- Email: [email protected]
Phenotypic spectrum of CHARGE syndrome with CHD7 mutations
PHENOTYPIC SPECTRUM OF CHARGE SYNDROME WITH CHD7 MUTATIONS MICHIHIKO ARAMAKI, MD, TORU UDAKA, MS, RIKA KOSAKI, MD, YOSHIO MAKITA, MD, NOBUHIKO OKAMOTO, MD, HIROSHI YOSHIHASHI, MD, HIROTAKA OKI, MD, KENJI NANAO, MD, NOBUKO MORIYAMA, MD, SHOZO OKU, MD, TOMONOBU HASEGAWA, MD, TAKAO TAKAHASHI, MD, YOSHIMITSU FUKUSHIMA, MD, HIROSHI KAWAME, MD, AND KENJIRO KOSAKI, MD
CHD7 gene mutations were identified in 17 (71%) of 24 children clinically diagnosed to have CHARGE syndrome (C, coloboma of the iris or retina; H, heart defects; A, atresia of the choanae; R, retardation of growth and/or development; G, genital anomalies; and E, ear abnormalities). Colobomata, hearing loss, laryngomalacia, and vestibulo-cochlear defect were prevalent. Molecular testing for CHD7 enables an accurate diagnosis and provides health anticipatory guidance and genetic counseling to families with CHARGE syndrome. (J Pediatr 2006;148:410-4)
wenty-five years ago, Hall1 documented in The Journal of Pediatrics that choanal atresia and ocular colomba can be associated with a specific pattern of malformations. Two years later, in the same journal, Pagon et al coined the term CHARGE to represent a constellation of non-randomly associated malformations: C, coloboma of the iris or retina; H, heart defects; A, atresia of the choanae; R, retardation of growth and/or development; G, genital anomalies; and E, ear abnormalities.2 Recently, Vissers et al3 identified the gene Chromodomain helicase DNAbinding protein-7 (CHD7) at chromosome 8q12.1 as a causative gene of CHARGE association. Now that the underlying cause is known in most cases, the entity is referred to as CHARGE syndrome rather than CHARGE association. According to their study, From the Department of Pediatrics, Keio 12 of 19 (63%) of their patients had heterozygous mutations in CHD7. Here, we further University School of Medicine, Tokyo, Japan; the Department of Clinical Genetics delineate the phenotypic spectrum of CHARGE syndrome patients with mutations in and Molecular Medicine, National ChilCHD7. dren’s Medical Center, Tokyo, Japan; the
T
METHODS The subjects were recruited from a group of patients with CHARGE syndrome who were regularly followed at the authors’ institutions at the time of study (August 2004 to March 2005). A diagnosis of CHARGE syndrome was made according to the criteria defined by Blake et al.4 Blake’s major criteria are coloboma or microphthalmia, choanal atresia or stenosis, typical ear anomalies, and cranial nerve dysfunction. Choanal atresia was diagnosed by CT scan. The minor criteria are genital hypoplasia, delayed development, cardiovascular malformations, growth deficiency, orofacial cleft, tracheoesophageal fistula, and distinctive face characteristics. Twenty-three patients who fulfilled Blake’s criteria (4 major or 3 major plus 3 or more minor criteria) and 1 patient who fulfilled 2 major and 6 minor criteria4 were included in the study: All the patients were unrelated and simplex case (ie, a single occurrence in a family). Each patient and family members were enrolled in the study after receiving their written informed consent, according to a protocol approved by an institutional review board. The entire CHD7 coding region (exons 2-38) was screened for mutations. The primer pairs, which were designed to amplify exons, exon-intron boundaries, and short flanking intronic sequences, are available on request. Polymerase chain reaction (PCR) amplicons from genomic DNA were scanned for mutations using DNA high-performance liquid chromatography (DHPLC, Transgenomic, Omaha, Nebraska), as described previously.5 PCR products corresponding to all variant elution profiles of the DHPLC were purified by using a desalting column and were sequenced bidirectionally by using the DHPLC
410
DNA high-performance liquid chromatography
PCR
Polymerase chain reaction
