Revealing the Function of a Novel Splice-Site Mutation of CHD7 in CHARGE Syndrome

Gene 576 (2016) 776–781

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Research paper Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome

Byeonghyeon Lee a,b,1, Mehmet Bugrahan Duz c,1, Borum Sagong a,b,AsumanKoparirc, Kyu-Yup Lee d, Jae Young Choi e, Mehmet Seven c, Adnan Yuksel f,Un-KyungKima,b,⁎, Mustafa Ozen c,f,g,⁎⁎ a Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea b School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, South Korea c Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey d Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea e Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, South Korea f Department of Medical Genetics, Biruni University Medical School, Istanbul, Turkey g Department of Pathology & Immunology, Baylor College of Medicine, Michael E. DeBakey VAMC, Houston, TX, United States article info abstract

Article history: Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of Received 24 June 2015 CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of Received in revised form 24 September 2015 CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and Accepted 4 November 2015 ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2ANG) Available online 10 November 2015 and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2ANG mutation at the transcriptional level, and found that it caused a com- Keywords: CHARGE syndrome plete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demon- CHD7 strating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2ANG mutation affects Splice-site mutation the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and Frameshift mutation coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes Turkish of CHARGE syndrome. © 2015 Elsevier B.V. All rights reserved.

1. Introduction asymptomatic or exhibit only mild phenotypes, whereas their children show more severe phenotypes (Zentner et al., 2010; Bergman et al., CHARGE syndrome occurs in approximately 1 in every 10,000 new- 2011). borns worldwide, and almost all cases of CHARGE syndrome occur CHD7, a gene located on chromosome 8q12.1 and encoding the sporadically (Bergman et al., 2011).Therecurrencerateisapproximately chromodomain helicase DNA-binding (CHD) protein 7, is a major 1% among sib-pairs, monozygotic twins, and 2-generation families causative gene of CHARGE syndrome (Vissers et al., 2004). CHD7 (Jongmans et al., 2006; Lalani et al., 2006; Delahaye et al., 2007; contains one non-coding and 37 coding exons, and encodes for the Jongmans et al., 2008; Vuorela et al., 2008; Wincent et al., 2008; Pauli 2997 amino acids CHD7 protein belonging to a family of nine CHD et al., 2009; Bergman et al., 2011). Familial CHARGE syndrome follows proteins that can modify chromatin structure (Vissers et al., 2004). an autosomal dominant inheritance with variable penetrance (Bergman Approximately 60%–70% of patients with CHARGE syndrome have et al., 2011). In parent-to-child cases, the parents are generally pathogenic mutations in CHD7 (Zentner et al., 2010). While approximately 70% of these mutations are truncating mutations, such as nonsense or frameshift mutations (Zentner et al., 2010), the incidence of splice-site or missense mutations is low (Zentner et al., 2010). The most likely Abbreviations: CHD, chromodomain helicase DNA-binding; PCR, polymerase chain re- cause of CHARGE syndrome is the haploinsufﬁciency of CHD7.Thisﬁnding action; ECHO, echocardiogram; ASD, atrial septal defect; CCA, common carotid artery; MRI, magnetic resonance imaging; 3D CT, three-dimensional computed tomography; ICA, in- is supported by mouse model studies in which homozygous Chd7 mice ternal carotid artery; RT-PCR, reverse transcription-PCR. with loss-of-function mutations showed embryonic lethality, while ⁎ Corresponding author at: Department of Biology, College of Natural Sciences, heterozygous Chd7 mice showed phenotypic features similar to those Kyungpook National University, Daegu, South Korea. associated with CHARGE syndrome (Bosman et al., 2005; Hurd et al., ⁎⁎ Corresponding author at: Department of Medical Genetics, Biruni University Medical 2007). School, Istanbul, Turkey. E-mail addresses: [email protected] (U.-K. Kim), [email protected] (M. Ozen). Because many of the symptoms and features of CHARGE syndrome 1 These authors contributed equally. are common to other syndromes, genetic analysis of CHD7 is necessary

