for analysis were from CJS-affected individuals reported by Temple et al.;3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar these comprised both eyelid tissue (sample 1-2, collected during an operation to repair a malformed upper eyelid with ectopic hairs) and fibroblasts from an affected skin biopsy (sample 1-4) from subject 1 (the individual originally described by Jones) and fibroblasts from affected skin of subjects 2 (sample 2-3) and 3 (sample 3-2), the latter of whom was the individual originally described by Curry. All fibroblast lines were analyzed between three and five passages. An additional fifth sample (4-1) was from the blood of subject 4, an unpublished individual with features overlapping CJS. The clinical features of these four individuals are summarized in Table 1.Table 1Clinical Features of Subjects Diagnosed with CJSSubjectGenderCraniosynostosisBrainDevelopmental AttainmentEyesSkinHairGI TractCutaneous SyndactylyPolydactylyTumorsOtherPreviously Reported?1 (SMO+)FRCpossible cyst in trigone of right ventriclenormalR amblyopia; skin overgrowthwaxy, streaky lesions; nevus sebaceusectopic patch of hair above R eyeGI bleeding; chronic constipationL 1/2/3 (H); R 2/3 (H)R trifid hallux–asymmetric facecase 4 in Temple et al.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar2 (SMO+)MLCACC; R HMEG; R VMEGmild delaynormalraised, linear white streaks; normal biopsyectopic patch of hair near eyesintestinal obstructionL 2/3 (H), 3/4 (F); R 2/3/4 (H), 2/3 (F)L and R broad thumbs, preaxial polydactyly (F)myofibromas of large bowellip pits; seizurescase 1 in Temple et al.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar3 (SMO+)MLCpartial ACC; asymmetric dilated ventriclesmild ID; IQ < 70L and R microphthalmia; R iris colobomaraised, scar-like pale lesions; non-specific biopsyectopic patch of hair above R eyeesophageal dysmotilityL and R 1/2/3/4 (H)L and R bifid halluces–asymmetric face; fused central incisor; oligodontia; freckled areas on soles of feet; esophageal dysmotilitycase 3 in Temple et al.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar4Mbicoronaldilated ventricles; choroid plexus cystmild delaycongenital glaucoma; secondary cataractpigmentary anomalies in Blashko's lines, most notable on limbschaotic hair patterning of lateral portions of eyebrowsdiarrheal episodesL and R 2/3/4 (F)broad halluces–cleft palateno5 (SMO+)Mnonemild ACC; L HMEG; R VMEG and PMG; occipital meningocele; Chiari I malformationmild to moderate developmental delayL mild colobomatous microphthalmia with unusually shaped pupilraised, linear streaks (L arm and leg, chin, and a few other areas)ectopic hair on cheekmalrotation; intestinal pseudo-obstruction; chronic constipation; subtotal colectomy for volvulus and obstipationL 1/2/3/4 (H), 3/4 (F); R 2/3 (H), 3/4 (F)L and R preaxial polydactyly (F); R thumb nubbindesmoplastic medulloblastoma of cerebellum; odontogenic keratocysts; benign colonic polyps and smooth muscle hamartomalarge anterior fontanelle; lip pitspatient 2 in Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar6 (SMO+)Mnonepartial ACC; MEG; VMEG; Chiari I malformationmild delay (1–2 years behind academically)L and R iris colobomashypopigmented streaky lesionsectopic patch of hair near eyesmalrotationL 1/2/3 (H), 1/2 (F); R 2/3 (H)L and R preaxial polydactyly (H), preaxial polydactyly (F)trichoblastoma; smooth muscle hamartomas of GI tractmildly asymmetric face; lip pitpatient 1 in Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar7 (SMO+)Mnonenormalnormalnormalwhite, patchy skin behind kneesabnormal hair growth on shoulders and limbsnormalR 1/2/3/4 (H)L and R preaxial polydactyly (F); R: broad thumb–plagiocephalyThomas et al.5Thomas E.R. Wakeling E.L. Goodman F.R. Dickinson J.C. Hall C.M. Brady A.F. Mild case of Curry-Jones syndrome.Clin. Dysmorphol. 