Department of Pediatrics, Asahikawa Medical College, Asahikawa, Japan; the Department of Planning and Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan; the Departments of Neonatology and Cardiology, Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo, Japan; the Department of Pediatrics, Yamato Municipal Hospital, Kanagawa, Japan; the Department of Pediatrics, Ibaraki Children’s, Ibaraki, Japan; the Department of Pediatrics, Kagoshima City Hospital, Kagoshima, Japan; and the Department of Medical Genetics, Shinshu University School of Medicine and Division of Clinical Genetics, Nagano Children’s Hospital, Nagano, Japan. Contract grant sponsors were The Ministry of Health, Labour, and Welfare of Japan. T.U. was supported by a research grant from Transgenomic, Inc, Omaha, Nebraska, whose machine was used to analyze the patients’ genomic DNA in the study. Submitted for publication Apr 5, 2005; last revision received Oct 2, 2005; accepted Oct 21, 2005. Reprint requests: Dr Kenjiro Kosaki, Division of Medical Genetics, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160 – 8582, Japan. 0022-3476/$ - see front matter Copyright © 2006 Elsevier Inc. All rights reserved. 10.1016/j.jpeds.2005.10.044
dideoxy sequencing method (BigDye Dideoxy sequencing kit; Applied BioSystems) and an automated sequencer (ABI3100; Applied Biosystems). When no mutations were detected, we further assessed the exon copy number using a recently published technique based on semiquantitative multiple PCR/ liquid chromatography.6
RESULTS We identified heterozygous CHD7 mutations in 17 (71%) of the 24 patients enrolled in the study: 7 frameshift mutations, 6 nonsense mutations, 3 splice-site mutations, and 1 intragenic deletion from exon 8 to 12 (Table). The clinical characteristics of the patients, including the presence or absence of the major and minor characteristics described by Blake et al,4 are listed in the Table and shown in the Figure. Only 3 patients had 4 of the major characteristics of CHARGE syndrome: 13 patients had 3 major and at least 3 minor characteristics and 1 patient had 2 major and 6 minor characteristics.
Coloboma The ocular colobomas occurred as isolated iris defects or in various combinations of iris, retina, and disc defects. Altogether, 15 of the 17 cases had coloboma of the eyes. However, only 2 patients had iridal coloboma and 5 patients had only disc coloboma, without iridal or retinal coloboma.
Genitalia Genital hypoplasia was observed in all of the 8 boys and 5 of the 9 girls. An endocrinologic and radiologic evaluation of the hypothalamic-pituitary-gonadal axis of the 3 boys revealed hypogonadotropic hypogonadism. Ears External ear abnormalities were present in all 17 patients. A typical “CHARGE ear” includes a web over the auricle or a snip-off defect (Figure). Severe hearing loss was a universal feature among all the patients. Hence, hearing should be promptly evaluated when a diagnosis of CHARGE syndrome is suspected in infancy. In 6 patients whose ears were studied using radiography, all the patients had a deficiency of one or more semicircular canals in combination with cochlear hypoplasia.
Others A square face with asymmetry, unusually shaped ears, unilateral facial palsy, square face, and malar flattening7 were observed (Figure). Oral clefts were present in 8 patients. Laryngomalacia was present in 14 patients, and tracheoesophageal fistula was present in 3 patients. The documentation of these 3 patients confirms that CHD7 mutations can lead to tracheo-esophageal abnormalities.8
DISCUSSION Heart Congenital heart disease was present in 13 cases. Defects of the aortic arch were relatively common: 3 patients had aortic valve stenosis, 2 had coarctation, 1 had an interrupted aortic arch, and 1 had a transposition of the great arteries. Eight of the 13 patients who had intracardiac defects also had patent ductus arteriosus. Choanal Atresia Choanal atresia/stenosis, though originally considered to be an important feature of CHARGE syndrome,1 was not common. Only 3 patients had choanal atresia, and 2 had choanal stenosis. The relatively low incidence of choanal atresia/stenosis (30%) in