http://dx.doi.org/10.1016/j.gene.2015.11.006 0378-1119/© 2015 Elsevier B.V. All rights reserved. B. Lee et al. / Gene 576 (2016) 776–781 777 for an accurate diagnosis of CHARGE syndrome (Corsten-Janssen et al., 3. Results 2013). In the present study, we have performed DNA-sequencing analysis of CHD7 in two Turkish patients with several clinical characteristics 3.1. Clinical features of CHARGE syndrome. Patient 1 was the third child born to a 34-year-old, gravida 3, para 3 mother; her parents were both healthy and were third-degree cousins 2. Materials and methods (Fig. 1A). Pregnancy was uncomplicated with a spontaneous vaginal delivery, at term, according to normal birth parameters. However, 2.1. Subjects and clinical examinations choanal atresia was discovered upon birth and an operation was performed immediately. The patient was followed-up in the neonatal in- This study included two Turkish patients who were diagnosed tensive care unit for twenty days. She was hypotonic at birth, and at 4 with CHARGE syndrome. Both of the patients were evaluated by clin- months old, her family realized she was not able to control her head. ical medical geneticist and detailed physical examinations were per- During a physical examination, a systolic murmur was determined. An formed covering all the systems and required tests were performed echocardiogram (ECHO) revealed a wide secundum atrial septal defect to confirm diagnosis. The mutations identified in these patients (ASD), increased right ventricular dimensions, right side located arcus were screened for in 48 other individuals who had no known genetic aorta and both truncus brachiocephalicus and the left common carotid defects. Before their participation in the study, written informed artery (CCA) originated from the same root. These cardiovascular ab- consent was obtained from all participants, and guardians when normalities were accompanied by a vascular ring, which was detected necessary. during a three-dimensional computed tomography (3D CT) chest examination. To elucidate these major vessel anomalies, intracranial angiography and cervical magnetic resonance imaging (MRI) angiography 2.2. Genetic analysis were used. A total occlusion of the left internal carotid artery (ICA) from its origin was detected and accompanied by collateral circulation by the Blood samples were collected from both the patients and their vertebral artery. There were no brain malformations observed in the family members, and genomic DNA was extracted using the EZ1 DNA cranial MRI. When she was referred to us at 5-year-old, her height, blood kit (Qiagen, Hilden, Germany). The 38 exons and exon–intron weight, and head circumference were 97 cm (b3rd percentile), 17 kg boundaries of CHD7 were amplified by polymerase chain reaction (b50–75th percentile) and 48.5 cm (3rd percentile), respectively. She (PCR) with h-Taq polymerase (Solgent, Daejeon, Korea) and specific had prominent eyes, a broad nasal root and bridge, a thin upper lip primers designed using Primer3 v0.4.0 (http://gmdd.shgmo.org/ and low-set, protruding, and posteriorly rotated ears. Autistic behaviors primer3). To eliminate unincorporated nucleotides and primers, the were also identified. The patient was referred to an otolaryngologist for PCR products were digested using shrimp alkaline phosphatase a hearing test and was diagnosed with hearing loss. Ophthalmologic (USB, Cleveland, OH, USA) and exonuclease I (USB). Sequences of the assessment determined choroidal coloboma in the left eye, adjacent to amplified products were identified by direct sequencing with the the optic disc. When she was 4.5 years old, she was evaluated by the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Denver Developmental Screening test with the results of severe Foster City, CA, USA). Subsequently, the amplified products were puri- mental-motor retardation. Her fine motor adaption, gross motor ability, fied using ethanol precipitation, and the purified products were personal social activities, and language were compatible with 20, 17, 10, sequenced using the 3130xl genetic analyzer (Applied Biosystems). and 10 months, respectively. The severe mental-motor retardation was Sequencing data were analyzed using ABI sequencing analysis software accompanied by autism spectrum symptoms, including stereotypy, re- v5.2 (Applied Biosystems) and Chromas pro v1.5 (Technelysium Pty stricted and repetitive behavior, and self-injury. She was given a high Ltd., Tewantin, QLD, Australia). Sequencing data of the patients with dosage of Risperdal for these symptoms, to no effect. She was also un- CHARGE syndrome were compared with reference sequence of CHD7 able to take solid foods due to tracheal compression. Clinical assessment gene (NM_017780.3) obtained from the National Center for Biotechnol- of the patient was compatible with CHARGE syndrome, including three ogy Information database (http://blast.ncbi.nlm.nih.gov). of the major diagnostic characteristic criteria of CHARGE syndrome including ocular coloboma, choanal atresia, and abnormal features of the outer ear. Four of the minor diagnostic characteristics of CHARGE syn- 2.3. Exon trapping analysis drome, consisting of cardiovascular malformation, growth deficiency, developmental delay, and distinctive facial appearance, were present An in vitro splicing assay was performed using the amplified exon 7 as well. of CHD7, and an approximately 300 bp intronic region flanking the 5′ Patient 2 was born at 40 weeks gestation, with a birth weight of and 3′ ends of exon 7, from the genomic DNA of patient 1 and a normal 3000 g. She was the second child born to a 24-year-old, gravida 2, control. The amplified products were digested using XhoI and BamHI para 2 mother. Her parents were both healthy and were third-degree and were inserted in pSPL3 splicing vectors containing exons A and B. cousins (Fig. 1C). In the postnatal period, she was not able to cry, was cy- The hybrid vectors were transfected into HeLa cells by using lipofecta- anotic and had dyspnea. The patient was hospitalized for twenty-five mine 2000 (Invitrogen, Carlsbad, CA, USA), and the cells were cultured days for these symptoms. After hospitalization, the condition of the pa- in Dulbecco's modified eagle's medium containing 10% fetal bovine tient had stabilized and there were no symptoms except for a flow of serum and 1% penicillin/streptomycin at 37 °C in 5% CO2.The transparent fluid from the nose and recurrent infections. During a rou- transfected cells were harvested after 24 h, and total RNA was extracted tine examination, atypical appearance and severe growth retardation using the RNeasy mini kit (Qiagen). Next, 1 μg of total RNA was reverse were identified when she was 7 months old. All growth parameters transcribed using the high capacity cDNA reverse transcription kit were under 3rd percentile. She had a square face, down-slanting palpe- (Applied Biosystems) to synthesize cDNA. This cDNA was amplified bral fissures, microphthalmia, a broad nasal root, hypoplasia of alae nasi, using the pSPL3 vector-specific primers SD6 (5′-TCTGAGTCACCTGGAC and cup-shaped and low-set ears. An ECHO revealed some cardiovascu- AACC-3′) and SA2 (5′-ATCTCAGTGGTATTTGTGAGC-3′). The size of the lar malformations, followed by diagnostic angiography which showed amplified products was determined by electrophoresis in 2% agarose ventricular septal defect, secundum ASD, pulmonary valvular stenosis, gel, and the products were purified using the DokDo-prep gel extraction and the continuation of the azygos vein. Balloon pulmonary kit (Elpis Biotech, Daejeon, Korea). Finally, the sequences of the PCR valvuloplasty was performed for the pulmonary valvular stenosis. The products were determined by direct sequencing. patient was referred to an otolaryngologist due to transparent runny 778 B. Lee et al. / Gene 576 (2016) 776–781

