Gorlin syndrome is an autosomal dominant disorder characterized by a wide range of developmental abnormalities and a predisposition to various tumors, and it is linked to the alteration of several causative genes, including PTCH1. We performed targeted resequencing using a next-generation sequencer to analyze genes associated with known clinical phenotypes in an 11-year-old male with sporadic jaw keratocysts. A novel duplication mutation (c.426dup) in PTCH1, resulting in a truncated protein, was identified

Imoto, Issei

Kohmoto, Tomohiro

Komori, Takahide

Naruto, Takuya

Okamoto, Nana

English

Tokushima University Institutional Repository

OPENDATA REPORTA novel PTCH1 mutation in a patient with Gorlin syndromeNana Okamoto1,5, Takuya Naruto2,5, Tomohiro Kohmoto3, Takahide Komori1 and Issei Imoto4Gorlin syndrome is an autosomal dominant disorder characterized by a wide range of developmental abnormalities and apredisposition to various tumors, and it is linked to the alteration of several causative genes, including PTCH1. We performed targetedresequencing using a next-generation sequencer to analyze genes associated with known clinical phenotypes in an 11-year-old malewith sporadic jaw keratocysts. A novel duplication mutation (c.426dup) in PTCH1, resulting in a truncated protein, was identified.Human Genome Variation (2014) 1, 14022; doi:10.1038/hgv.2014.22; published online 13 November 2014Basal cell nevus syndrome (BCNS; MIM #109400) or Gorlinsyndrome,1 also known as nevoid basal cell carcinoma syndrome(NBCCS) or Gorlin–Goltz syndrome, is a rare autosomal dominantdisorder with almost 100% penetrance and variable expressivity.Gorlin syndrome is characterized by a wide range of develop-mental abnormalities and a predisposition to neoplasms. Muta-tions in the PTCH1 gene (MIM #601309) are the main moleculardefects associated with Gorlin syndrome,2,3 although mutations inother genes involved in the hedgehog pathway, such as PTCH2(MIM #603673) and SUFU (MIM #607035),4,5 have also beenreported in patients with this syndrome. Here, we report a novelPTCH1 gene duplication mutation (c.426dup) that was detected ina patient with a history of multiple keratocystic odontogenictumors (KCOTs, Figure 1a) of the jaw, which is the most consistentand common manifestation of Gorlin syndrome.6An 11-year-old Chinese male with no family history ofconsanguinity presented to our clinic with plantar pits(Figure 1b), mild macrocephaly, a coarse face and melanoticmacules. His psychomotor development was appropriate for hisage, and the results of neurological and ophthalmologicalexaminations were normal. Imaging tests (magnetic resonanceimaging and orthopantomography) revealed multiple odonto-genic cysts (Figure 1a). A radiograph of the skull revealedbilamellar calcifications of the falx cerebri and bulging of thesella turcica (Figure 1c). Histopathological examination of theodontogenic cysts confirmed the diagnosis of KCOTs. Over 2 yearsof follow-up, new black pit lesions appeared on his right cheek.However, neither basal cell carcinoma nor bifid, fused or markedlysplayed ribs were observed.On the basis of the presence of three major criteria for clinicaldiagnosis,7 the patient was diagnosed with Gorlin syndrome. Themolecular diagnosis was performed using genomic DNA extractedfrom the patient’s whole blood after obtaining informed consent.The study was approved by the ethical committees of KobeUniversity and The University of Tokushima. Because variousalterations, including mutations and copy number alterations(CNAs)8 of three genes with numerous exons (PTCH1, PTCH2 andSUFU) can be responsible for Gorlin syndrome, we first used aMiseq bench-top sequencer (Illumina, San Diego, CA, USA) toperform next-generation sequencing with a TruSight OneSequencing Panel (Illumina) for the simultaneous targetedsequencing of the exon