the present study may be due to the high frequency of cleft palate (53%), since choanal atresia/ stenosis is uncommon in the presence of cleft palate, particularly cleft palate associated with bilateral cleft lip (6 of the 9 patients did not have choanal atresia or stenosis). Retardation of Growth and Development Most had postnatal growth retardation, and a short stature (height ⬍ ⫺2 SD) was observed in all but 3 patients at the time of examination. All the patients had developmental delays. However, it is difficult to predict their ultimate development at present, since 13 of the 17 patients were less than 6.5 years of age and they had concurrent visual and hearing impairments. Phenotypic Spectrum Of CHARGE Syndrome With CHD7 Mutations
We have verified the validity of the existing diagnostic criteria for CHARGE syndrome. In view of the results of the mutation analysis, we wish to address two issues regarding the clinical diagnostic workup when a diagnosis of CHARGE syndrome is suspected. First, a detailed examination of the retina and the optic disc must be performed, even in the absence of iridal coloboma. Further, the absence of coloboma does not rule out the possible presence of a CHD7 mutation, since two of the patients in this study did not have coloboma of any type. Second, imaging studies of the vestibulo-cochlear system should be performed. When we combined our data with those reported by Vissers et al,3 vestibulo-cochlear defects were present in 14 of the 14 cases in which appropriate studies were performed. These data support the notion that vestibulo-cochlear defects should be included as a major criteria of CHARGE syndrome, as proposed by Amiel 9 and Verloes.10 All the mutations identified in the present study belonged to the same category: heterozygous mutations leading to truncated proteins. However, the specific combinations of malformations differed among the patients. Hence, no apparent genotype-phenotype correlations were noted among the 17 patients. Two clinical questions must next be answered. (a) What additional findings might be part of the phenotypic spectrum of CHD7 mutation? We observed several atypical features such as hypothyroidism (cases 1, 8, and 12 in the Table), tibial hypoplasia (case 5), thyroid tumor (case 14), and duodenal atresia (case 15). Molecular analyses of patients with 411
412
Aramaki et al
The Journal of Pediatrics • March 2006
⫺4.4 ⫺4.9 ⫺4.0 1295 delA
⫺5.0 ⫺5.9 ⫺6.0 4480C⬎T
D EX
2.66 46.4 NA 5 mo
2.45 48 33.5 5 mo
R1494X A EX
2/F 37
1/F 37
4/F 38
5/M 40
6/M 41
TEF
NA
LM
8/M 41
9/M 39
CP LM
CP LM
LM
CP LM
2.38 49.5 34.5 5y 3 mo ⫺3.3 ⫺2.3 ⫺2.0 IVS26 ⫹1G ⬎T
3.45 50 34.0 5y 5 mo ⫺4.7 ⫺2.3 ⫺0.9 6991 A⬎T
12/M 37
3180 48 34 6y 5 mo ⫺1.5 ⫺0.7 ⫺1.0 6998 delC
13/F 42
2.07 46.5 31 13 y
15/F 34
LM
-
⬎90/ ⬎90 LH
R,D EX
⫺4.3 ⫺7.1 ⫺2.4 ⫺3.6 ⫺3.0 ⫺5.0 6228 deletion del G of exon 8-12
2.92 48.2 35.0 12 y
14/F 40
K2331X fs2334X R,D R,D I,R,D R EX/ EX/CO EX/CO EX CO 90/70 ⬎90/ 120/100 50/50 90/90 105/105 100/60 ⬎100/ ⬎100 ⬎100 MP/CO MP/CO MP/ MP/ MP LH CO CO PDA/ TGA/ VSD PDA/ PDA AS/ VSD ASD/ bicuspid ASD VSD aortic valve CLP CLP CLP CLP CLP LM LM LM LM/ LM LM LM TEF R EX
⫺5.5 ⫺3.4 ⫺3.0 IVS25 ⫺7G ⬎A
2.71 48 31 5y
10/M 11/M 40 38
2.88 46 32.5 18 y
17/F 37
CLP LM/TEF
-
-
LH
LH VSD/ PDA
100/100
90/90
⫺2.9 ⫺2.5 ⫺1.5 ⫺0.7 ⫺3.0 ⫺2.0 7486550 7487 C⬎T insGC fs2503X Q184X R,D D EX/CO EX
2.81 50 32.0 17 y
16/F 39
Coloboma: iris (I), retina (R), disc (D), or microphthalmia (M); choanal atresia: atresia (A) or stenosis (S); ear abnormalities: external ear defects (EX), cochlear defects (CO); genital hypoplasia: micropenis (MP), cryptorchidism (CO), or labial hypoplasia (LH); orofacial cleft: cleft lip and palate (CLP) or cleft palate only (CP); tracheoesophageal defects: tracheoesophageal-fistula (TEF) or laryngomalacia (LM). Cardiovascular malformations: endocardial cushion defect; (ECD), aortic stenosis (AS), patent ductus arteriosus (PDA), ventricular septal defect (VSD), coarctation of aorta (CoA), atrial septal defect (ASD), transposition of great arteries (TGA).
