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Identification of three novel mutations in the CHD7 gene in patients with clinical signs of typical or atypical CHARGE syndrome
International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Case report
Identification of three novel mutations in the CHD7 gene in patients with clinical signs of typical or atypical CHARGE syndrome Angela Michelucci a, Paolo Ghirri c,*, Paola Iacopetti b, Maria Elena Conidi a, Antonella Fogli a, Fulvia Baldinotti a, Sara Lunardi c, Francesca Forli d, Francesca Moscuzza c, Stefano Berrettini d, Antonio Boldrini c, Paolo Simi a, Silvia Pellegrini b a
Mother and Child Department, Cytogenetics and Molecular Genetics Unit, University Hospital of Pisa, Pisa, Italy Department of Experimental Pathology, Medical Biotechnologies, Infectious Diseases and Epidemiology, University of Pisa, Pisa, Italy c Mother and Child Department, Neonatology Unit and Section of Neonatal Endocrinology and Dysmorphology, University Hospital of Pisa, Pisa, Italy d Division of ENT, Department of Neuroscience, University of Pisa, Pisa, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 June 2010 Received in revised form 4 September 2010 Accepted 6 September 2010 Available online 12 October 2010
CHARGE syndrome is an autosomal dominant disorder characterized by features represented in its acronym: Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness. We report two patients with a diagnosis of typical CHARGE syndrome and one with atypical clinical diagnosis. All the three patients had uni- or bilateral choanal atresia and sensorineural hearing loss. The patients were screened for CHD7 gene mutations. Three novel occurring de novo heterozygous mutations were identified: a mutation in the donor splice site of intron 24, a missense mutation in exon 2 and a deletion in exon 11. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: CHARGE syndrome CHD7 gene Novel mutations Splicing defect
1. Introduction CHARGE (Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness) syndrome (OMIM 214800) is a rare disorder with an incidence of about one every 8500–10000 births [1,2]. Diagnostic criteria for CHARGE syndrome are based on a nonrandom association of the following congenital anomalies: coloboma of the eye, heart defects, choanal atresia, retarded growth and development, genital hypoplasia, ear anomalies and/or deafness [1,3]. Severe hearing loss and semicircular canals hypoplasia were found in almost all patients evaluated [4–7] and Sanlaville et al. [8] proposed semicircular canals hypoplasia as a major diagnostic criteria of the syndrome. The CHARGE syndrome gene, CHD7, was identified in 2004 [9] and belongs to the gene family coding for the Chromodomain Helicase DNA binding proteins. During early human development, CHD7 is expressed in the undifferentiated neuroepithelium and in mesenchyme of neural crest origin [8]. CHD7 is thought to play a role in regulating the expression of important
* Corresponding author at: Mother and Child Department, Neonatology Unit and Section of Neonatal Endocrinology and Dysmorphology, University Hospital of Pisa, Via Roma 55, 56126 Pisa, Italy. Tel.: +39 050993452; fax: +39 050992529. E-mail address: [email protected] (P. Ghirri). 0165-5876/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2010.09.006
developmental genes in mesenchymal cells derived from the cephalic neural crest, by chromatin remodeling [8]. Here we describe three patients affected by CHARGE syndrome carrying three novel de novo mutations in the CHD7 gene. 2. Case reports Patient 1 was a female infant, one of dizygotic twins born at 34 weeks of gestation by caesarian section to non-consanguineous parents. After birth the patient developed cyanosis and bradycardia, requiring resuscitation. Apgar scores were 4 at 1 min and 6 at 5 min. At birth weight was 1925 g (25th centile), length 43 cm (25th centile). She met three out of four major and five out of seven minor clinical diagnostic criteria (Table 1 and Fig. 1A–C) proposed for CHARGE syndrome. MRI and CT studies showed inner ear anomalies with semicircular canals hypoplasia. Her twin brother had a normal phenotype. Patient 2 was a male infant born at 42 weeks of gestation to non-consanguineous parents. Pregnancy was complicated by polyhydramnios during the eighth month of gestation. At birth weight was 3400 g (25–50th centile), length of 50 cm (25th centile). The patient met all the four major and six minor criteria for CHARGE syndrome. MRI and CT studies showed inner ear anomalies with semicircular canals hypoplasia (Table 1 and Fig. 1D).
