CHARGE syndrome is a rare autosomal dominant developmental disorder involving multiple organs. CHD7 is a major causative gene of CHARGE syndrome. We performed targeted-exome sequencing using a next-generation sequencer for molecular diagnosis of a 4-month-old male patient who was clinically suspected to have CHARGE syndrome, and report a novel monoallelic mutation in CHD7, NM_017780.3(CHD7_v001):c.2966del causing a reading frameshift [p.(Cys989Serfs*3)]

Imoto, Issei

Kagami, Shoji

Kohmoto, Tomohiro

Masuda, Kiyoshi

Nakagawa, Ryuji

Naruto, Takuya

Shono, Miki

Suga, Ken-ichi

Watanabe, Miki

English

Tokushima University Institutional Repository

OPENDATA REPORTA novel frameshift mutation of CHD7 in a Japanese patientwith CHARGE syndromeTomohiro Kohmoto1,2,4, Miki Shono3,4, Takuya Naruto1, Miki Watanabe1,2, Ken-ichi Suga3, Ryuji Nakagawa3, Shoji Kagami3,Kiyoshi Masuda1 and Issei Imoto1CHARGE syndrome is a rare autosomal dominant developmental disorder involving multiple organs. CHD7 is a major causative geneof CHARGE syndrome. We performed targeted-exome sequencing using a next-generation sequencer for molecular diagnosis of a4-month-old male patient who was clinically suspected to have CHARGE syndrome, and report a novel monoallelic mutation inCHD7, NM_017780.3(CHD7_v001):c.2966del causing a reading frameshift [p.(Cys989Serfs*3)].Human Genome Variation (2016) 3, 16004; doi:10.1038/hgv.2016.4; published online 7 April 2016CHARGE syndrome (OMIM 214800) is an autosomal dominantcongenital multi-system disorder occurring in an estimated1:10,000 births worldwide and displaying broad clinicalvariability.1,2 Hall3 and Hittner et al.4 were the first to indepen-dently describe a non-random cluster of malformations that waslater referred to as CHARGE association, named by the acronymsummarizing the six major clinical features: ocular coloboma, heartmalformations, atresia of the choanae, retardation of growth,genital hypoplasia and ear abnormalities.5 The clinical diagnosis ofCHARGE syndrome with major and minor criteria was thenproposed by Blake et al.6 and updated by Verloes.7 As CHARGEsyndrome can closely resemble several other genetic andteratogenic conditions, genetic testing should be emphasized asa useful tool in patients with clinical suspicion.8 Monoallelicmutations or deletions of CHD7 (OMIM 608892), encoding thechromodomain helicase DNA-bindingprotein 7 at chromosome8q12, are present in 65–70% of patients clinically diagnosed withCHARGE syndrome.9 CHD7mutations in typical CHARGE syndromepatients occur de novo in the vast majority of the cases.8 There isalso evidence that CHARGE syndrome can be caused by mutationsin SEMA3E (OMIM 608166) at chromosome 7q21, although onlyone case has been reported.10The difficulty in clinical diagnosis of CHARGE syndrome lies indifferentiating it from various other diseases sharing its clinicalfeatures, such as 22q deletion syndrome, Kabuki syndrome,velo-cardio-facial syndrome, cat eye syndrome, retinoic acidembryopathy, VACTERL association and PAX2 abnormalities.8Molecular diagnosis is the most accurate and definitive methodfor differential diagnosis.11,12 Conventional Sanger sequencing hasroutinely been used to identify disease-causing mutations forgenetic diseases, but is laborious, expensive and time-consumingfor large genes with numerous exons, such as CHD7 (37 exonscovering 8,994 bp of the coding sequence), as well as formultiple candidate genes. Recently, targeted-exome sequencingfor candidate or known disease-associated genes using next-generation sequencing (NGS) has been used to diagnose patientswith various types of disorders including CHARGE syndrome.13Here, we report a novel monoallelic frameshift mutation ofCDH7, NM_017780.3(CHD7 v001):c.2966delG, detected in