2006; 15: 115-117Crossref PubMed Scopus (5) Google Scholar8 (SMO+)FRCpartial ACC; moderate cerebral asymmetry consistent with L HMEG; PMGmild delaynormal; dysmorphic R eyelidlinear areas of hypopigmented skin atrophy (extremities and trunk)–malrotation; intermittent pseudo-obstruction; serosal nodules in the appendix, mesentery, and duodenumL and R variable 1/2/3 (H and F)L and R duplicated thumbs and hallucesscalp lymphangiomas: benign hamartomatous lesion with features of lymphangioma and nevus sebaceus; serosal hamartomas in duodenum, appendix, and mesenteryaccessory bone at anterior fontanelle; wormian bones of L posterior skull; L leg longer than R; lumbar scoliosisno9FnoneACC; dilated ventricles; macrocephalypsychomotor delaybilateral colobomas of iris, retina, and choroid; strabismus; nystagmushypopigmented patch on trunkhigh frontal hairlinechronic constipationL and R 3/4 (H)L and R duplicated thumbs and halluceslipomyelomeningocelerudimentary sacral vertebraeno10 (SMO+)FLCACC; abnormal cortical dysplasia of R perirolandic regionpsychomotor delaycorneal clouding; eyelid irregularity and ectopic hairsmostly R-sided hypo- and hyperpigmented swirling skin lesions; increased hair in hyperpigmented areasectopic hairs; hirsutismdysmotility; malrotation; multiple hamartomas of small intestineR 1/2 (H)L and R duplicated thumbs and hallucesmultiple mesenteric hamartomas with smooth muscle bundles with inter-myenteric gangliagrade 2 hydronephrosisnoAbbreviations are as follows: F, female; M, male; LC, left coronal; RC, right coronal; R, right; L, left; ACC, agenesis of the corpus callosum; MEG, megalencephaly; HMEG, hemimegalencephaly; VMEG, ventriculomegaly; PMG, polymicrogyria; ID, intellectual disability; GI, gastrointestinal; H, hands; and F, feet. Open table in a new tab Abbreviations are as follows: F, female; M, male; LC, left coronal; RC, right coronal; R, right; L, left; ACC, agenesis of the corpus callosum; MEG, megalencephaly; HMEG, hemimegalencephaly; VMEG, ventriculomegaly; PMG, polymicrogyria; ID, intellectual disability; GI, gastrointestinal; H, hands; and F, feet. After DNA extraction, we performed WES by using the SeqCap EZ Human Exome Library v.2.0 (NimbleGen) on a HiSeq 2000 (Illumina). Paired-end reads (100 bp) were mapped to hs37d5 with Stampy v.1.0.22,7Lunter G. Goodson M. Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads.Genome Res. 2011; 21: 936-939Crossref PubMed Scopus (765) Google Scholar and after removal of artifacts, the average coverage was >30× over 82% of the exome. We used Platypus8Rimmer A. Phan H. Mathieson I. Iqbal Z. Twigg S.R. Wilkie A.O. McVean G. Lunter G. WGS500 ConsortiumIntegrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications.Nat. Genet. 2014; 46: 912-918Crossref PubMed Scopus (619) Google Scholar to call variants, and assuming that CJS is caused by very rare autosomally located variant(s), we prioritized the data by excluding common variants present in either our in-house database of solved cases or the NHLBI Exome Sequencing Project (ESP) Exome Variant Server (later revised to the Exome Aggregation Consortium [ExAC] Browser for reanalysis). We compared across CJS individuals for hits in the same gene. This analysis did not yield any genes with rare coding variants shared by three or four individuals, and only three genes had rare coding variants shared by two individuals, but none were strong candidates (Table S1). As an alternative approach, we compared the data from the two tissues separately sequenced from subject 1, given that mosaicism for a pathogenic variant could be detectable through differences in allele frequencies between datasets. Variants were ranked according to a somatic p value score generated by the software tool MiG,9McGowan S.J. Hughes J.R. Han Z.P. Taylor S. MIG: Multi-Image Genome viewer.Bioinformatics. 