Orofacial cleft Tracheoesophageal defects
ABR
7/F 39
3 3.66 2.35 3.37 2.48 3.06 2.75 51 50 41.3 50.6 47.5 48.5 48.5 36.5 34.5 32.5 36.0 34.8 31 30.5 1y 2y 2y 3y 3y 4y 4y 3 mo 4 mo 6 mo 8 mo 8 mo ⫺5.6 ⫺4.5 ⫺5.5 ⫺1.9 ⫺3.3 ⫺6.0 ⫺0.9 ⫺4.5 ⫺2.6 ⫺3.7 ⫺1.7 ⫺1.3 ⫺4.7 ⫺0.9 ⫺2.1 ⫺2.0 NA ⫺0.2 ⫺0.7 ⫺4.0 ⫺0.1 56763106 2500IVS18⫹1G⬎T 2839 4015 1683 5677 C⬎T 2504 C⬎T C⬎T delC insT delTCTTA fs462X R1036X fs848X R947X R1339X fs563X D I,R R D M D A S S A EX EX EX EX/CO EX EX/CO EX
3/M 40
⬎100/ 80/80 100/80 NA 70/90 ⬎100 Genital hypoplasia LH MP/ MP/CO MP/CO CO Cardiovascular ECD/AS/ VSD CoA PDA/AS/ PDA/ASD IAA/ malformations CoA/PDA/ Ebstein AP-Window/ hypo-LV anomaly PDA
Amino acid Coloboma Choanal atresia Ear abnormalities
Case No./sex Gestational age (wk) Birth parameters Wt (kg) Ht (cm) OFC (cm) Age at examination Height (SD) Weight (SD) OFC (SD) Mutation Nucleotide
Table. Clinical features of 17 patients with CHD7 mutation
Figure. Clinical features of the patients with CHD7 mutations. A, Patient 1; B, patient 5; C, patient 6; D, patient 9; E, patient 11; F, patient 15.
atypical features in addition to the classic features of CHARGE syndrome would help to answer this question. (b) What are the minimum findings indicating a diagnosis of CHARGE syndrome? Because we selected patients on the basis of the existing criteria, we were unable to determine whether patients who do not meet these criteria may have CHD7 mutations. Molecular analyses of patients with partial features of the existing criteria would help to answer this question. Possible reasons for the absence of detectable mutations among these 7 patients include the following explanations: Some of the 7 patients may have promoter mutations or intragenic rearrangements, both of which cannot be detected by a mutation screening strategy covering only the coding region of the gene. Indeed, a mutation detection rate of ⬃70% is comparable to that of other well-defined mendelian disorders. In addition, the sensitivity of DHPLC approaches 100% but is not 100%. Hence, we could have missed several true-positive cases. Alternatively, mutation-negative cases may constitute a clinically distinctive Phenotypic Spectrum Of CHARGE Syndrome With CHD7 Mutations
group from those with mutations. In particular, all of the 4 mutation-negative patients whose temporal bones were evaluated by CT had a normal vestibulo-cochlear system, whereas all of the 6 mutation-positive patients whose temporal bones were evaluated by CT had a hypoplastic vestibulo-cochlear system. In the present study, we confirmed that mutations in CHD7 are present in the majority of patients with CHARGE syndrome. The identification of CHD7 as the causative gene of CHARGE syndrome is of importance to pediatric practice. Molecular genetic testing of CHD7 will be helpful to pediatricians caring for a child who looks like he or she may have CHARGE syndrome because the discovery of such a mutation in a patient would confirm the diagnosis. Once an accurate diagnosis is made, appropriate health anticipatory guidance can be provided to patients and families to deal with prevalent problems in auditory, respiratory, and neurodevelopmental aspects. Confirmation of the CHD7 mutation also enables pediatricians to provide accurate genetic counseling to the families. 413
The authors are greatly indebted to the patients and families for generously donating samples and clinical information. We thank Ms K. Shinohara for secretarial assistance.
REFERENCES 1. Hall B. Choanal atresia and associated multiple anomalies. J Pediatr 1979;95:395-8. 2. Pagon RA, Graham JMJ, Zonana J, Yong SL. Coloboma, congenital heart disease, and choanal atresia with multiple anomalies: CHARGE association. J Pediatr 1981;99:223-7. 3. Vissers LE, van Ravenswaaij CM, Admiraal R, Hurst JA, de Vries BB, Janssen IM, et al. Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat Genet 2004;36:955-7. 4. Blake KD, Davenport SL, Hall BD, Hefner MA, Pagon RA, Williams MS, et al. CHARGE association: an update and review for the primary pediatrician. Clin Pediatr (Phila) 1998;37:159-73.
414
Aramaki et al
5. Kosaki K, Suzuki T, Muroya K, Hasegawa T, Sato S, Matsuo N, et al. PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) mutations in seven Japanese patients with Noonan syndrome. J Clin Endocrinol Metab 2002;87:3529-33. 6. Dehainault C, Lauge A, Caux-Moncoutier V, Pages-Berhouet S, Doz F, Desjardins L, et al. Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene. Nucleic Acids Res 2004;32:e139. 7. Oley CA, Baraitser M, Grant DB. A reappraisal of the CHARGE association. J Med Genet 1988;25:147-56. 8. Kutiyanawala M, Wyse RK, Brereton RJ, Spitz L, Kiely EM, Drake D, et al. CHARGE and esophageal atresia. J Pediatr Surg 1992;27:558-60. 9. Amiel J, Attiee-Bitach T, Marianowski R, Cormier-Daire V, Abadie V, Bonnet D, et al. Temporal bone anomaly proposed as a major criteria for diagnosis of CHARGE syndrome. Am J Med Genet 2001;99:124-7. 10. Verloes A. Updated diagnostic criteria for CHARGE syndrome: a proposal. Am J Med Genet A 2005;133:306-8.