[(Fig._1)TD$IG]
A. Michelucci et al. / International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
1442
Fig. 1. Clinical features of the patients with CHD7 mutations.
Table 1 Clinical features and mutations of the three patients. Patient 1
Patient 2
Patient 3
Age at diagnosis Sex Atresia of choanae Coloboma
1 month F Bilateral –
2 years, 5 months F Bilateral –
Cranial nerve dysfunction
Unilateral right facial palsy, sensorineural hearing loss, swallowing problems. Decreased cartilaginous folds and triangular concha. Inner ear anomalies. Squared-shaped, asymmetric facies with narrow bifrontal diameter, broad nasal bridge, small mouth and inverted V-shaped upper lip.
1 year, 9 months M Right Bilateral, involving choroid and retina (and iris on right eye). Sensorineural hearing loss.
Ear anomalies
Characteristic facial features
Congenital cardiovascular malformations Developmental delay
Ventricular septal defect Hypotonia and developmental delay.
Genital anomalies
Hypoplastic labia
Growth deficiency
Growth failure
Orofacial cleft
–
Tracheo-esophageal fistula CHD7 Mutation phenotype
– c.5300+1G ! T Exon 24 skipping (P.K1737D, p.1738_1767del)
Asymmetric pinna. Inner ear anomalies. Squa red-shaped, asymmetric facies with narrow bifrontal diameter, broad nasal bridge, small mouth and inverted V-shaped upper lip, orofacial cleft. Transposition of the great arteries Hypotonia, delayed motor milestones, delayed relationship development. Bilateral cryptorchidism and genital hypoplasy. Weight, lenght and CC below the third centile. Surgical correction was performed. – c.925 > T Premature stop codon (P.Q309X)
Sensorineural (and conductive) hearing loss. Low set, not typical ears. Inner ear imaging has not been performed yet. Square-shaped facies with narrow bifrontal diameter, broad nasal bridge, inverted V-shaped upper lip, anteverted nostrils, short neck, and synophrys. Aberrant right succlavia
Major criteria
Minor criteria
Delayed motor milestones, delayed relationship development, language delay. Hypoplastic labia Weight and lenght below the third centile. – – c.2954_2956delACA p.N958del
Mutations
[(Fig._2)TD$IG]
A. Michelucci et al. / International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
1443
Fig. 2. Sequence analysis of novel mutations in CHD7 gene.
Patient 3 was a female infant born at 36 weeks of gestation to non-consanguineous parents. Pregnancy was complicated by polyhydramnios and gestational diabetes. She was born by caesarian section because of fetal distress. At birth weight was 2170 g (10–25th centile), length of 46 cm (25–50th centile). The patient showed only two major criteria for CHARGE syndrome but several minor criteria (Table 1 and Fig. 1E–G). Radiological imaging to reveal the presence of agenesis/ hypoplasia of semicircular canals has not been performed yet. If this malformation, almost patognomonic for this syndrome, was present, this case should be considered no longer clinically atypical. 3. CHD7 mutation analysis The entire CHD7 coding sequence was examined by DHPLC and all the potential alterations were further investigated by bidirectionally direct sequencing. The sample was mixed with a known wild type and then completely denatured and re-annealed by heating up to 95 8C, then slowly cooling to 45 8C. Both samples that are heterozygous or homozygous for a polymorphic site will form heteroduplexes. All coding exons and exon–intron boundaries of the CHD7 gene (Ensembl Gene ID: ENSG00000171316) were amplified with 39 primer pairs according to the conditions
indicated in the ‘‘Multiple Malformation Syndromes’’ website (http://www.dhplc.jp). Haplotype analysis has not been performed because no literature data suggested this kind of analysis and also because each of the three families was not large enough to perform haplotype analysis. Considering the fact that none of the patients’ parents carried the mutations, only the paternity test has been performed. 4. Results In the three cases a heterozygous novel mutation was identified in CHD7 (Table 1) and the analysis of parental samples showed that all were de novo mutations. In patient 1 a G ! T transversion (Fig. 2A) at the donor splice site (c.5300 + 1G ! T) was found. The analysis of patient’s CHD7 mRNA detected an additional, shorter PCR product whose molecular weight corresponded to that expected with exon 24 skipping (Fig. 2B). Direct sequencing confirmed that the shorter band lacked exon 24 (Fig. 2C). Exon 24 skipping results in the loss of 30 codons and an amino acid change at the in-frame junction between exon 23 and 25 (p.K1737D, p.1738_1767del). In patient 2 a nonsense mutation was found: a C ! T transition (Fig. 2D) at the nucleotide 925 (c.925C > T) causing a premature stop codon (p.Q309X) in exon 2.