Fig. 1. Mutation analysis of CHD7 in the family of patient 1 and 2. (A) Pedigree of the family of patient 1: a three generation pedigree of 13 members is presented. The filled and open symbols indicate the affected and unaffected individuals, respectively. The double horizontal line indicates consanguineous marriage. The arrow indicates the proband. (B) Partial nucleotide sequences of intron 6 to exon 7 of CHD7 show the c.2443-2ANG mutation in the proband (III-3). This mutation is not detected in the parents of the proband (II-3 and II-4). The red arrow indicates the location of the nucleotide substitution. (C) Pedigree of the family of patient 2: a three generation pedigree of 8 members is presented. The filled and open symbols indicate affected and unaffected individuals, respectively. The double horizontal line indicates consanguineous marriage. The arrow indicates the proband. (D) Partial nucleotide sequences of intron 7 to exon 8 of CHD7 show the c.2504_2508delATCTT mutation in the proband (III-2). This mutation is not detected in the parents of the proband (II-1 and II-2). The red dotted lines indicate the location of the nucleotides deletion. nose, and she was diagnosed with choanal atresia and hearing loss dur- identified in patient 2 was a 5-bp deletion from nucleotides 2504 to ing this examination. She was referred to an ophthalmologist to evalu- 2508 in exon 8 (c.2504_2508delATCTT; Fig. 1D). This deletion changed ate the microphthalmia, and bilateral optic nerve coloboma and tyrosine to serine at amino acid position 835, and created a stop nystagmus were also determined. Clinical assessment of the patient codon after the 14th amino acid (p.Y835SfsX14); this mutation was pre- was compatible with CHARGE syndrome. This included three of the viously identified by Jongmans et al., (2006). The heterozygous frame- major diagnostic characteristics of CHARGE syndrome, including ocular shift mutation was not detected in the patient's parents, suggesting a coloboma, choanal atresia, and abnormal features of the outer ear. She de novo mutation. also exhibited four of the minor diagnostic characteristics of CHARGE syndrome, including cardiovascular malformation, growth deficiency, 3.3. In vitro exon trapping analysis developmental delay, and distinctive facial appearance. To determine the pathogenic effect of this splice-site mutation 3.2. Mutation analysis (c.2443-2ANG), we investigated the RNA splicing pattern of the mutant gene in transfected HeLa cells by performing a splicing assay with the All of the coding regions and the exon-intron boundaries of CHD7 pSPL3-CHD7 minigene. Exon 7 (56 bp) from both wild-type and mutant from the two CHARGE syndrome patients were sequenced. This identi- CHD7, along with the flanking intron sequences, was inserted between fied two mutations: a splice-site mutation in patient 1 and a frameshift exons A and B of the pSPL3 splicing vector (Fig. 2A). The resultant tran- mutation in patient 2. The splice-site mutation identified in patient 1 scripts were analyzed by reverse transcription-PCR (RT-PCR), and the was a single nucleotide change from adenine to guanine at the splice ac- amplified products were separated by agarose gel electrophoresis ceptor site position −2 of intron 6 (c.2443-2ANG; Fig. 1B).Thisisa (Fig. 2B). Analysis of the PCR products showed that the cDNA of the novel mutation which is not registered in the dbSNP (build 142) data- minigene containing the wild-type exon 7 produced a normal-sized base (http://www.ncbi.nlm.nih.gov), the human genome mutation (319 bp) band that included exons A and B of the pSPL3 vector and database (http://www.hgmd.cf.ac.uk/ac/index.php), the 1000 Genomes exon 7 of CHD7. The cDNA of the minigene containing the mutant Project database (http://www.1000genomes.org/), the Exome Variant exon 7 produced two bands of different sizes: one type (a) band Project (http://evs.gs.washington.edu/EVS/), the Exome Aggregation whose size was similar to that of the wild-type cDNA band, and one Consortium (http://exac.broadinstitute.org/) and in-house exome type (b) band (containing exons A and B of the pSPL3 vector, but not database and was not detected in the parents of patient 1 and in the exon 7 of CHD7) with a size of approximately 263 bp (Fig. 2C). To 48 unrelated normal control individuals. The frameshift mutation confirm these results, we performed direct sequencing of the RT-PCR B. Lee et al. / Gene 576 (2016) 776–781 779