The Journal of Pediatrics • March 2006
CHD7 gene mutations were identified in 17 (71%) of 24 children clinically diagnosed to have CHARGE syndrome (C, coloboma of the iris or retina; H, heart defects; A, atresia of the choanae; R, retardation of growth and/or development; G, genital anomalies; and E, ear abnormalities). Colobomata, hearing loss, laryngomalacia, and vestibulo-cochlear defect were prevalent. Molecular testing for CHD7 enables an accurate diagnosis and provides health anticipatory guidance and genetic counseling to families with CHARGE syndrome. (J Pediatr 2006;148:410-4)
wenty-five years ago, Hall1 documented in The Journal of Pediatrics that choanal atresia and ocular colomba can be associated with a specific pattern of malformations. Two years later, in the same journal, Pagon et al coined the term CHARGE to represent a constellation of non-randomly associated malformations: C, coloboma of the iris or retina; H, heart defects; A, atresia of the choanae; R, retardation of growth and/or development; G, genital anomalies; and E, ear abnormalities.2 Recently, Vissers et al3 identified the gene Chromodomain helicase DNAbinding protein-7 (CHD7) at chromosome 8q12.1 as a causative gene of CHARGE association. Now that the underlying cause is known in most cases, the entity is referred to as CHARGE syndrome rather than CHARGE association. According to their study, From the Department of Pediatrics, Keio 12 of 19 (63%) of their patients had heterozygous mutations in CHD7. Here, we further University School of Medicine, Tokyo, Japan; the Department of Clinical Genetics delineate the phenotypic spectrum of CHARGE syndrome patients with mutations in and Molecular Medicine, National ChilCHD7. dren’s Medical Center, Tokyo, Japan; the
T
METHODS The subjects were recruited from a group of patients with CHARGE syndrome who were regularly followed at the authors’ institutions at the time of study (August 2004 to March 2005). A diagnosis of CHARGE syndrome was made according to the criteria defined by Blake et al.4 Blake’s major criteria are coloboma or microphthalmia, choanal atresia or stenosis, typical ear anomalies, and cranial nerve dysfunction. Choanal atresia was diagnosed by CT scan. The minor criteria are genital hypoplasia, delayed development, cardiovascular malformations, growth deficiency, orofacial cleft, tracheoesophageal fistula, and distinctive face characteristics. Twenty-three patients who fulfilled Blake’s criteria (4 major or 3 major plus 3 or more minor criteria) and 1 patient who fulfilled 2 major and 6 minor criteria4 were included in the study: All the patients were unrelated and simplex case (ie, a single occurrence in a family). Each patient and family members were enrolled in the study after receiving their written informed consent, according to a protocol approved by an institutional review board. The entire CHD7 coding region (exons 2-38) was screened for mutations. The primer pairs, which were designed to amplify exons, exon-intron boundaries, and short flanking intronic sequences, are available on request. Polymerase chain reaction (PCR) amplicons from genomic DNA were scanned for mutations using DNA high-performance liquid chromatography (DHPLC, Transgenomic, Omaha, Nebraska), as described previously.5 PCR products corresponding to all variant elution profiles of the DHPLC were purified by using a desalting column and were sequenced bidirectionally by using the DHPLC
410
DNA high-performance liquid chromatography
PCR
Polymerase chain reaction
Department of Pediatrics, Asahikawa Medical College, Asahikawa, Japan; the Department of Planning and Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan; the Departments of Neonatology and Cardiology, Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo, Japan; the Department of Pediatrics, Yamato Municipal Hospital, Kanagawa, Japan; the Department of Pediatrics, Ibaraki Children’s, Ibaraki, Japan; the Department of Pediatrics, Kagoshima City Hospital, Kagoshima, Japan; and the Department of Medical Genetics, Shinshu University School of Medicine and Division of Clinical Genetics, Nagano Children’s Hospital, Nagano, Japan. Contract grant sponsors were The Ministry of Health, Labour, and Welfare of Japan. T.U. was supported by a research grant from Transgenomic, Inc, Omaha, Nebraska, whose machine was used to analyze the patients’ genomic DNA in the study. Submitted for publication Apr 5, 2005; last revision received Oct 2, 2005; accepted Oct 21, 2005. Reprint requests: Dr Kenjiro Kosaki, Division of Medical Genetics, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160 – 8582, Japan. 0022-3476/$ - see front matter Copyright © 2006 Elsevier Inc. All rights reserved. 10.1016/j.jpeds.2005.10.044
dideoxy sequencing method (BigDye Dideoxy sequencing kit; Applied BioSystems) and an automated sequencer (ABI3100; Applied Biosystems). When no mutations were detected, we further assessed the exon copy number using a recently published technique based on semiquantitative multiple PCR/ liquid chromatography.6
RESULTS We identified heterozygous CHD7 mutations in 17 (71%) of the 24 patients enrolled in the study: 7 frameshift mutations, 6 nonsense mutations, 3 splice-site mutations, and 1 intragenic deletion from exon 8 to 12 (Table). The clinical characteristics of the patients, including the presence or absence of the major and minor characteristics described by Blake et al,4 are listed in the Table and shown in the Figure. Only 3 patients had 4 of the major characteristics of CHARGE syndrome: 13 patients had 3 major and at least 3 minor characteristics and 1 patient had 2 major and 6 minor characteristics.