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A. Michelucci et al. / International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
In patient 3 a three nucleotide deletion c.2954_2956delACA (Fig. 2E) causing the loss of one amino acid (p.N985del) was found in exon 11. 5. Discussion CHARGE syndrome is an autosomal dominant disorder. Mutations in CHD7 gene have been detected in about 70% of cases, most frequently as de novo mutations [4,5,10]. In our study, patients 1 and 2 had a definite clinical diagnosis of CHARGE syndrome based on Blake et al. [1] and Verloes [3] criteria including semicircular canals hypoplasia which is a malformation almost patognomonic for this syndrome. Patient 3 did not strictly fit the classic criteria even if radiological studies to reveal inner ear anomalies have not been performed yet. If agenesis/hypoplasia of semicircular canals, was present, this case should be considered no longer clinically atypical. However in our experience parents often refuse to submit neonates or infants to radiological studies as CT scans. The occurrence of several minor criteria in addition to the two major ones suggested a possible diagnosis of CHARGE syndrome. In each patient a de novo heterozygous mutation in CHD7 gene was identified and all were novel: the first affected the donor splice site of exon 24, the second produced a stop codon in exon 2, and the third caused the loss of an Asparagine in exon 11. CHD7 mutations account for the majority of the cases with definite CHARGE syndrome but there is no clear correlation between the severity of clinical symptoms and the type of mutation. In fact, a high variability in the expression of CHARGE syndrome among family members carrying the same CHD7 mutation has been described [11]. The mutation of exon 24 donor splice site detected in patient 1 caused typical CHARGE syndrome without coloboma, one of the major criteria of both Blake and Verloes, and without early hypothalamo-hypophyseal dysfunction although it led to the loss of 30 codons and to an amino acid change. These 30 amino acid do not belong to any known functional domain of the protein. A less severe phenotype (atypical CHARGE) was observed for the patient carrying the 3 bp deletion in exon 11, determining the loss of one amino acid. The most severe symptoms were exhibited by the patient with the mutation in exon 2, which probably produced a truncated protein. The presence of a premature stop codon might also lead to nonsense-mediated mRNA decay, further reinforcing the hypothesis that this mutation determined the complete lack of a functional protein, in line with the more severe phenotype observed in the patient.
In conclusion, we report three novel mutations in the CHD7 gene in two patients with a definite clinical diagnosis of typical CHARGE syndrome and in one patient with atypical clinical diagnosis. The protein produced in the presence of the c.2954_2956delACA deletion (patient 3) might be at least partially functional, while the c.5300 + 1G ! T transversion (patient 1) and the stop codon in exon 2 carried by patient 2 seem to completely abolish Chd7 function, in line with the severity of the observed symptoms. The sequence analysis of the CHD7 gene is important to define the molecular basis of the classical clinical CHARGE phenotype and to increase diagnostic accuracy when the clinical phenotype is atypical. Acknowledgments We thank Chiara Domenici M.D. for assisting in preparing the English form of the manuscript. References [1] K.D. Blake, S.L. Davenport, B.D. Hall, M.A. Hefner, R.A. Pagon, M.S. Williams, et al., CHARGE association: an update and review for the primary paediatrician, Clin. Pediatr. (Phila.) 37 (1998) 159–173. [2] K.A. Issekutz, J.M. Graham Jr., C. Prasad, I.M. Smith, Blake KD, An epidemiological analysis of CHARGE syndrome: preliminary results from a Canadian study, Am. J. Med. Genet. A 133 (2005) 309–317. [3] A. Verloes, Updated diagnostic criteria for CHARGE syndrome: a proposal, Am. J. Med. Genet. A 133 (2005) 306–308. [4] M.C. Jongmans, R.J. Admiraal, K.P. van der Donk, L.E. Vissers, A.F. Baas, L. Kapusta, et al., CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene, J. Med. Genet. 