atargeted-exome panel for the coding regions of 4,813 clinicalphenotype-associated genes including CHARGE syndrome-associated genes in a Japanese patient with CHARGE syndrome.A 4-month-old male infant was the second child born to non-consanguineous Japanese healthy parents with an unremarkablefamily history, after uncomplicated pregnancy and delivery at alocal hospital. He was born at 38 weeks and 2 days of gestationwith a birth weight of 3,086 g (−0.1 s.d.), a length of 47 cm (−1.1 s.d.)and an occipitofrontal circumference of 33.4 cm (+0.1 s.d.). Apgarscores were 9 and 9 at 1 and 5 min, respectively. At birth, heshowed cleft lip and palate resulting in poor feeding, and requiredtube feeding as well as oxygen; he was transferred to our hospitalat the age of 11 days. Results of repeatedly performed auditorybrainstem response test showed bilateral profound deafnesswith auditory brainstem response thresholds 495 dB. Computedtomography revealed bilateral hypoplastic semicircular canalsand filled tympanic cavities. Two-dimensional color-Dopplerechocardiography showed incomplete atrioventricular septaldefect, patent ductus arteriosus, aortic valve stenosis and bicuspidaortic valve. As the child had ventilation dysfunction due toglossoptosis and upper airway obstruction detected by multi-detector row computed tomography were observed, he requiredartificial ventilation with endotracheal intubation at 14 days, andwas subjected to a tracheotomy owing to difficulties of extubationat 81 days. He still required nasogastric tube feeding owingto swallowing difficulty. Symptoms of heart failure due to theprogression of aortic valve stenosis developed at 90 days.He showed dysmorphic features, including choanal stenosis,bilateral low-set ears, micropenis and cryptorchidism. His motordevelopment, such as head control and rolling over, was almostnormal. Karyotype analysis revealed a normal male karyotype of46,XY. Biochemical and gas chromatography-mass spectrometrymetabolic disorder tests were negative, with no evidence ofmetabolic disease. No clinical characteristics of CHARGE syndromewere detected in the patient’s parents. Although coloboma was1Department of Human Genetics, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan; 2Student Laboratory, Faculty of Medicine,Tokushima University, Tokushima, Japan and 3Department of Pediatrics, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.Correspondence: I Imoto (issehgen@tokushima-u.ac.jp)4These authors contributed equally to this work.Received 16 November 2015; revised 19 January 2016; accepted 19 January 2016Citation: Human Genome Variation (2016) 3, 16004; doi:10.1038/hgv.2016.4Official journal of the Japan Society of Human Genetics 2054-345X/16www.nature.com/hgvnot observed, the patient was suspected to have CHARGEsyndrome because two of four major characteristics (choanalatresia and characteristic outer and inner ears, and deafness) andthree minor characteristics (cardiovascular malformation, genitalhypoplasia and cleft lip and palate) of CHARGE syndrome wereobserved (Table 1).8Molecular diagnosis was performed using genomic DNAextracted from a blood sample, after informed consent wasobtained from the parents. The study was approved by the ethicscommittees of Tokushima University. To screen known disease-associated genes including CHD7, we first used a MiSeq bench-topsequencer (Illumina, San Diego, CA, USA) to perform NGS with aTruSight One Sequencing Panel (Illumina), and our pipelinefor NGS data analysis as described elsewhere.14,15 To identifypresumably pathogenic single-nucleotide variants (SNVs), weexcluded sequence variants with minor allele frequency 40.05from the 1000 Genomes Project database (http://www.1000genomes.org/), NHLBI Grand Opportunity Exome Sequencing