Coloboma The ocular colobomas occurred as isolated iris defects or in various combinations of iris, retina, and disc defects. Altogether, 15 of the 17 cases had coloboma of the eyes. However, only 2 patients had iridal coloboma and 5 patients had only disc coloboma, without iridal or retinal coloboma.
Genitalia Genital hypoplasia was observed in all of the 8 boys and 5 of the 9 girls. An endocrinologic and radiologic evaluation of the hypothalamic-pituitary-gonadal axis of the 3 boys revealed hypogonadotropic hypogonadism. Ears External ear abnormalities were present in all 17 patients. A typical “CHARGE ear” includes a web over the auricle or a snip-off defect (Figure). Severe hearing loss was a universal feature among all the patients. Hence, hearing should be promptly evaluated when a diagnosis of CHARGE syndrome is suspected in infancy. In 6 patients whose ears were studied using radiography, all the patients had a deficiency of one or more semicircular canals in combination with cochlear hypoplasia.
Others A square face with asymmetry, unusually shaped ears, unilateral facial palsy, square face, and malar flattening7 were observed (Figure). Oral clefts were present in 8 patients. Laryngomalacia was present in 14 patients, and tracheoesophageal fistula was present in 3 patients. The documentation of these 3 patients confirms that CHD7 mutations can lead to tracheo-esophageal abnormalities.8
DISCUSSION Heart Congenital heart disease was present in 13 cases. Defects of the aortic arch were relatively common: 3 patients had aortic valve stenosis, 2 had coarctation, 1 had an interrupted aortic arch, and 1 had a transposition of the great arteries. Eight of the 13 patients who had intracardiac defects also had patent ductus arteriosus. Choanal Atresia Choanal atresia/stenosis, though originally considered to be an important feature of CHARGE syndrome,1 was not common. Only 3 patients had choanal atresia, and 2 had choanal stenosis. The relatively low incidence of choanal atresia/stenosis (30%) in the present study may be due to the high frequency of cleft palate (53%), since choanal atresia/ stenosis is uncommon in the presence of cleft palate, particularly cleft palate associated with bilateral cleft lip (6 of the 9 patients did not have choanal atresia or stenosis). Retardation of Growth and Development Most had postnatal growth retardation, and a short stature (height ⬍ ⫺2 SD) was observed in all but 3 patients at the time of examination. All the patients had developmental delays. However, it is difficult to predict their ultimate development at present, since 13 of the 17 patients were less than 6.5 years of age and they had concurrent visual and hearing impairments. Phenotypic Spectrum Of CHARGE Syndrome With CHD7 Mutations
We have verified the validity of the existing diagnostic criteria for CHARGE syndrome. In view of the results of the mutation analysis, we wish to address two issues regarding the clinical diagnostic workup when a diagnosis of CHARGE syndrome is suspected. First, a detailed examination of the retina and the optic disc must be performed, even in the absence of iridal coloboma. Further, the absence of coloboma does not rule out the possible presence of a CHD7 mutation, since two of the patients in this study did not have coloboma of any type. Second, imaging studies of the vestibulo-cochlear system should be performed. When we combined our data with those reported by Vissers et al,3 vestibulo-cochlear defects were present in 14 of the 14 cases in which appropriate studies were performed. These