regions of 4,813 clinically relevant genes.The sequencing reads were aligned to the human referencegenome (hg19) using Burrows–Wheeler aligner (BWA, version0.7.8; http://bio-bwa.sourceforge.net/).9 The alignments wereconverted from a sequence alignment map (SAM) format to asorted and indexed binary alignment map files (SAMtools version0.1.19; http://samtools.sourceforge.net). Duplicate reads wereremoved using Picard (http://picard.sourceforge.net). Local rea-lignment around the indels, base quality score recalibration andUnifiedGenotyper calls were performed using Genome AnalysisToolkit software (GATK, version 3.1-1; http://www.broadinstitute.org/gatk/).10 All sequence variants were annotated usingAnnovar.11 To identify single-nucleotide variations, we excludedsequence variants with minor allele frequency 40.05 from the1000 Genomes Project databases (http://www.1000genomes.org/),NHLBI GO Exome Sequencing Project (ESP6500, http://evs.gs.washington.edu/EVS/) and the Human Genetic Variation Database(HGVD, http://www.genome.med.kyoto-u.ac.jp/SnpDB/). To com-plement the single-nucleotide variations and indel analyses, a CNAdetection algorithm was applied to the alignment map files toidentify large CNAs as follows: (1) binary alignment map files wereconverted into files covering target regions using GATK, (2) logcoverage ratios and Z scores were calculated between the caseand other samples and (3) regions with an abnormal copy numberwere detected using circular binary segmentation with DNAcopy(R/Bioconductor; http://bioconductor.org).12 These analyses iden-tified one heterozygous frameshift mutation NM_000264.3(PTCH1_v001):c.426dup in exon 3 of the PTCH1 gene, whichcaused a frameshift at codon 143, resulting in the introduction of astop codon at position 155 (NM_000264.3 (PTCH1_i001):p.(Thr143Tyrfs*12)). This resulted in the loss of most of the codingregion, which resulted in a loss of protein function; thus, thismutation is the disease-causing alteration in our patient. No otheralterations, including CNAs, were observed around the threegenes. The alteration was then confirmed using PCR-based directSanger sequencing with a BigDye Terminator v3.1 Cycle Sequen-cing Kit (Applied Biosystems, Foster City, CA, USA) on a 31301Department of Oral and Maxillofacial Surgery, Kobe University Graduate School of Medicine, Kobe, Japan; 2Department of Stress Science, Institute of Health Biosciences, TheUniversity of Tokushima Graduate School, Tokushima, Japan; 3Student Lab, Faculty of Medicine, The University of Tokushima, Tokushima, Japan and 4Department of HumanGenetics, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.Correspondence: I Imoto (issehgen@tokushima-u.ac.jp)5These authors contributed equally to this work.Received 14 August 2014; revised 10 September 2014; accepted 11 September 2014Citation: Human Genome Variation (2014) 1, 14022; doi:10.1038/hgv.2014.22© 2014 The Japan Society of Human Genetics All rights reserved 2054-345X/14www.nature.com/hgvFigure 1. (a) Dental orthopantomography of a patient affected by multiple keratocystic odontogenic tumors (KCOTs). (b) Numerous tiny blackpits over the right plantar area (arrows). (c) Radiograph of the skull revealing bulging of the sella turcica (arrow).Normal alleleMutated allele‘T’ duplicationWild type(1447 AA)p.(Thr143Tyrfs*12)(153 AA)Exon 31 4 0 1 4 41 4 1 1 4 31 4 2Figure 2. Electropherogram of the PTCH1 exon 3 sequence showing the heterozygous germline duplication of a thymine (T) in the patientDNA. The DNA and corresponding amino-acid sequences of wild-type and mutant PTCH1 alleles are also shown. The affected transcript andprotein were labeled NM_000264.3 (PTCH1_v001):c.426dup and NM_000264.3 (PTCH1_i001):p.