Fig. 2. Exon trapping analysis of exon 7 of CHD7 in transfected HeLa cells. (A) Construction of pSPL3-CHD7 minigenes to identify the splicing of exon 7 and ﬂanking intronic sequences in the wild-type gene and mutant gene containing the c.2443-2ANG mutation. (B) RT-PCR products of the hybrid minigene transcripts. Splicing of wild-type exon 7 (WT lane), mutant exon 7 (a and b of MT lane), and a pSPL3 mock vector (mock lane) produced different sized bands on agarose gel. (C) Construction of splicing processes. The arrow indicates the location of nucleotide substitution (c.2443-2ANG). The asterisk indicates the cryptic splice site in exon 7 of CHD7. SDc represents the cryptic splicing donor site of the pSPL3 vector. (D) Partial nucleotide sequence of CHD7 showing the spliced transcripts in wild-type (WT), mutant types a and b (MT a and MT b), and pSPL3 mock vectors (pSPL3 mock). The red rectangle and dotted lines indicate the location of nucleotide skipping in exon 7. (E) Multiple alignments of the CHD7 partial region. Amino acid sequence of human CHD7 was aligned with that of other species. The asterisk represents amino acid position 815 in human CHD7. products of a pSPL3 mock vector, a wild-type exon 7-containing 7-containing minigene had 316 base pairs, of which 92 and 171 base minigene, and a mutant exon 7-containing minigene (Fig. 2D). The re- pairs belonged to exons A and B of the pSPL3 vector, respectively, and sults showed that the sequence of type (a) cDNA of the mutant exon 53 base pairs belonged to exon 7, excluding the ﬁrst 3 base pairs 780 B. Lee et al. / Gene 576 (2016) 776–781