data support the notion that vestibulo-cochlear defects should be included as a major criteria of CHARGE syndrome, as proposed by Amiel 9 and Verloes.10 All the mutations identified in the present study belonged to the same category: heterozygous mutations leading to truncated proteins. However, the specific combinations of malformations differed among the patients. Hence, no apparent genotype-phenotype correlations were noted among the 17 patients. Two clinical questions must next be answered. (a) What additional findings might be part of the phenotypic spectrum of CHD7 mutation? We observed several atypical features such as hypothyroidism (cases 1, 8, and 12 in the Table), tibial hypoplasia (case 5), thyroid tumor (case 14), and duodenal atresia (case 15). Molecular analyses of patients with 411
412
Aramaki et al
The Journal of Pediatrics • March 2006
⫺4.4 ⫺4.9 ⫺4.0 1295 delA
⫺5.0 ⫺5.9 ⫺6.0 4480C⬎T
D EX
2.66 46.4 NA 5 mo
2.45 48 33.5 5 mo
R1494X A EX
2/F 37
1/F 37
4/F 38
5/M 40
6/M 41
TEF
NA
LM
8/M 41
9/M 39
CP LM
CP LM
LM
CP LM
2.38 49.5 34.5 5y 3 mo ⫺3.3 ⫺2.3 ⫺2.0 IVS26 ⫹1G ⬎T
3.45 50 34.0 5y 5 mo ⫺4.7 ⫺2.3 ⫺0.9 6991 A⬎T
12/M 37
3180 48 34 6y 5 mo ⫺1.5 ⫺0.7 ⫺1.0 6998 delC
13/F 42
2.07 46.5 31 13 y
15/F 34
LM
-
⬎90/ ⬎90 LH
R,D EX
⫺4.3 ⫺7.1 ⫺2.4 ⫺3.6 ⫺3.0 ⫺5.0 6228 deletion del G of exon 8-12
2.92 48.2 35.0 12 y
14/F 40
K2331X fs2334X R,D R,D I,R,D R EX/ EX/CO EX/CO EX CO 90/70 ⬎90/ 120/100 50/50 90/90 105/105 100/60 ⬎100/ ⬎100 ⬎100 MP/CO MP/CO MP/ MP/ MP LH CO CO PDA/ TGA/ VSD PDA/ PDA AS/ VSD ASD/ bicuspid ASD VSD aortic valve CLP CLP CLP CLP CLP LM LM LM LM/ LM LM LM TEF R EX
⫺5.5 ⫺3.4 ⫺3.0 IVS25 ⫺7G ⬎A
2.71 48 31 5y
10/M 11/M 40 38
2.88 46 32.5 18 y
17/F 37
CLP LM/TEF
-
-
LH
LH VSD/ PDA
100/100
90/90
⫺2.9 ⫺2.5 ⫺1.5 ⫺0.7 ⫺3.0 ⫺2.0 7486550 7487 C⬎T insGC fs2503X Q184X R,D D EX/CO EX
2.81 50 32.0 17 y
16/F 39
Coloboma: iris (I), retina (R), disc (D), or microphthalmia (M); choanal atresia: atresia (A) or stenosis (S); ear abnormalities: external ear defects (EX), cochlear defects (CO); genital hypoplasia: micropenis (MP), cryptorchidism (CO), or labial hypoplasia (LH); orofacial cleft: cleft lip and palate (CLP) or cleft palate only (CP); tracheoesophageal defects: tracheoesophageal-fistula (TEF) or laryngomalacia (LM). Cardiovascular malformations: endocardial cushion defect; (ECD), aortic stenosis (AS), patent ductus arteriosus (PDA), ventricular septal defect (VSD), coarctation of aorta (CoA), atrial septal defect (ASD), transposition of great arteries (TGA).
Orofacial cleft Tracheoesophageal defects
ABR
7/F 39
3 3.66 2.35 3.37 2.48 3.06 2.75 51 50 41.3 50.6 47.5 48.5 48.5 36.5 34.5 32.5 36.0 34.8 31 30.5 1y 2y 2y 3y 3y 4y 4y 3 mo 4 mo 6 mo 8 mo 8 mo ⫺5.6 ⫺4.5 ⫺5.5 ⫺1.9 ⫺3.3 ⫺6.0 ⫺0.9 ⫺4.5 ⫺2.6 ⫺3.7 ⫺1.7 ⫺1.3 ⫺4.7 ⫺0.9 ⫺2.1 ⫺2.0 NA ⫺0.2 ⫺0.7 ⫺4.0 ⫺0.1 56763106 2500IVS18⫹1G⬎T 2839 4015 1683 5677 C⬎T 2504 C⬎T C⬎T delC insT delTCTTA fs462X R1036X fs848X R947X R1339X fs563X D I,R R D M D A S S A EX EX EX EX/CO EX EX/CO EX
3/M 40
⬎100/ 80/80 100/80 NA 70/90 ⬎100 Genital hypoplasia LH MP/ MP/CO MP/CO CO Cardiovascular ECD/AS/ VSD CoA PDA/AS/ PDA/ASD IAA/ malformations CoA/PDA/ Ebstein AP-Window/ hypo-LV anomaly PDA
Amino acid Coloboma Choanal atresia Ear abnormalities
Case No./sex Gestational age (wk) Birth parameters Wt (kg) Ht (cm) OFC (cm) Age at examination Height (SD) Weight (SD) OFC (SD) Mutation Nucleotide
Table. Clinical features of 17 patients with CHD7 mutation
Figure. Clinical features of the patients with CHD7 mutations. A, Patient 1; B, patient 5; C, patient 6; D, patient 9; E, patient 11; F, patient 15.