(Thr143Tyrfs*12), respectively, using the HumanGenome Variation Society (HGVS) nomenclature version 2.0 (Mutalyzer 2.0.beta-32, https://mutalyzer.nl/). The arrow indicates theduplication point.Novel PTCH1 mutation in Gorlin syndromeN Okamoto et al2Human Genome Variation (2014) 14022 © 2014 The Japan Society of Human GeneticsGenetic Analyzer (Applied Biosystems) in both directions (Figure 2,primer information is available on request). This mutation has notbeen previously reported in any patients with Gorlin syndrome(Human Gene Mutation Database professional 2014.2, http://www.hgmd.org/ and ClinVar, http://www.ncbi.nlm.nih.gov/clinvar/) andis not present in the 1000 Genomes, ESP6500 or HGVD databases.Because parental DNA was not available, this mutation could notbe confirmed to be de novo.The human PTCH1 gene contains 23 coding exons that span~ 70 kb and encode a protein composed of 1,447 amino acids with12 transmembrane-spanning domains and two large extracellularloops.3 PTCH1 is the ligand-binding component of the sonichedgehog receptor complex. Aberrant activation of the sonichedgehog signaling cascade owing to haploinsufficiency of PTCH1is believed to cause Gorlin syndrome.13 As germline mutations ofPTCH1 in patients with Gorlin syndrome were first reported by twogroups in 1996,2,3 4300 different mutations covering almost allexons, with no hot spots and various CNAs, have been reported.Most of these are frameshift or nonsense mutations that lead tothe synthesis of a truncated protein. In a series of 313 PTCH1mutations reported in the Human Gene Mutation Database, mostof which were detected in cases of Gorlin syndrome, 33.9% weremissense or nonsense mutations, 47.6% were small insertionsand/or deletions and 8.3% were gross deletions, insertions orduplications. In the current study, we reported a novel germlineduplication (insertion) mutation of PTCH1, c.426dup, in a Chinesepatient with sporadic Gorlin syndrome. This mutation created apremature termination codon in the mutant allele that resulted inthe truncation of PTCH1. This truncation of PTCH1 is one of theshortest forms reported to date; this truncation lacks 11 of the12 transmembrane-spanning domains. Because a prematuretermination codon leads to mRNA degradation via nonsense-mediated mRNA decay,14 the haploinsufficiency of PTCH1 likelyhad an important etiological role in this case.A combination of clinical and molecular screening tools couldbe useful for the identification of Gorlin syndrome in patients whowere previously diagnosed with multiple KCOTs of the jaw. Thesepatients could be identified by retrospectively evaluating thepathological records.15 Although this case presented with severalsymptoms that fulfilled the criteria for the diagnosis of Gorlinsyndrome, identifying the specific pathogenic mutation is usefulfor genetic counseling. Therefore, a proper diagnosis is essential,particularly during the early stages of life. Gorlin syndrome isprimarily caused by mutations in PTCH1. However, no hot-spotmutations within the 23 coding exons have been reported in thisgene; CNAs are also known to be responsible for Gorlin syndrome.Genetic alterations in PTCH2 (22 coding exons) and SUFU(12 coding exons) have also been identified, although they arerarely observed. Previous studies have revealed that there were nogenotype–phenotype correlations between mutations in PTCH1or other genes and the major clinical features of Gorlinsyndrome.16,17 Target resequencing of the possible disease-causing genes and other genes associated with various knownclinical phenotypes using next-generation sequencing technologyenables the simultaneous evaluation of mutations and CNAs.Therefore, this could form a simple, efficient and economicalgenetic test for Gorlin syndrome.HGV DATABASEThe relevant data from this Data Report are hosted at theHuman Genome Variation Database at http://dx.doi.org/10.6084/m9.figshare.hgv.513.ACKNOWLEDGEMENTSWe thank the patient and his family for their participation in