2013; 29: 2477-2478Crossref PubMed Scopus (11) Google Scholar which compares variant and reference read counts between two datasets (Figure S1). The top hit was a nonsynonymous substitution in SMO (MIM: 601500; GenBank: NM_005631.4): c.1234C>T (p.Leu412Phe), present in 44 of 83 sequence reads from fibroblast sample 1-4. Notably, this variant was not called in the exome data from eyelid sample 1-2, although a low level of the same variant (2 of 40 sequence reads) was apparent on manual examination of the reads (Figure 2A). The fortuitous presence of two heterozygous flanking SNPs (rs2228617 and rs2735842; dbSNP137) in this individual enabled the two SMO alleles to be easily distinguished. We noted that when sequence reads included both the c.1234 position and one of the flanking SNPs, the mutant c.1234T reads were always in cis with the variant allele of the SNP in both samples. However, for the eyelid sample 1-2, the converse was not true, supporting the conclusion that the c.1234C>T mutation was mosaic in this sample (see Figure 2). SMO encodes smoothened (SMO), a frizzled G-protein-coupled receptor that plays a key role in transducing Hh signaling. Hh binding relieves patched-mediated suppression of SMO to allow transduction of the signal, probably mediated by conformational changes within the 7-transmembrane bundle.11Wang C. Wu H. Katritch V. Han G.W. Huang X.P. Liu W. Siu F.Y. Roth B.L. Cherezov V. Stevens R.C. Structure of the human smoothened receptor bound to an antitumour agent.Nature. 2013; 497: 338-343Crossref PubMed Scopus (390) Google Scholar Hence, the variant matched all three criteria (autosomal, mosaic, and affecting Hh signaling) for the characteristics of a candidate CJS-associated gene on the basis of clinical deduction.3Temple I.K. Eccles D.M. Winter R.M. Baraitser M. Carr S.B. Shortland D. Jones M.C. Curry C. Craniofacial abnormalities, agenesis of the corpus callosum, polysyndactyly and abnormal skin and gut development--the Curry Jones syndrome.Clin. Dysmorphol. 1995; 4: 116-129Crossref PubMed Scopus (14) Google Scholar, 6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar Adding further weight to the conclusion that this was the pathogenic variant, the Catalogue of Somatic Mutations in Cancer (COSMIC) revealed that SMO c.1234C>T is a known mutation hotspot in multiple tumor types (discussed further below). Dideoxy-sequencing of SMO exon 6 in sample 1-4 confirmed the presence of the C>T variant in an apparently heterozygous state, but the mutant peak was barely visible in sample 1-2 (Figure 2B). In light of this finding, we scrutinized the exome sequence data for SMO from the other three CJS individuals in greater detail. The identical c.1234C>T variant was present in 2.7% (6 of 219) of reads from sample 2-3 (Figure S2) but was absent in the other two samples (0 of 57 reads from sample 3-2 and 0 of 179 reads from sample 4-1); no other suspicious SMO variant was detected in the exome sequences of subjects 3 or 4. To gather further evidence that mosaic SMO mutations cause CJS, we collected and analyzed additional tissue samples from subjects 1–4 together with a further six individuals with CJS (subjects 5 and 6 reported by Grange et al.,6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar subject 7 reported by Thomas et al.,5Thomas E.R. Wakeling E.L. Goodman F.R. Dickinson J.C. Hall C.M. Brady A.F. Mild case of Curry-Jones syndrome.Clin. Dysmorphol. 