43 (2006) 306–314. [5] S.R. Lalani, A.M. Safiullah, S.D. Fernbach, K.G. Harutyunyan, C. Thaller, L.E. Peterson, et al., Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation, Am. J. Hum. Genet. 78 (2006) 303– 314. [6] A.K. Morimoto, R.H. Wiggings III, P.A. Hudgins, G.L. Hedlund, B. Hamilton, S.K. Mukherji, et al., Absent semicircular canals in charge syndrome: radiologic spectrum of findings, Am. J. Neuroradiol. 27 (2006) 1663–1671. [7] Y. Asakura, Y. Toyota, K. Muroya, K. Kurosawa, K. Fujita, N. Aida, et al., Endocrine and radiological studies in patients with molecularly confirmed charge syndrome, J. Clin. Endocrinol. Metab. 93 (2008) 920–924. [8] D. Sanlaville, H.C. Etchevers, M. Gonzales, J. Martinovic, M. Cle´ment-Ziza, A.L. Delezoide, et al., Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development, J. Med. Genet. 43 (2006) 211–217. [9] L.E. Vissers, C.M. van Ravenswaaij, R. Admiraal, J.A. Hurst, B.B. de Vries, I.M. Janssen, et al., Mutations in a new member of the chromodomain gene family cause CHARGE syndrome, Nat. Genet. 36 (2004) 955–957. [10] P. Vuorela, S. Ala-Mello, C. Saloranta, M. Penttinen, M. Po¨yho¨nen, K. Huoponen, et al., Molecular analysis of the CHD7 gene in CHARGE syndrome: identification of 22 novel mutations and evidence for a low contribution of large CHD7 deletions, Genet. Med. 9 (2007) 690–694. [11] A. Delahaye, SznajerY, S. Lyonnet, M. Elmaleh-Berge`s, I. Delpierre, S. Audollent, et al., Familial CHARGE syndrome because of CHD7 mutation: clinical intra- and interfamilial variability, Clin. Genet. 72 (2007) 112–121.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Case report
Identification of three novel mutations in the CHD7 gene in patients with clinical signs of typical or atypical CHARGE syndrome Angela Michelucci a, Paolo Ghirri c,*, Paola Iacopetti b, Maria Elena Conidi a, Antonella Fogli a, Fulvia Baldinotti a, Sara Lunardi c, Francesca Forli d, Francesca Moscuzza c, Stefano Berrettini d, Antonio Boldrini c, Paolo Simi a, Silvia Pellegrini b a
Mother and Child Department, Cytogenetics and Molecular Genetics Unit, University Hospital of Pisa, Pisa, Italy Department of Experimental Pathology, Medical Biotechnologies, Infectious Diseases and Epidemiology, University of Pisa, Pisa, Italy c Mother and Child Department, Neonatology Unit and Section of Neonatal Endocrinology and Dysmorphology, University Hospital of Pisa, Pisa, Italy d Division of ENT, Department of Neuroscience, University of Pisa, Pisa, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 June 2010 Received in revised form 4 September 2010 Accepted 6 September 2010 Available online 12 October 2010
CHARGE syndrome is an autosomal dominant disorder characterized by features represented in its acronym: Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness. We report two patients with a diagnosis of typical CHARGE syndrome and one with atypical clinical diagnosis. All the three patients had uni- or bilateral choanal atresia and sensorineural hearing loss. The patients were screened for CHD7 gene mutations. Three novel occurring de novo heterozygous mutations were identified: a mutation in the donor splice site of intron 24, a missense mutation in exon 2 and a deletion in exon 11. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: CHARGE syndrome CHD7 gene Novel mutations Splicing defect
1. Introduction CHARGE (Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness) syndrome (OMIM 214800) is a rare disorder with an incidence of about one every 8500–10000 births [1,2]. Diagnostic criteria for CHARGE syndrome are based on a nonrandom association of the following congenital anomalies: coloboma of the eye, heart defects, choanal atresia, retarded growth and development, genital hypoplasia, ear anomalies and/or deafness [1,3]. Severe hearing loss and semicircular canals hypoplasia were found in almost all patients evaluated [4–7] and Sanlaville et al. [8] proposed semicircular canals hypoplasia as a major diagnostic criteria of the syndrome. The CHARGE syndrome gene, CHD7, was identified in 2004 [9] and belongs to the gene family coding for the Chromodomain Helicase DNA binding proteins. During early human development, CHD7 is expressed in the undifferentiated neuroepithelium and in mesenchyme of neural crest origin [8]. CHD7 is thought to play a role in regulating the expression of important