(Fig. 2D, MT a panel). The splicing acceptor site between exon A and facial appearance). We screened CHD7 for mutations in these patients, exon 7 was the cryptic splice site that was predicted using the splice and identified a novel splice-site mutation in patient 1 and a previously site prediction tools. The sequence of type (b) cDNA of the mutant reported frameshift mutation in patient 2. The c.2504_2508delATCTT exon 7-containing minigene had only 263 base pairs, as exon 7 was mutation identified in patient 2 has been reported six times in previous completely skipped (Fig. 2D, MT b panel). studies (Aramaki et al., 2006; Jongmans et al., 2006; Janssen et al., 2012; Husu et al., 2013), and a clinical summary of CHARGE syndrome pa- 4. Discussion tients with heterozygous c.2504_2508delATCTT mutation is presented in Table 1. Although the number of patients with this mutation is insuf- The CHD family of proteins is one of four major families of ATP- ficient to investigate a genotype-phenotype correlation, a comparison of dependent chromatin remodeling proteins (Martens and Winston, phenotypes in these patients revealed that coloboma, cardiovascular 2003; Corona and Tamkun, 2004; Denslow and Wade, 2007; Conaway malformation, growth retardation, external ear anomalies and hearing and Conaway, 2009). Several studies have reported the functions of loss were diagnosed in all patients. The protein encoded by CHD7 with CHD proteins, such as methylated histone binding, DNA binding, this mutation contained only the chromodomain p.Y835SfsX14, unlike transcriptional regulation, cell cycle regulation, apoptosis regulation, the normal CHD7 protein, which contains 2997 amino acids. This mu- chromatin remodeling, and embryonic stem cell pluripotency regula- tant protein cannot perform its normal functions, such as DNA mainte- tion (Stokes and Perry, 1995; Bouazoune et al., 2002; Surapureddi nance, repair, replication, transcription, and recombination, because of et al., 2002; Flanagan et al., 2005; Shur and Benayahu, 2005; Ishihara the absence of the SWI/SNF2 and helicase domains, which are required et al., 2006; Lutz et al., 2006; Stockdale et al., 2006; Surapureddi et al., for these functions. 2006; Bagchi et al., 2007; Denslow and Wade, 2007; Yuan et al., 2007; The c.2443-2ANG mutation detected in patient 1 is a splice acceptor Biswas et al., 2008; Thompson et al., 2008; Gaspar-Maia et al., 2009; site mutation that has not been previously reported. To elucidate the Nishiyama et al., 2009; Rodriguez-Paredes et al., 2009; Schnetz et al., pathogenic effect of this mutation on RNA splicing, we performed an 2009). All nine members of the CHD protein family are characterized in vitro splicing assay. The results of this assay verified that the by the presence of chromodomains (chromatin organization modifiers) c.2443-2ANG mutation disturbed the normal splicing of exon 7, and SNF2-like ATPase/helicase domains. The CHD7 protein contains two resulting in exon skipping or a splicing error that removed part of chromodomains, one SWI/SNF2 domain, one helicase domain, and two exon 7. Open reading frame analysis showed that the mRNA of CHD7 BRK domains. The chromodomains recognize DNA targets and histone containing a frameshift mutation (p.K815LfsX17) and an in-frame dele- tails with methylated lysine residues, and regulate the chromatin struc- tion (p.K815del) encoded a truncated protein. In fact, the CHD7 protein ture (Brehm et al., 2004). The SWI/SNF2 domain is an ATP-dependent encoded by the gene containing the p.K815LfsX17 mutation contained chromatin-remodeling complex that is involved in DNA transcription, only the chromodomain. The in-frame p.K815del is located in a region repair, replication, and recombination (Dirscherl and Krebs, 2004). that is highly conserved among mammals (Fig. 2E), and is predicted as The helicase domain maintains DNA strand separation during replica- one of the causes of CHARGE syndrome. In the present study, this mutation, repair, recombination, and transcription. The function of the BRK tion occurred de novo and was not detected in the 48 unrelated normal domains is unknown at present. control individuals. These results suggested that this splice-site muta- Previous studies have reported that mutations in CHD7 are the major tion had a pathogenic effect on the normal function of CHD7. cause of CHARGE syndrome (Vissers et al., 2004). CHD7 localizes in Recently, Vatta et al. reported a patient with CHARGE syndrome several tissues that are affected by CHARGE syndrome, including the whose genetic analysis showed exon 7 deletion in CHD7 (Vatta et al., developing ear, eye, and olfactory system (Bosman et al., 2005; Lalani 2013). Similarly, our patient had two different splicing alternatives, one et al., 2006; Sanlaville et al., 2006; Adams et al., 2007; Hurd et al., of which was the skip of exon 7 in CHD7. To investigate any clinical resem- 2007; Layman et al., 2009). Zentner et al. reported that the most com- blance, we have compared these two patients in Table 1.Thepatientwith mon clinical features of patients with CHD7 mutations were temporal a deletion of exon 7 had a temporal bone abnormality; however, our pa- bone anomalies, external ear malformations, and hearing loss, and tient had severe mental-motor retardation and a total occlusion of the that approximately three-fourths of these patients had coloboma, ICA, which is a very rare symptom of CHARGE syndrome (Martin et al., heart malformations, and/or delayed growth and development 2006; Chang et al., 2010). Additionally, there was no genotype– (Zentner et al., 2010). Thus, the major criteria of patients with CHD7 phenotype correlation presented in the literature. Although these two pa- mutations are similar to those with CHARGE syndrome. In this study, tients' mutations caused similar effects on the mRNA level, there may be we reported the cases of two Turkish patients with typical features several modulating factors that regulate these processes and result in dif- of CHARGE syndrome, including 3 major clinical characteristics ferent clinical severity and different positive criteria. (i.e., ocular coloboma, choanal atresia, and abnormal features of the In summary, we identified a novel splice-site mutation, c.2443-2ANG, outer ear) and 4 minor clinical characteristics (i.e., cardiovascular and a previously reported frameshift mutation, c.2504_2508delATCTT, malformation, growth deficiency, developmental delay, and distinctive in two Turkish patients with CHARGE syndrome. CHD7 containing