atypical features in addition to the classic features of CHARGE syndrome would help to answer this question. (b) What are the minimum findings indicating a diagnosis of CHARGE syndrome? Because we selected patients on the basis of the existing criteria, we were unable to determine whether patients who do not meet these criteria may have CHD7 mutations. Molecular analyses of patients with partial features of the existing criteria would help to answer this question. Possible reasons for the absence of detectable mutations among these 7 patients include the following explanations: Some of the 7 patients may have promoter mutations or intragenic rearrangements, both of which cannot be detected by a mutation screening strategy covering only the coding region of the gene. Indeed, a mutation detection rate of ⬃70% is comparable to that of other well-defined mendelian disorders. In addition, the sensitivity of DHPLC approaches 100% but is not 100%. Hence, we could have missed several true-positive cases. Alternatively, mutation-negative cases may constitute a clinically distinctive Phenotypic Spectrum Of CHARGE Syndrome With CHD7 Mutations
group from those with mutations. In particular, all of the 4 mutation-negative patients whose temporal bones were evaluated by CT had a normal vestibulo-cochlear system, whereas all of the 6 mutation-positive patients whose temporal bones were evaluated by CT had a hypoplastic vestibulo-cochlear system. In the present study, we confirmed that mutations in CHD7 are present in the majority of patients with CHARGE syndrome. The identification of CHD7 as the causative gene of CHARGE syndrome is of importance to pediatric practice. Molecular genetic testing of CHD7 will be helpful to pediatricians caring for a child who looks like he or she may have CHARGE syndrome because the discovery of such a mutation in a patient would confirm the diagnosis. Once an accurate diagnosis is made, appropriate health anticipatory guidance can be provided to patients and families to deal with prevalent problems in auditory, respiratory, and neurodevelopmental aspects. Confirmation of the CHD7 mutation also enables pediatricians to provide accurate genetic counseling to the families. 413
The authors are greatly indebted to the patients and families for generously donating samples and clinical information. We thank Ms K. Shinohara for secretarial assistance.
REFERENCES 1. Hall B. Choanal atresia and associated multiple anomalies. J Pediatr 1979;95:395-8. 2. Pagon RA, Graham JMJ, Zonana J, Yong SL. Coloboma, congenital heart disease, and choanal atresia with multiple anomalies: CHARGE association. J Pediatr 1981;99:223-7. 3. Vissers LE, van Ravenswaaij CM, Admiraal R, Hurst JA, de Vries BB, Janssen IM, et al. Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat Genet 2004;36:955-7. 4. Blake KD, Davenport SL, Hall BD, Hefner MA, Pagon RA, Williams MS, et al. CHARGE association: an update and review for the primary pediatrician. Clin Pediatr (Phila) 1998;37:159-73.
414
Aramaki et al
5. Kosaki K, Suzuki T, Muroya K, Hasegawa T, Sato S, Matsuo N, et al. PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) mutations in seven Japanese patients with Noonan syndrome. J Clin Endocrinol Metab 2002;87:3529-33. 6. Dehainault C, Lauge A, Caux-Moncoutier V, Pages-Berhouet S, Doz F, Desjardins L, et al. Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene. Nucleic Acids Res 2004;32:e139. 7. Oley CA, Baraitser M, Grant DB. A reappraisal of the CHARGE association. J Med Genet 1988;25:147-56. 8. Kutiyanawala M, Wyse RK, Brereton RJ, Spitz L, Kiely EM, Drake D, et al. CHARGE and esophageal atresia. J Pediatr Surg 1992;27:558-60. 9. Amiel J, Attiee-Bitach T, Marianowski R, Cormier-Daire V, Abadie V, Bonnet D, et al. Temporal bone anomaly proposed as a major criteria for diagnosis of CHARGE syndrome. Am J Med Genet 2001;99:124-7. 10. Verloes A. Updated diagnostic criteria for CHARGE syndrome: a proposal. Am J Med Genet A 2005;133:306-8.
The Journal of Pediatrics • March 2006