* Corresponding author at: Mother and Child Department, Neonatology Unit and Section of Neonatal Endocrinology and Dysmorphology, University Hospital of Pisa, Via Roma 55, 56126 Pisa, Italy. Tel.: +39 050993452; fax: +39 050992529. E-mail address: [email protected] (P. Ghirri). 0165-5876/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2010.09.006
developmental genes in mesenchymal cells derived from the cephalic neural crest, by chromatin remodeling [8]. Here we describe three patients affected by CHARGE syndrome carrying three novel de novo mutations in the CHD7 gene. 2. Case reports Patient 1 was a female infant, one of dizygotic twins born at 34 weeks of gestation by caesarian section to non-consanguineous parents. After birth the patient developed cyanosis and bradycardia, requiring resuscitation. Apgar scores were 4 at 1 min and 6 at 5 min. At birth weight was 1925 g (25th centile), length 43 cm (25th centile). She met three out of four major and five out of seven minor clinical diagnostic criteria (Table 1 and Fig. 1A–C) proposed for CHARGE syndrome. MRI and CT studies showed inner ear anomalies with semicircular canals hypoplasia. Her twin brother had a normal phenotype. Patient 2 was a male infant born at 42 weeks of gestation to non-consanguineous parents. Pregnancy was complicated by polyhydramnios during the eighth month of gestation. At birth weight was 3400 g (25–50th centile), length of 50 cm (25th centile). The patient met all the four major and six minor criteria for CHARGE syndrome. MRI and CT studies showed inner ear anomalies with semicircular canals hypoplasia (Table 1 and Fig. 1D).
[(Fig._1)TD$IG]
A. Michelucci et al. / International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
1442
Fig. 1. Clinical features of the patients with CHD7 mutations.
Table 1 Clinical features and mutations of the three patients. Patient 1
Patient 2
Patient 3
Age at diagnosis Sex Atresia of choanae Coloboma
1 month F Bilateral –
2 years, 5 months F Bilateral –
Cranial nerve dysfunction
Unilateral right facial palsy, sensorineural hearing loss, swallowing problems. Decreased cartilaginous folds and triangular concha. Inner ear anomalies. Squared-shaped, asymmetric facies with narrow bifrontal diameter, broad nasal bridge, small mouth and inverted V-shaped upper lip.
1 year, 9 months M Right Bilateral, involving choroid and retina (and iris on right eye). Sensorineural hearing loss.
Ear anomalies
Characteristic facial features
Congenital cardiovascular malformations Developmental delay
Ventricular septal defect Hypotonia and developmental delay.
Genital anomalies
Hypoplastic labia
Growth deficiency
Growth failure
Orofacial cleft
–
Tracheo-esophageal fistula CHD7 Mutation phenotype
– c.5300+1G ! T Exon 24 skipping (P.K1737D, p.1738_1767del)
Asymmetric pinna. Inner ear anomalies. Squa red-shaped, asymmetric facies with narrow bifrontal diameter, broad nasal bridge, small mouth and inverted V-shaped upper lip, orofacial cleft. Transposition of the great arteries Hypotonia, delayed motor milestones, delayed relationship development. Bilateral cryptorchidism and genital hypoplasy. Weight, lenght and CC below the third centile. Surgical correction was performed. – c.925 > T Premature stop codon (P.Q309X)
Sensorineural (and conductive) hearing loss. Low set, not typical ears. Inner ear imaging has not been performed yet. Square-shaped facies with narrow bifrontal diameter, broad nasal bridge, inverted V-shaped upper lip, anteverted nostrils, short neck, and synophrys. Aberrant right succlavia
Major criteria
Minor criteria
Delayed motor milestones, delayed relationship development, language delay. Hypoplastic labia Weight and lenght below the third centile. – – c.2954_2956delACA p.N958del
Mutations
[(Fig._2)TD$IG]
A. Michelucci et al. / International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
1443
Fig. 2. Sequence analysis of novel mutations in CHD7 gene.
