Gene 517 (2013) 164–168

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Genetic analysis of the CHD7 gene in Korean patients with CHARGE syndrome☆

Hyun-Ju Cho a,1, Mee Hyun Song b,1, Soo-Young Choi c, Jeongho Kim a, Jinwook Lee a, Un-Kyung Kim a,Jinwoong Bok d,e,f,⁎, Jae Young Choi e,f,⁎⁎a Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Koreab Department of Otorhinolaryngology, Kwandong University College of Medicine, Myongji Hospital, Goyang, South Koreac Department of Medicine, University of Pennsylvania, Philadelphia, PA, USAd Department of Anatomy, Yonsei University College of Medicine, Seoul, South Koreae Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, South Koreaf BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea

Abbreviations: CHD7, chromodomain helicase DNAColoboma of the eye, Heart malformations, Atresia of cand development, Genital hypoplasia, and Ear anomaliecomputed tomography; MRI, magnetic resonance imagPCR, polymerase chain reaction.☆ The authors declare no competing interest.⁎ Correspondence to: J. Bok, Department of Anatomy

Medicine, Seoul 120-752, South Korea. Tel.: +82 2 2228⁎⁎ Correspondence to: J.Y. Choi, Department of OtorhinCollege of Medicine, Seoul 120-752, South Korea. Tel.: +8

E-mail addresses: bokj@yuhs.ac (J. Bok), jychoi@yuh1 These authors contributed equally to this work.

0378-1119/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.gene.2013.01.010

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 4 January 2013Available online 17 January 2013

Keywords:CHARGE syndromeClinical diagnosisCHD7 geneMutationHearing loss

CHARGE syndrome is an autosomal dominant congenital disorder known to be caused by the haploinsufficiencyof the CHD7 gene. Heterozygous mutations in the CHD7 gene have been identified in approximately 60–70% ofpatients clinically diagnosedwith CHARGE syndrome. Although there have beenmany reports on themutationalspectrum of the CHD7 gene in patients with CHARGE syndrome worldwide, little is known about this syndromein the Korean population. In this study, three Korean patientswith CHARGE syndrome including one patientwithPatau syndromewere evaluated for genetic analysis of the CHD7 gene using direct sequencing of all 38 exons andtheflanking intronic regions. One nonsense and twonovelmissensemutationswere identified in the CHD7 gene.Clinical symptoms caused by the missense mutations were much milder compared to the nonsense mutation,confirming the previously determined genotype–phenotype correlation in CHARGE syndrome. Our study dem-onstrates the importance of mutational screening of CHD7 in patients who have been diagnosed with other syn-dromes but display clinical features of CHARGE syndrome.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

CHARGE syndrome (OMIM 214800) is an autosomal dominant con-genital malformation estimated to occur in 1:10,000 births worldwide(Issekutz et al., 2005; Sanlaville and Verloes, 2007). CHARGE associationwas first described by Hall and Hittner in children withmultiple congen-ital abnormalities including choanal atresia (Hall, 1979), and the acronymCHARGEwas subsequently proposed by Pagon et al. (1981) based on thefollowing diagnostic criteria and clinical characteristics: Coloboma of theeye, Heart malformations, Atresia of choanae, Retardation of growth anddevelopment, Genital hypoplasia, and Ear anomalies. The clinical diagno-sis of CHARGE syndromewithmajor andminor criteriawas proposed andupdated by Blake et al. (1998) and Verloes (2005). The diagnostic criteriaproposed by Verloes, which reinforced the importance of semicircular

-binding protein 7; CHARGE,hoanae, Retardation of growths; SCC, semicircular canal; CT,ing; CI, cochlear implantation;

, Yonsei University College of1650; fax: +82 2 365 0700.olaryngology, Yonsei University2 2 2228 3603.s.ac (J.Y. Choi).

l rights reserved.

canal (SCC) hypoplasia or aplasia, are currently widely used for the diag-nosis of CHARGE syndrome (Verloes, 2005).

