Osteopetrosis is a heritable disorder of the skeleton that is characterized by increased bone density on radiographs caused by defects in osteoclast formation and function. Mutations in >10 genes are identified as causative for this clinically and genetically heterogeneous disease in humans. We report two novel missense variations in a compound heterozygous state in the CLCN7 gene, detected through targeted exome sequencing, in a 15-year-old Japanese female with intermediate autosomal recessive osteopetrosis

Imoto, Issei

Kohmoto, Tomohiro

Komori, Takahide

Masuda, Kiyoshi

Naruto, Takuya

Okamoto, Nana

English

Tokushima University Institutional Repository

OPENDATA REPORTNovel CLCN7 compound heterozygous mutationsin intermediate autosomal recessive osteopetrosisNana Okamoto1, Tomohiro Kohmoto2, Takuya Naruto2, Kiyoshi Masuda2, Takahide Komori1 and Issei Imoto2Osteopetrosis is a heritable disorder of the skeleton that is characterized by increased bone density on radiographs caused bydefects in osteoclast formation and function. Mutations in 410 genes are identified as causative for this clinically and geneticallyheterogeneous disease in humans. We report two novel missense variations in a compound heterozygous state in the CLCN7 gene,detected through targeted exome sequencing, in a 15-year-old Japanese female with intermediate autosomal recessiveosteopetrosis.Human Genome Variation (2017) 4, 17036; doi:10.1038/hgv.2017.36; published online 17 August 2017Osteopetrosis is a heritable, clinically, and genetically hetero-geneous group of conditions that share the hallmark, observableon radiographs, of increased bone density caused by a failure ofosteoclast development or function.1 The Nosology Group of theInternational Skeletal Dysplasia Society classifies these osteope-trotic conditions into several distinct entities based on clinicalfeatures, mode of inheritance, and underlying molecular andpathogenetic mechanisms.2 Mutations in at least 10 genes havebeen identified as causative in humans, accounting for 70% of allcases.3 These conditions can be inherited as autosomal recessive,dominant or X-linked traits, with the most severe forms beingautosomal recessive. In addition to the paradigmatic recessivemalignant and dominant benign forms, an intermediate groupexists in which the defect can be inherited as either a recessive ora dominant trait. These intermediate cases often represent adiagnostic challenge, although diagnosis is mostly based onclinical and radiographic evaluation. Genetic testing can be usedto confirm the diagnosis and to differentiate among the differentsubtypes of osteopetrosis, to provide additional informationregarding the natural history and to predict likely responses tospecific treatments and recurrence risks for adapted geneticcounseling.1Here, we present a case of intermediate autosomal recessiveosteopetrosis (IARO; MIM 259710) with novel compound hetero-zygous mutations of the CLCN7 gene (MIM 602727), detectedusing a targeted exome sequencing (TES)-based approach todifferentially cover possible causative genes for osteopetrosis,although IARO is generally caused by either homozygous/compound heterozygous mutations of CLCN7 or PLEKHM1.1A 15-year-old Japanese female was the first child of non-consanguineous, healthy parents who had no notable familydisease history (Figure 1a). Her first visit to the Department of Oraland Maxillofacial Surgery of Kobe University Hospital was at theage of 9 years. She had a history of fracture of the leg at the age of5 years and was tentatively diagnosed with IARO based on clinicalhistories and signs at another hospital. Her early motor milestonesand language development were normal, and she possessednormal intelligence. The patient presented with spontaneousfractures in the first years of life and a generalized increase inbone density. On presentation, her height and weight were146.7 cm (−2.1 s.d.) and 50 kg (−0.2 s.d.), respectively. Her headcircumference (55.9 cm) did not suggest any abnormality. She hadnormal hearing, but she had mild bilateral visual impairment dueto atrophy of the bilateral optic nerves. Asymmetrical deformity ofthe fractured leg had not significantly progressed. She had aheavy malocclusion with hypodontia, several impacted teeth,enamel dysplasia, and malformed teeth (Figure 1b). Skeletalradiographs showed a diffuse increase in bone density withvarious characteristic signs, such as a ‘sandwich’ appearance in thevertebrae and a ‘bone-within-bone’ appearance of the iliac wings(Figure 1c–f). No hepatosplenomegaly was noted during thephysical examination. Blood counts and phosphocalcic parameterswere normal, and her karyotype was normal. No causativemutations were reported by sequence analyzes of all codingexons of TCIRG1 and CLCN7 performed at another hospital. Herclinical and radiological features were compatible with those ofIARO, but the patient remained molecularly undiagnosed.After informed consent was obtained from