Project(ESP6500, http://evs.gs.washington.edu/EVS/) and the HumanGenetic Variation Database (HGVD, http://www.genome.med.kyoto-u.ac.jp/SnpDB/). Copy number aberration (CNA) analysiswas also performed as described elsewhere.16 These analysesidentified a monoallelic, one-base (guanine) deletion of CHD7,NM_017780.3(CHD7_v001):c.2966del (NM_017780.3:n.3458del),which was confirmed by Sanger sequencing (Figure 1a). Primersequences are available upon request. This mutation causes areading frameshift starting from residue Cys989, with the newreading frame ending with a premature stop codon twocodons downstream, NM_017780.3(CHD7_i001):p.(Cys989Serfs*3).As shown in Figure 1, the mutation is located in exon 12, encodingthe SNF2 family N-terminal (SNF2 N) and DEAD-like helicasesuperfamily including an ATP-binding (DEXDc) domains.9,17 Thismutation is not present in dbSNP138, 1000 Genomes Project,ESP6500 and HGVD. The mutation is also not present in theHuman Gene Mutation Database professional 2015.2 (HGMD,http://www.hgmd.org/) or ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/), suggesting it to be novel. No other potentiallypathogenic SNVs and CNAs were detected in genes possiblyresponsible for CHARGE syndrome or other related diseases.17The mutation was not confirmed as de novo because of theunavailability of parental DNA.In HGMD professional 2015.2, 615 CHD7 mutations werereported in CHARGE syndrome. The majority of these mutationsare nonsense, frameshift or splice site mutations, which arepredicted to be loss-of-function mutations, likely leading to anaberrant messenger RNA targeted for degradation via nonsense-mediated decay (NMD).18 Therefore, haploinsufficiency for CHD7 isthe most likely pathogenic mechanism underlying CHARGEsyndrome.18 Even if the mutated transcript escapes NMD, thetruncated CHD7 protein predicted in this patient contains only theN-terminal tandem chromodomains, which are incapable ofperforming the protein’s functions owing to the loss of chromatinremodeling activity (Figure 1b).NGS emergence for the use in diagnostic laboratories willfacilitate the characterization of new sequence variants in patientswith genetic disorders. In a diagnostic setting for CHARGEsyndrome, CHD7 mutation analysis using Sanger sequencingmay miss genetic alterations causing CHARGE syndrome orphenocopies of CHARGE syndrome, such as gross deletions ordeep intronic mutations of CHD7, alterations in SEMA3E, uniquechromosomal imbalances and 22q11.2 deletion syndrome.17Given that targeted-exome or whole-exome sequencing candetect both exonic SNVs and CNAs in disease-causinggenes,15,16 those platforms can facilitate differential diagnosis,appropriate therapy and genetic counseling for patients with thisTable 1. Symptoms of the patient compared with major and minor diagnostic criteria in CHARGE patients8Diagnostic criteria Symptoms of the patient Frequency in CHARGE patients8MajorOcular coloboma − 80–90%Choanal atresia or stenosis + 50–60%Cranial nerve dysfunction Unclear (VIII, IX/X) Frequent (VIII) or 70–90% (IX/X)Characteristic CHARGE syndrome ear + (Characteristic outer and inner ears anddeafness)80–100%MinorGenital hypoplasia due to congenital hypogonadotropichypogonadism+ (Micropenis, cryptorchidism) 50–60% (male; micropenis,cryptorchidism)Developmental delay − ⩽ 100%Cardiovascular malformations + 75–85%Growth deficiency − 70–80%Orofacial cleft + 15–20%Tracheoesophageal fistula − 15–20%Distinctive facial feature − 70–80%Figure 1. (a) Electropherogram of CHD7 (NM_017780.3) exon 12 andflanking intron 11 sequences showing the monoallelic germlinemutation NM_017780.3:c.2966del (arrowhead) in the patient’s DNA.The DNA and corresponding amino acid sequences of the wild-typeand mutant CHD7 alleles are also shown. (b) A