In 2004, Vissers et al. found that haploinsufficiency of the CHD7gene encoding the chromodomain helicase DNA binding protein 7was the molecular basis of CHARGE syndrome (Vissers et al., 2004).CHD7 with a genomic size of 188 kb is located at chromosome 8q12and includes 38 exons. The encoded protein is composed of 2997amino acids and belongs to a family of nine CHDproteins that common-ly have the ability to utilize ATP hydrolysis to alter nucleosome struc-ture (Marfella and Imbalzano, 2007). These alterations of chromatinstructure are involved in gene expression, DNA repair, replication andrecombination, and play important roles in embryonic development(Vissers et al., 2004).

Heterozygous mutations and deletions of the CHD7 gene have beenidentified in approximately 60–70% of patients clinically diagnosed withCHARGE syndrome (Jongmans et al., 2006; Lalani et al., 2006; Zentneret al., 2010). Genetic analysis of the CHD7 gene is a major contributor tothe diagnosis of CHARGE syndrome today, because some other syn-dromes share the clinical features of CHARGE syndrome, which maylead to incorrect diagnosis. For example, Patau syndrome,which is causedby the presence of an extra copy of chromosome 13, is one of such syn-dromes showing overlapping clinical features with CHARGE syndrome,including multiple congenital anomalies of the central nervous system,eye, heart, urogenital system, limbs, and ears (Patau et al., 1960). In addi-tion, since some patients with CHD7 mutations demonstrate only mild

165H.-J. Cho et al. / Gene 517 (2013) 164–168

symptoms, as seen in familial cases of CHARGE syndrome, genetic analy-sis of the CHD7 gene is required for the accurate diagnosis of CHARGEsyndrome (Bergman et al., 2011). In this study, we performed geneticanalysis of the CHD7 gene in Korean patients with variable clinical fea-tures of CHARGE syndrome including one patient with combined Patausyndrome.

2. Material and methods

2.1. Subjects

Three of thepatientswhowere enrolled in the auditory rehabilitationprogram at Severance Hospital demonstrating profound sensorineuralhearing loss and SCC aplasia or hypoplasia on the temporal bone com-puted tomography (CT) were included in this study. All three patientswere females and had received cochlear implantation (CI) for auditoryrehabilitation. One of the patients (patient 1) had been diagnosed withPatau syndrome at another institution (Table 1). Audiologic evaluationsincluding auditory brainstem response, otoacoustic emissions, and audi-tory steady state response were performed in all patients, and pure toneaudiometry was tested when possible. Temporal bone CT and temporalmagnetic resonance imaging (MRI) were performed in all patients aspreviously described (Song et al., 2011). Combined disabilities andothermedical conditionswere thoroughly reviewed for clinical diagnosisof CHARGE syndrome.Written informed consentwas obtained frompar-ticipating individuals, and this study was approved by the InstitutionalReview Board of the Yonsei University College of Medicine.

2.2. Genetic analysis

Genomic DNA from the three patients and 200 unrelated normalhearing controls was isolated from peripheral blood using a FlexiGeneDNA extraction kit (Qiagen, Hilden, Germany). For sequencing analysisof the CHD7 gene, all 38 exons and exon-intron boundary regions wereamplified by polymerase chain reaction (PCR) using H-Taq polymerase(Solgent, Daejeon, South Korea) and appropriate primer sets as previous-ly described (Song et al., 2011). After confirming PCR products in the 2%agarose gel, amplified fragments were purified and subsequently se-quenced using an ABI PRISM Big Dye Terminator Cycle Sequencing Kit(V3.1) and an ABI PRISM3130XL DNA analyzer (Applied Biosystems,Foster City, CA, USA). The sequencing data were then analyzed usingABI Sequencing Analysis (v.5.0) and Lasergene–SeqMan software. Identi-fied novel missense mutations were also analyzed in the 200 unrelatednormal controls who demonstrated normal hearing on pure tone audi-ometry. Multiple sequence alignment was performed using ClustalW2(http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Table 1Clinical diagnosis of CHARGE syndrome following Verloes's diagnostic criteria.