the patient’sparents, molecular diagnosis was performed on genomic DNAextracted from a blood sample. The study was approved by theEthics Committees of Kobe University and Tokushima University.To simultaneously screen multiple osteopetrosis-causing genes,we first performed TES for the proband using a TruSight OneSequencing Panel (Illumina, San Diego, CA, USA), which allows thetargeted sequencing of the exon regions of 4813 clinically relevantgenes, and a MiSeq sequencer (Illumina) with our pipeline for nextgeneration sequencing data analysis, as described elsewhere,4,5with a minor modification due to a software update specific for abioinformatics pipeline.6 To identify presumably pathogenicsingle-nucleotide variants, we excluded minor sequence variantswith relatively high-allele frequencies, i.e., 40.01, included in the1000 Genomes Project database (http://www.1000genomes.org/),the NHLBI GO Exome Sequencing Project (ESP6500, http://evs.gs.washington.edu/EVS/), the Human Genetic Variation Database(HGVD, http://www.genome.med.kyoto-u.ac.jp/SnpDB/) and theIntegrative Japanese Genome Variation Database (iJGVD, https://1Department of Oral and Maxillofacial Surgery, Kobe University Graduate School of Medicine, Kobe, Japan and 2Department of Human Genetics, Graduate School of BiomedicalSciences, Tokushima University, Tokushima, Japan.Correspondence: I Imoto (issehgen@tokushima-u.ac.jp)Received 5 June 2017; revised 29 June 2017; accepted 4 July 2017Citation: Human Genome Variation (2017) 4, 17036; doi:10.1038/hgv.2017.36Official journal of the Japan Society of Human Geneticswww.nature.com/hgvijgvd.megabank.tohoku.ac.jp/). The detection of copy-numbervariations (CNV) using TES data with a resolution of a single exonto several exons, depending on the exon size, was performed aspreviously described.6 These analyzes detected two differentsingle-base substitutions, NM_001287.5(CLCN7_v001):c.1505G4Aand c.1729G4A, in exons 17 and 19, respectively, and thesubstitutions in the exons were confirmed by Sanger sequencing(Figure 2a). The patient’s mother and father were heterozygouscarriers of the c.1505G4A and the c.1729G4A variations,respectively, suggesting that these variations were present inthe patient in a compound heterozygous state. These variationstransformed the codon for cysteine 502 into that for tyrosine[NM_001287.5(CLCN7_i001):p.(Cys502Tyr)] and the codon forvaline 577 into that for methionine [p.(Val577Met)], respectively.These variants are not present in the Human Gene MutationDatabase (HGMD, Professional 2017.1, http://www.hgmd.org/) orClinVar (http://www.ncbi.nlm.nih.gov/clinvar/). No cases with p.(Cys502Tyr) are present in the publicly available variationdatabases, whereas one heterozygous case (carrier) with p.(Val577Met) was found in the Exome Aggregation Consortium(ExAC, http://exac.broadinstitute.org/) database. In multiple bioin-formatics tools, including MutationTaster (http://www.mutationtaster.org/index.html), PolyPhen-2 version 2.2.2r405 (http://genetics.bwh.harvard.edu/pph2/index.shtml) and PROVEAN (http://provean.jcvi.org), p.(Cys502Tyr) was predicted to be deleterious byall three tools, whereas p.(Val577Met) was predicted to bedeleterious by MutationTaster and Polyphen-2 and to be neutralby PROVEAN. p.(Cys502Tyr) and p.(Val577Met) are located withinone of the helical transmembrane domains (TMDs) and within theloop between two TMDs, respectively. Multiple sequence align-ment of CLCN7 orthologous proteins from different metazoanspecies revealed that these missense variants affected highlyconserved residues (Figure 2b). No possible disease-causingvariants, including CNVs, were detected within the potentialcandidate genes causing osteopetrosis in the TruSight OneSequencing Panel. As a result of the molecular diagnosis and re-evaluation of the clinical features of the affected patient and herunaffected parents having one of the mutations, the patientpresented here was diagnosed with IARO caused by novelpathogenic missense mutations of CLCN7 in a compoundheterozygous state. Because no possible features associated withosteopetrosis were observed in the patient’s sister, her allelestatus as a potential carrier was not determined through testing.Mutations in the CLCN7 gene encoding the chloride channel ofthe osteoclast membrane are associated with 75% of autosomaldominant osteopetrosis (ADO) cases, 10–15% of autosomalrecessive osteopetrosis (ARO) cases and all cases of theintermediate form.7 Mutations in the CLCN7 gene affect thefunction of osteoclast-mediated extracellular acidification, result-ing in the disturbed dissolution of the bone inorganic matrix and aFigure 1. (a) Family pedigree; arrow shows the proband (P). (b) Orthopantomography showing impacted teeth, enamel hypoplasia andmalformed premolars and roots. (c) Lateral radiograph of the skull showing diffuse thickening of the calvarium. (d) Lateral radiograph of thespine showing a ‘sandwich’ vertebrae appearance (arrows). (e) Radiograph of the lower limb and pelvis showing diffuse thickening of bones,with a ‘bone-within-bone’ appearance (arrows). (f) Radiograph of hands showing the ‘bone-within-bone’ appearance in metacarpals (arrows)and phalanges.Novel CLCN7 mutations in IARON Okamoto et al2Human Genome Variation (2017) 17036 Official journal of the Japan Society of Human Geneticsseries of clinical features.8 One of the mutations, p.