this study. We also thankDr Hiroshi Nagai for his technical assistance. This work was supported by JSPSKAKENHI Grant Numbers 60645216 (NO) and 26293304 (II) from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan.COMPETING INTERESTSThe authors declare no conflict of interest.REFERENCES1 Gorlin RJ, Goltz RW. Multiple nevoid basal-cell epithelioma, jaw cysts and bifid rib:a syndrome. N Engl J Med 1960; 262: 908–912.2 Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley S, Chidambaram Aet al. Mutations of the human homolog of Drosophila patched in the nevoid basalcell carcinoma syndrome. Cell 1996; 85: 841–851.3 Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM et al. Humanhomolog of patched, a candidate gene for the basal cell nevus syndrome. Science1996; 272: 1668–1671.4 Fan Z, Li J, Du J, Zhang H, Shen Y, Wang CY et al. A missense mutation in PTCH2underlies dominantly inherited NBCCS in a Chinese family. J Med Genet 2008; 45:303–308.5 Pastorino L, Ghiorzo P, Nasti S, Battistuzzi L, Cusano R, Marzocchi C et al.Identification of a SUFU germline mutation in a family with Gorlin syndrome. Am JMed Genet A 2009; 149A: 1539–1543.6 Gorlin RJ. Nevoid basal-cell carcinoma syndrome. Medicine (Baltimore) 1987; 66:98–113.7 Lo Muzio L. Nevoid basal cell carcinoma syndrome (Gorlin syndrome). Orphanet JRare Dis 2008; 3: 32.8 Fujii K, Ishikawa S, Uchikawa H, Komura D, Shapero MH, Shen F et al. High-densityoligonucleotide array with sub-kilobase resolution reveals breakpoint informationof submicroscopic deletions in nevoid basal cell carcinoma syndrome. Hum Genet2007; 122: 459–466.9 Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheelertransform. Bioinformatics 2009; 25: 1754–1760.10 McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A et al. TheGenome Analysis Toolkit: a MapReduce framework for analyzing next-generationDNA sequencing data. Genome Res 2010; 20: 1297–1303.11 Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of geneticvariants from high-throughput sequencing data. Nucleic Acids Res 2010;38: e164.12 Olshen AB, Venkatraman ES, Lucito R, Wigler M. Circular binary segmentationfor the analysis of array-based DNA copy number data. Biostatistics 2004; 5:557–572.13 Ingham PW, McMahon AP. Hedgehog signaling in animal development:paradigms and principles. Genes Dev 2001; 15: 3059–3087.14 Holbrook JA, Neu-Yilik G, Hentze MW, Kulozik AE. Nonsense-mediated decayapproaches the clinic. Nat Genet 2004; 36: 801–808.15 Pastorino L, Pollio A, Pellacani G, Guarneri C, Ghiorzo P, Longo C et al.Novel PTCH1 mutations in patients with keratocystic odontogenic tumorsscreened for nevoid basal cell carcinoma (NBCC) syndrome. PLoS ONE 2012; 7:e43827.16 Wicking C, Shanley S, Smyth I, Gillies S, Negus K, Graham S et al. Most germ-linemutations in the nevoid basal cell carcinoma syndrome lead to a prematuretermination of the PATCHED protein, and no genotype–phenotype correlationsare evident. Am J Hum Genet 1997; 60: 21–26.17 Bale AE, Gailani MR, Leffell DJ. The Gorlin syndrome gene: a tumor suppressoractive in basal cell carcinogenesis and embryonic development. Proc Assoc AmPhys 1995; 107: 253–257.This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. The images orother third party material in this article are included in the article’s Creative Commonslicense, unless indicatedotherwise in the credit line; if thematerial is not included underthe Creative Commons license, users will need to obtain permission from the licenseholder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/Novel PTCH1 mutation in Gorlin syndromeN Okamoto et al3© 2014 The Japan Society of Human Genetics Human Genome Variation (2014) 14022

Novel PTCH1 mutation in Gorlin syndrome

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Novel PTCH1 mutation in Gorlin syndrome

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