Patient 1 Patient 2 Patient 3

MajorOcular coloboma +Choanal atresiaHypoplastic semicircular canals + + +

MinorRhombencephalic dysfunction + + +Hypothalamohypophyseal dysfunctionAbnormal middle/external ear + +Malformation of mediastinal organs + +Mental retardation + +

OthersUrogenital anomalies +Cleft palate/lipLimb abnormalities + +Facial dysmorphia +CHARGE syndrome Atypical Possible Typical

3. Results

3.1. Clinical diagnosis of CHARGE syndrome

Three unrelated Korean patients suspected of CHARGE syndromebased on the clinical criteria proposed by Verloes (2005) were ana-lyzed in this study (Table 1). All patients showed hypoplastic SCCand rhomencephalic dysfunction (sensorineural hearing loss) as com-mon clinical features. Patient 1 exhibited one major (SCC hypoplasia)and three minor (rhombencephalic dysfunction, abnormal middle/external ear, and mental retardation) criteria to be classified as atypicalCHARGE syndrome. This patient had been diagnosed with translocationtype of trisomy 13 (Patau syndrome) caused by 46,XX,der(14)t(13:14)(q21.2:p13). In addition to the features of CHARGE syndrome, facialdysmorphia and limb abnormalities were observed in this patient. Forpatient 2, cardiac malformation was the only definite clinical manifesta-tion of CHARGE syndromeother than sensorineural hearing loss and SCChypoplasia. Although this patient did not satisfy the diagnostic criteria ofCHARGE syndrome proposed by Verloes, genetic analysis for CHD7wasperformed since SCC hypoplasia/aplasia combined with profound sen-sorineural hearing loss has been reported to be very common manifes-tations of CHARGE syndrome (Song et al., 2011; Verloes, 2005).Furthermore, cardiac malformation is one of the mediastinal organ de-fects commonly observed in CHARGE syndrome. Based on these find-ings, we classified patient 2 as ‘possible’ CHARGE syndrome. Patient 3showed two major (ocular coloboma and SCC aplasia) and four minor(rhombencephalic dysfunction, abnormal middle/external ear, malfor-mation of mediastinal organs, and mental retardation) criteria and wasdiagnosed as typical CHARGE syndrome. Polycystic kidney disease andlimb abnormalities were also combined clinical features in patient 3.

On high resolution temporal bone CT, patient 1 demonstrated SCChypoplasia mainly involving the horizontal SCC and dilated vestibule aswell as cystic type of cochlear hypoplasia (type II) (Fig. 1) (Sennaroglu,2010). Patient 2 exhibited the mildest inner ear anomaly including hori-zontal SCC dysplasia and nearly normal cochlea. In patient 3, cochlear hy-poplasia (type III) and complete aplasia of SCCs were seen (Sennaroglu,2010).

3.2. Genetic analysis of the CHD7 gene

Three different mutations (one nonsense and two novel missense) inthe CHD7 gene were identified in three patients diagnosed with CHARGEsyndrome (Table 2; Fig. 2). A novelmissensemutation, a thymine to gua-nine transition at nucleotide position 4764 (c.4764T>G) resulting in anamino acid change from cysteine to tryptophan (p.C1588W) was identi-fied in exon 21 in patient 1. Another novel missense mutation, a guanineto adenine transversion at position 5473 (c.5473G>A) leading to substi-tution of aspartate with asparagine (p.D1825N), was identified in patient2. These twomissensemutationswere not found in the 200 controlswithnormal auditory thresholds and theywere not in the 1000 Genome data-base. The conservation of each amino acid was analyzed by multiplealignments with vertebrate homologs such as Homo sapiens, Pan troglo-dytes, Mus musculus, Rattus norvegicus, Vicugna vicugna, Gallus gallus andDanio rerio (Fig. 2). The aspartate of p.D1825N was found to be highlyconserved among vertebrate species. The cysteine of p.C1588W wasshown to be less conserved than p.D1825N and its conservation was re-stricted to mammals. The nonsense mutation in exon 30 (c.6079C>T)resulting from substitution of the arginine to a stop codon (p.R2027X)was a recurrent mutation that has also been identified in seven other pa-tients (Bartels et al., 2010; Jongmans et al., 2006) (CHD7 database; http://www.chd7.org).

4. Discussion

Two studies were previously reported on the clinical and geneticanalyses of CHARGE syndrome in Korea (Lee et al., 2009; Song et al.,

Fig. 1. Temporal bone computed tomography findings of patients with CHARGE syndrome. In patient 1 and patient 2, semicircular canal hypoplasia mainly involving the horizontalcanal (black empty arrowhead) was seen, while complete semicircular canal aplasia was observed in patient 3. The superior (white thick arrow) and posterior (white arrowhead)semicircular canals could be found in patient 1 and patient 2, but not in patient 3. Cochlear hypoplasia of cystic type (type II) and less than two turns (type III) was seen in patient 1and patient 3, respectively, whereas nearly normal cochlea was observed in patient 2 (cochlea marked by white arrows).