schematicrepresentation of the CHD7 protein. Domains are depictedapproximately to the scale adapted from human reference sequence(NM_017780.3(CHD7_i001)) and reference 17. The arrowhead showsthe position corresponding to the mutation. BRK, Brahma andKismet domain; Chromo, chromodomain; DEXDC, DEAD-like heli-case superfamily including an ATP-binding domain; Helicase C,helicase superfamily C-terminal domain; SANT, switching-defectiveprotein 3, adaptor 2, nuclear receptor corepressor, transcriptionfactor IIIB domain; SNF2 N, SNF2 family N-terminal domain.Novel CHD7 mutation in CHARGE syndromeT Kohmoto et al2Human Genome Variation (2016) 16004 © 2016 The Japan Society of Human Geneticssyndrome and related diseases in a cost-effective manner.Coverage of all genetic alterations causing CHARGE andCHARGE-like diseases will be extended with whole-genomesequencing when the cost of sequencing is lowered.HGV DATABASEThe relevant data from this Data Report are hosted at the HumanGenome Variation Database at http://dx.doi.org/10.6084/m9.figshare.hgv.777.ACKNOWLEDGEMENTSAuthors would like to thank the patient and his family for participation in this study.This work was supported by JSPS KAKENHI Grant Numbers 26293304 (II) and15K19620 (TN) from the Ministry of Education, Culture, Sports, Science andTechnology, Japan.COMPETING INTERESTSThe authors declare no conflict of interest.REFERENCES1 Issekutz KA, Graham JM Jr, Prasad C, Smith IM, Blake KD. An epidemiologicalanalysis of CHARGE syndrome: preliminary results from a Canadian study. Am JMed Genet A 2005; 133: 309–317.2 Sanlaville D, Verloes A. CHARGE syndrome: an update. Eur J Hum Genet 2007; 15:389–399.3 Hall BD. Choanal atresia and associated multiple anomalies. J Pediatr 1979; 95:395–398.4 Hittner HM, Hirsch NJ, Kreh GM, Rudolph AJ. Colobomatous microphthalmia,heart disease, hearing loss, and mental retardation—a syndrome. J PediatrOphthalmol Strabismus 1979; 16: 122–128.5 Pagon RA, Graham JM Jr, Zonana J, Yong SL. Coloboma, congenital heart disease,and choanal atresia with multiple anomalies: CHARGE association. J Pediatr 1981;99: 223–227.6 Blake KD, Davenport SL, Hall BD, Hefner MA, Pagon RA, Williams MS et al. CHARGEassociation: an update and review for the primary pediatrician. Clin Pediatr (Phila)1998; 37: 159–173.7 Verloes A. Updated diagnostic criteria for CHARGE syndrome: a proposal. 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Identification of one novel CHD7mutation in a patient from China with atypical CHARGE syndrome. Gene 2015;571: 298–302.14 Okamoto N, Naruto T, Kohmoto T, Komori T, Imoto I. A novel PTCH1 mutation in apatient with Gorlin syndrome. Hum Genome Variation 2014; 1: 14022.15 Naruto T, Okamoto N, Masuda K, Endo T, Hatsukawa Y, Kohmoto T et al. Deepintronic GPR143 mutation in a Japanese family with ocular albinism. Sci Rep 2015;5: 11334.16 Morita K, Naruto T, Tanimoto K, Yasukawa C, Oikawa Y, Masuda K et al.Simultaneous detection of both single nucleotide variations and copy numberalterations by next-generation sequencing in Gorlin syndrome. PLoS ONE 2015;10: e0140480.17 Janssen N, Bergman JE, Swertz MA, Tranebjaerg L, Lodahl M, Schoots J et al.Mutation update on the CHD7 gene involved in CHARGE syndrome. Hum Mutat2012; 33: 1149–1160.18 Zentner GE, Layman WS, Martin DM, Scacherin PC. Molecular and phenotypicaspects of CHD7 mutation in CHARGE syndrome. Am J Med Genet A 2010; 152A:674–686.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/Novel CHD7 mutation in CHARGE syndromeT Kohmoto et al3© 2016 The Japan Society of Human Genetics Human Genome Variation (2016) 16004

Novel CHD7 mutation in CHARGE syndrome

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Novel CHD7 mutation in CHARGE syndrome

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