Patient 3 was a female infant born at 36 weeks of gestation to non-consanguineous parents. Pregnancy was complicated by polyhydramnios and gestational diabetes. She was born by caesarian section because of fetal distress. At birth weight was 2170 g (10–25th centile), length of 46 cm (25–50th centile). The patient showed only two major criteria for CHARGE syndrome but several minor criteria (Table 1 and Fig. 1E–G). Radiological imaging to reveal the presence of agenesis/ hypoplasia of semicircular canals has not been performed yet. If this malformation, almost patognomonic for this syndrome, was present, this case should be considered no longer clinically atypical. 3. CHD7 mutation analysis The entire CHD7 coding sequence was examined by DHPLC and all the potential alterations were further investigated by bidirectionally direct sequencing. The sample was mixed with a known wild type and then completely denatured and re-annealed by heating up to 95 8C, then slowly cooling to 45 8C. Both samples that are heterozygous or homozygous for a polymorphic site will form heteroduplexes. All coding exons and exon–intron boundaries of the CHD7 gene (Ensembl Gene ID: ENSG00000171316) were amplified with 39 primer pairs according to the conditions
indicated in the ‘‘Multiple Malformation Syndromes’’ website (http://www.dhplc.jp). Haplotype analysis has not been performed because no literature data suggested this kind of analysis and also because each of the three families was not large enough to perform haplotype analysis. Considering the fact that none of the patients’ parents carried the mutations, only the paternity test has been performed. 4. Results In the three cases a heterozygous novel mutation was identified in CHD7 (Table 1) and the analysis of parental samples showed that all were de novo mutations. In patient 1 a G ! T transversion (Fig. 2A) at the donor splice site (c.5300 + 1G ! T) was found. The analysis of patient’s CHD7 mRNA detected an additional, shorter PCR product whose molecular weight corresponded to that expected with exon 24 skipping (Fig. 2B). Direct sequencing confirmed that the shorter band lacked exon 24 (Fig. 2C). Exon 24 skipping results in the loss of 30 codons and an amino acid change at the in-frame junction between exon 23 and 25 (p.K1737D, p.1738_1767del). In patient 2 a nonsense mutation was found: a C ! T transition (Fig. 2D) at the nucleotide 925 (c.925C > T) causing a premature stop codon (p.Q309X) in exon 2.
1444
A. Michelucci et al. / International Journal of Pediatric Otorhinolaryngology 74 (2010) 1441–1444
In patient 3 a three nucleotide deletion c.2954_2956delACA (Fig. 2E) causing the loss of one amino acid (p.N985del) was found in exon 11. 5. Discussion CHARGE syndrome is an autosomal dominant disorder. Mutations in CHD7 gene have been detected in about 70% of cases, most frequently as de novo mutations [4,5,10]. In our study, patients 1 and 2 had a definite clinical diagnosis of CHARGE syndrome based on Blake et al. [1] and Verloes [3] criteria including semicircular canals hypoplasia which is a malformation almost patognomonic for this syndrome. Patient 3 did not strictly fit the classic criteria even if radiological studies to reveal inner ear anomalies have not been performed yet. If agenesis/hypoplasia of semicircular canals, was present, this case should be considered no longer clinically atypical. However in our experience parents often refuse to submit neonates or infants to radiological studies as CT scans. The occurrence of several minor criteria in addition to the two major ones suggested a possible diagnosis of CHARGE syndrome. In each patient a de novo heterozygous mutation in CHD7 gene was identified and all were novel: the first affected the donor splice site of exon 24, the second produced a stop codon in exon 2, and the third caused the loss of an Asparagine in exon 11. CHD7 mutations account for the majority of the cases with definite CHARGE syndrome but there is no clear correlation between the severity of clinical symptoms and the type of mutation. In fact, a high variability in the expression of CHARGE syndrome among family members carrying the same CHD7 mutation has been described [11]. The mutation of exon 24 donor splice site detected in patient 1 caused typical CHARGE syndrome without coloboma, one of the major criteria of both Blake and Verloes, and without early hypothalamo-hypophyseal dysfunction although it led to the loss of 30 codons and to an amino acid change. These 30 amino acid do not belong to any known functional domain of the protein. A less severe phenotype (atypical CHARGE) was observed for the patient carrying the 3 bp deletion in exon 11, determining the loss of one amino acid. The most severe symptoms were exhibited by the patient with the mutation in exon 2, which probably produced a truncated protein. The presence of a premature stop codon might also lead to nonsense-mediated mRNA decay, further reinforcing the hypothesis that this mutation determined the complete lack of a functional protein, in line with the more severe phenotype observed in the patient.