(Val577Met),observed in the present case, was predicted to be a ‘neutral’change by one of the three bioinformatics tools used in this study.With regard to the findings that the complete loss of function ofthe CLCN7 chloride channel causes malignant ARO, whereasdominant negative missense CLCN7 mutations are responsible forADO,9 this result supports the hypothesis that the intermediatephenotype observed in IARO results from CLCN7 mutations thatonly mildly reduce the capacity of Cl− conductance.10 As noted byPang et al.,11 a genetic analysis with prediction software would beinsufficient to fully predict the functions of novel mutations inIARO due to the relatively small number of IARO cases and thepossible partial residual capacity of Cl− conductance associatedwith mutated CLCN7 proteins in IARO. Our case contributes toenriching the database of CLCN7 mutations and improves ourunderstanding of the clinical and biological aspects of IARO.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.1399 and http://dx.doi.org/10.6084/m9.figshare.hgv.1402.ACKNOWLEDGEMENTSWe thank the patient and her family for their participation in this study. We alsothank our Orthopedics colleagues of Kobe University Hospital for their technicalassistance. This work was partly performed in the Cooperative Research ProjectProgram of the Medical Institute of Bioregulation, Kyushu University. This work wassupported by the Japan Society for the Promotion of Science (16K15618, 15K19620,26861783) and the Japan Agency for Medical Research and Development(16kk0205012h001, 16ek0109151h002).COMPETING INTERESTSThe authors declare no conflict of interest.PUBLISHER'S NOTESpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Figure 2. (a) Partial sequence chromatograms of exons 17 and 19 of CLCN7 in a patient and both parents. The nucleotide and correspondingamino acid sequences of the wild-type and mutant CLCN7 genes are also shown. (b) Multiple amino acid sequence alignment of the CLCN7orthologous proteins from different metazoan species around the mutated amino acid. Arrowheads denote the mutated amino acid. Aminoacids matching those in Homo sapiens are shaded.Novel CLCN7 mutations in IARON Okamoto et al3Official journal of the Japan Society of Human Genetics Human Genome Variation (2017) 17036REFERENCES1 Stark Z, Savarirayan R. Osteopetrosis. Orphanet J Rare Dis 2009; 4: 5.2 Superti-Furga A, Unger S. Nosology and classification of genetic skeletal disorders:2006 revision. Am J Med Genet A 2007; 143A: 1–18.3 Sobacchi C, Schulz A, Coxon FP, Villa A, Helfrich MH. Osteopetrosis: genetics, treatmentand new insights into osteoclast function. Nat Rev Endocrinol 2013; 9: 522–536.4 Okamoto N, Naruto T, Kohmoto T, Komori T, Imoto I. A novel PTCH1 mutation in apatient with Gorlin syndrome. Hum Genome Var 2014; 1: 14022.5 Watanabe M, Hayabuchi Y, Ono A, Naruto T, Horikawa H, Kohmoto T et al.Detection of 1p36 deletion by clinical exome-first diagnostic approach. HumGenome Var 2016; 3: 16006.6 Watanabe M, Nakagawa R, Naruto T, Kohmoto T, Suga K, Goji A et al. A novelmissense mutation of COL5A2 in a patient with Ehlers-Danlos syndrome. HumGenome Var 2016; 3: 16030.7 Balemans W, van Wesenbeeck L, van Hul W. A clinical and molecular overview ofthe human osteopetroses. Calcif Tissue Int 2005; 77: 263–274.8 Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, Schulz A et al. Loss of theClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001; 104:205–215.9 Cleiren E, Benichou O, Van Hul E, Gram J, Bollerslev J, Singer FR et al.Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) resultsfrom mutations in the CLCN7 chloride channel gene. Hum Mol Genet 2001; 10:2861–2867.10 Campos-Xavier AB, Saraiva JM, Ribeiro LM, Munnich A, Cormier-Daire V. Chloridechannel 7 (CLCN7) gene mutations in intermediate autosomal recessive osteo-petrosis. Hum Genet 2003; 112: 186–189.11 Pang Q, Chi Y, Zhao Z, Xing X, Li M, Wang O et al. Novel mutations of CLCN7 causeautosomal dominant osteopetrosis type II (ADO-II) and intermediate autosomalrecessive osteopetrosis (IARO) in Chinese patients. Osteoporos Int 2016; 27:1047–1055.This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International 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/4.0/© The Author(s) 2017Novel CLCN7 mutations in IARON Okamoto et al4Human Genome Variation (2017) 17036 Official journal of the Japan Society of Human Genetics

Novel CLCN7 mutations in IARO

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Novel CLCN7 mutations in IARO

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