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2011). Lee et al. reported four novel CHD7mutations in Korean patientswith typical and atypical CHARGE syndromes, all of whom demonstrat-ed aplasia of SCCs and congenital heart disease (Lee et al., 2009). Song etal. identified eight CHD7 mutations in Korean patients with CHARGEsyndrome and described the radiologic findings of the temporal boneincluding SCC aplasia, cochlear hypoplasia (type III), and narrow inter-nal auditory canals (Song et al., 2011). The outcome of auditory rehabil-itation in patients with CHARGE syndrome was also reported (Songet al., 2011).

In the present study, we identified one nonsense and two novel mis-sense mutations in the CHD7 gene in three Korean patients with clinicalfeatures of CHARGE syndrome, one of whom had also been diagnosedwith Patau syndrome. Patient 1 and patient 2, who hadmissense muta-tions in the CHD7 gene, showed the SCC hypoplasia mainly in the hori-zontal SCC with relatively intact superior and posterior SCCs (Fig. 1).Since these patients displayed milder phenotypes and fewer criteria ofCHARGE syndrome, they were diagnosed as an atypical (patient 1) anda ‘possible’ (patient 2) CHARGE patient. The cochlear phenotype wasalso largely normal in patient 2. Although patient 1 demonstrated co-chlear hypoplasia of cystic type (type II), we attribute this phenotypeto the combined symptoms with Patau syndrome. Consistently, Songet al. reported that patients with missense mutations in the CHD7 genehadmilder symptoms compared to other CHARGE patients carrying dif-ferent types of mutations (Song et al., 2011). In contrast, patient 3, whocarried a nonsense mutation in CHD7, was diagnosed with typicalCHARGE syndrome because the clinical findings included complete

Table 2CHD7 gene screening results of three Korean patients with CHARGE syndrome.

PatientNo.

Clinicaldiagnosis

Exon Nucleotidechange

Amino acidchange

Type Reference

1 Atypical 21 c.4764T>G p.C1588W Missense Novel2 Possible 26 c.5473G>A p.D1825N Missense Novel3 Typical 30 c.6079C>T p.R2027X Nonsense (Bartels et al.,

2010; Jongmanset al., 2006)

aplasia of all SCCs, cochlear hypoplasia (type III), ossicular deformity,and oval window atresia (Song et al., 2011). These observations confirmthe previously determined genotype–phenotype correlation in CHARGEsyndrome.

Despite the broad spectrum of inner ear anomalies, our genetic anal-ysis identified mutations in the CHD7 gene in all three patients. Thishigh mutation rate is consistent with previous reports showing thatthe hypoplastic or aplastic SCC is present in more than 80% of scannedCHARGE children and thus is an important criterion for the diagnosisof CHARGE syndrome (Morgan et al., 1993; Verloes, 2005). Interestinglyhowever, none of the patients showed choanal atresia, confirming a lowincidence of this major criterion of CHARGE syndrome in Korean popu-lation (Lee et al., 2009; Song et al., 2011).

Patau syndrome, which is caused by trisomy 13, shows a variety ofinner ear abnormalities including shortened cochlear coils, modiolar de-formity, incomplete partition, unusually wide cochlear aqueduct, dys-plastic or absent horizontal and/or superior SCC, abnormalities of theseventh and eighth cranial nerves and their ganglions, and degenerationof the organ of Corti, stria vascularis and saccule (Fukushima et al., 2008;Suga et al., 1976). Although partial trisomy for chromosome 13 due totranslocation, as seen in patient 1 in our study, often results in pheno-typic features different from the typical Patau syndrome, it has beenshown that the partial trisomy can also produce the majority of the tri-somy 13 syndrome features (Rivas et al., 1984). As the clinical manifes-tations of trisomy 13 syndrome considerably overlap with the clinicalfeatures of CHARGE syndrome, the anomalies found in patient 1, whowas diagnosed with both Patau and CHARGE syndromes, appeared toresult from the combined effect of partial trisomy 13 and the CHD7mu-tation. However, due to the lack of previous literature on the detailed de-scription of temporal bone anomalies associatedwith the partial trisomy13, it is difficult to delineate the effect of each genetic aberration in pa-tient 1.