2006; 15: 115-117Crossref PubMed Scopus (5) Google Scholar and three unpublished individuals; clinical features are summarized in Table 1). Material from affected regions was prioritized and included archival formalin-fixed paraffin-embedded (FFPE) sections, such as those illustrated for affected skin and gut hamartoma in Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar (Table S2). We used the Ion Torrent PGM to perform deep sequencing of SMO exon 6 in a total of 37 different tissue samples from the ten CJS subjects (including the previously sequenced samples) and three control samples. Table S3 lists the primer sequences and conditions used for SMO (GenBank: NG_023340.1) amplification and sequencing. Deep sequencing of DNA from subject 1 confirmed a ∼50% frequency for c.1234C>T in fibroblast sample 1-4 and showed that the variant was present at an 11% frequency in eyelid sample 1-2 (Figure 3 and Table S2). Surprisingly, we did not identify any mutant c.1234T alleles in the affected skin sample (1-3) from which the fibroblasts constituting sample 1-4 were derived, possibly indicating positive selection of mutant cells during growth in culture. In subject 2, the exome finding suggesting that the tested sample (2-3) was positive for an SMO mutation was validated, and c.1234C>T variant frequencies ranging from 4%–25% were detected in three of five samples, confirming mosaicism for the identical SMO mutation in this individual. Deep sequencing of SMO in the samples used for exome sequencing (3-2 and 4-1) confirmed that the c.1234C>T mutation was essentially undetectable, given that the data were indistinguishable (by Fisher's exact test) from the control samples (Table S2). However, a new skin-biopsy sample from subject 3 was positive (14%), and a low level (1.6%) was detected in saliva. Furthermore, five of the six additional CJS individuals (subjects 5–8 and 10) were also mosaic for c.1234C>T (Figure 3 and Table S2). FFPE material from an occipital meningocele and cerebellar medulloblastoma were available from subject 5 (corresponding to patient 2 in Grange et al.6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar), and analysis showed relatively high levels of mosaicism for the c.1234T allele at 20% and 43%, respectively. Similarly, the abdominal smooth muscle tumor sections from subject 6 (patient 1 in Grange et al.;6Grange D.K. Clericuzio C.L. Bayliss S.J. Berk D.R. Heideman R.L. Higginson J.K. Julian S. Lind A. Two new patients with Curry-Jones syndrome with trichoblastoma and medulloblastoma suggest an etiologic role of the sonic hedgehog-patched-GLI pathway.Am. J. Med. Genet. A. 2008; 146A: 2589-2597Crossref PubMed Scopus (9) Google Scholar samples 6-3 and 6-4) contained the c.1234T allele at 35%–37%. In subject 7, a mildly affected individual,5Thomas E.R. Wakeling E.L. Goodman F.R. Dickinson J.C. Hall C.M. Brady A.F. Mild case of Curry-Jones syndrome.Clin. Dysmorphol. 2006; 15: 115-117Crossref PubMed Scopus (5) Google Scholar we detected c.1234T at 3%–4% in skin (sample 7-1, taken from a hairy area of the inner leg) and associated fibroblasts (7-2), as well as a femoral bone marrow sample (7-5, 1%). Finally, we identified the c.1234T variant in samples from two unpublished individuals, subjects 8 (Figures 1A–1D and 1G and Supplemental Note) and 10 (Table 1). FFPE samples from the colon (8-2) and cecum (8-3) collected from subject 8 during investigation of intestinal malrotation and mesenteric masses (which contained hamartomatous nodules consisting of disorganized bundles of mature smooth muscle intermixed with nerve fibers and ganglion cells) contained the c.1234T allele at levels of 8%–9%. In subject 10, an affected skin sample and derived fibroblasts contained c.1234T at 33% and 16%, respectively (Figure 3). In total, we identified the identical SMO c.1234C>T mutation in tissues from eight unrelated CJS-affected individuals, including the two originally described by Curry and Jones. In all but o
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