Table 1 Comparison of phenotypic details of CHARGE syndrome patients with similar mutations.

Vatta et al. Presented case 1 Janssen et al. Aramaki et al. Husu et al. Presented case 2

Exon 7 c.2504_2508 c.2504_2508 c.2504_2508 c.2504_2508 c.2504_2508 c.2504_2508 c.2443-2ANG Mutation deletion delATCTT delATCTT delATCTT delATCTT delATCTT delATCTT

Coloboma + + NR + NR + + + 6/6 Heart defect + + + + + + + + 8/8 Chonal atresia + + + NR NR + − + 5/6 Retarded growth NR + NR NR NR + + + 4/4 Genital abnormalities −− −NR NR + + − 2/6 Ear anomalies + + + + NR + + + 7/7 Semicircular Hypoplasia + − +NRNRNRNR − 2/4 Hearing loss + + + NR NR + + + 6/6 Cleft lip and/or palate −− NR NR + −− −1/6

Abbreviations: NR, not reported. B. Lee et al. / Gene 576 (2016) 776–781 781 these mutations encodes a truncated protein that contains only the Janssen, N., Bergman, J.E., Swertz, M.A., Tranebjaerg, L., Lodahl, M., Schoots, J., Hofstra, R.M., van Ravenswaaij-Arts, C.M., Hoefsloot, L.H., 2012. Mutation update on the chromodomain. The clinical features of the two patients included in CHD7 gene involved in CHARGE syndrome. Hum. Mutat. 33, 1149–1160. the present study were highly similar, with negligible differences. The Jongmans, M.C., Admiraal, R.J., van der Donk, K.P., Vissers, L.E., Baas, A.F., Kapusta, L., van findings of our study reveal the function of a novel splice-site mutation Hagen, J.M., Donnai, D., de Ravel, T.J., Veltman, J.A., Geurts van Kessel, A., De Vries, B.B., Brunner, H.G., Hoefsloot, L.H., van Ravenswaaij, C.M., 2006. CHARGE syndrome: the detected in CHD7 and provides new insights on the genetic causes of phenotypic spectrum of mutations in the CHD7 gene. J. Med. Genet. 43, 306–314. CHARGE syndrome. However, future studies are needed to confirm Jongmans, M.C., Hoefsloot, L.H., van der Donk, K.P., Admiraal, R.J., Magee, A., van de Laar, I., and identify other causes of this syndrome. Hendriks, Y., Verheij, J.B., Walpole, I., Brunner, H.G., van Ravenswaaij, C.M., 2008. Familial CHARGE syndrome and the CHD7 gene: a recurrent missense mutation, intrafamilial recurrence and variability. Am J Med Genet A 146A, 43–50. Acknowledgments Lalani, S.R., Safiullah, A.M., Fernbach, S.D., Harutyunyan, K.G., Thaller, C., Peterson, L.E., McPherson, J.D., Gibbs, R.A., White, L.D., Hefner, M., Davenport, S.L., Graham, J.M., Bacino, C.A., Glass, N.L., Towbin, J.A., Craigen, W.J., Neish, S.R., Lin, A.E., Belmont, We are grateful to the families and individuals for their participation J.W., 2006. Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome in this study, and to the physicians and staff members of the Depart- and genotype-phenotype correlation. 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