Five-hundred twenty eight different pathogenicmutations have beenassociatedwith CHARGE syndrome to date, which are equally distributedalong the coding region of the CHD7 gene. Truncating mutations includ-ing nonsense and frameshift mutations are most frequently observed

Fig. 2. Three mutations of the CHD7 gene identified in Korean patients with CHARGE syndrome. Direct sequencing analysis of the CHD7 gene revealed one nonsense mutation andtwo missense mutations. (A) c.4764T>G and c.5473G>A, two missense mutations lead to one amino acid change and resulted in p.C1588W and p.D1825N, respectively. The othernonsense mutation, p.R2027X, resulted in conversion of an arginine to a stop codon and resulted in a truncated protein. (B) The two amino acids in which missense mutations wereidentified are well conserved among many vertebrate species.

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(~78%), followedbymissense and splice sitemutations (~19%). Completeor partial deletions/duplications and translocations are rarely identified(~3%) (Janssen et al., 2012). Most mutations are sporadic and not repeat-ed in other patients, except for 94 recurrent mutations that often replacearginine with stop codons. To date, 15 mutations in the CHD7 gene havebeen identified in Korean CHARGE patients (Fig. 3) (Lee et al., 2009; Songet al., 2011). Nonsensemutations (6 cases)were themost prevalent type,followed by frameshift (4 cases), missense (3 cases), and splice-site mu-tations (2 cases). Themutationswere randomly scattered throughout theentire coding region and splice sites of the CHD7 gene, and none of themutations were replicated among patients. The analysis of mutationaldata from the Korean population shows that the most common muta-tions are truncating mutations and supports previous studies that didnot find any evidence of a hotspot for mutations in CHD7 (Janssen et al.,2012). The frequency of the missense mutation was shown to be higherin the Korean population (20%) than other populations (8%). The threemissense mutations of the CHD7 gene found in the Korean populationincluding the two novel mutations identified in this study were locatedin the highly conserved middle exons of the gene and not in the first

Fig. 3. Schematic overview of chromodomain helicase DNA-binding protein 7 and summaridentified in Korean patients with CHARGE syndrome are shown. Six nonsense mutationsare spread over the CHD7 protein. The three mutations identified in this study are indicate

seven or last five exons, which is consistent with most of the previouslyreportedpathogenicmissensemutations (Janssen et al., 2012).Moreover,the locations of the three missense mutations identified in the Koreanpopulation were highly conserved among vertebrates (p.D1825N andp.R2027X) and mammals (p.C1588W), supporting the pathogenicity ofthemutations (Janssen et al., 2012; Song et al., 2011). A number of recur-rent mutations identified in CHARGE patients, including the nonsensemutation c.6079C>T (p.R2027X) in patient 3, change an arginine to astop codon (Bartels et al., 2010; Janssen et al., 2012). This frequentmuta-tion can be explained by previous observations that the CG-nucleotidepair is hypermutable to TG leading to the conversion of an arginine to astop codon (Antonarakis et al., 2000).

In conclusion, we have identified one nonsense and two novelmissense mutations in the CHD7 gene in three Korean patients withCHARGE syndrome. Clinical symptoms were much milder in patientswith missense mutations compared to a patient with a nonsense mu-tation, confirming the genotype–phenotype correlation in CHARGEsyndrome. The identification of a CHD7 mutation in a patient withPatau syndrome demonstrates the importance of genetic screening

y of CHD7 mutations. The CHD7 structure with functional domains and 15 mutations, four frameshift mutations, three missense mutations, and two splice site mutationsd in red.

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of the CHD7 gene in individuals diagnosed with other syndromes butdisplaying some clinical features of CHARGE syndrome.

Acknowledgments

This studywas supported by a grant of the Korean Health TechnologyR&D Project, Ministry for Health, Welfare & Family Affairs, Republic ofKorea (A100493) and by the Basic Science Research Program throughthe National Research Foundation of Korea (NRF) funded by theMinistryof Education, Science and Technology (2011-0028066).

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