In conclusion, we report three novel mutations in the CHD7 gene in two patients with a definite clinical diagnosis of typical CHARGE syndrome and in one patient with atypical clinical diagnosis. The protein produced in the presence of the c.2954_2956delACA deletion (patient 3) might be at least partially functional, while the c.5300 + 1G ! T transversion (patient 1) and the stop codon in exon 2 carried by patient 2 seem to completely abolish Chd7 function, in line with the severity of the observed symptoms. The sequence analysis of the CHD7 gene is important to define the molecular basis of the classical clinical CHARGE phenotype and to increase diagnostic accuracy when the clinical phenotype is atypical. Acknowledgments We thank Chiara Domenici M.D. for assisting in preparing the English form of the manuscript. References [1] K.D. Blake, S.L. Davenport, B.D. Hall, M.A. Hefner, R.A. Pagon, M.S. Williams, et al., CHARGE association: an update and review for the primary paediatrician, Clin. Pediatr. (Phila.) 37 (1998) 159–173. [2] K.A. Issekutz, J.M. Graham Jr., C. Prasad, I.M. Smith, Blake KD, An epidemiological analysis of CHARGE syndrome: preliminary results from a Canadian study, Am. J. Med. Genet. A 133 (2005) 309–317. [3] A. Verloes, Updated diagnostic criteria for CHARGE syndrome: a proposal, Am. J. Med. Genet. A 133 (2005) 306–308. [4] M.C. Jongmans, R.J. Admiraal, K.P. van der Donk, L.E. Vissers, A.F. Baas, L. Kapusta, et al., CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene, J. Med. Genet. 43 (2006) 306–314. [5] S.R. Lalani, A.M. Safiullah, S.D. Fernbach, K.G. Harutyunyan, C. Thaller, L.E. Peterson, et al., Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation, Am. J. Hum. Genet. 78 (2006) 303– 314. [6] A.K. Morimoto, R.H. Wiggings III, P.A. Hudgins, G.L. Hedlund, B. Hamilton, S.K. Mukherji, et al., Absent semicircular canals in charge syndrome: radiologic spectrum of findings, Am. J. Neuroradiol. 27 (2006) 1663–1671. [7] Y. Asakura, Y. Toyota, K. Muroya, K. Kurosawa, K. Fujita, N. Aida, et al., Endocrine and radiological studies in patients with molecularly confirmed charge syndrome, J. Clin. Endocrinol. Metab. 93 (2008) 920–924. [8] D. Sanlaville, H.C. Etchevers, M. Gonzales, J. Martinovic, M. Cle´ment-Ziza, A.L. Delezoide, et al., Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development, J. Med. Genet. 43 (2006) 211–217. [9] L.E. Vissers, C.M. van Ravenswaaij, R. Admiraal, J.A. Hurst, B.B. de Vries, I.M. Janssen, et al., Mutations in a new member of the chromodomain gene family cause CHARGE syndrome, Nat. Genet. 36 (2004) 955–957. [10] P. Vuorela, S. Ala-Mello, C. Saloranta, M. Penttinen, M. Po¨yho¨nen, K. Huoponen, et al., Molecular analysis of the CHD7 gene in CHARGE syndrome: identification of 22 novel mutations and evidence for a low contribution of large CHD7 deletions, Genet. Med. 9 (2007) 690–694. [11] A. Delahaye, SznajerY, S. Lyonnet, M. Elmaleh-Berge`s, I. Delpierre, S. Audollent, et al., Familial CHARGE syndrome because of CHD7 mutation: clinical intra- and interfamilial variability, Clin. Genet. 72 (2007) 112–121.