Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation

Abstract

CHARGE syndrome is a well-established multiple-malformation syndrome with distinctive consensus diagnostic criteria. Characteristic associated anomalies include ocular coloboma, choanal atresia, cranial nerve defects, distinctive external and inner ear abnormalities, hearing loss, cardiovascular malformations, urogenital anomalies, and growth retardation. Recently, mutations of the chromodomain helicase DNA-binding protein gene CHD7 were reported to be a major cause of CHARGE syndrome. We sequenced the CHD7 gene in 110 individuals who had received the clinical diagnosis of CHARGE syndrome, and we detected mutations in 64 (58%). Mutations were distributed throughout the coding exons and conserved splice sites of CHD7. Of the 64 mutations, 47 (73%) predicted premature truncation of the protein. These included nonsense and frameshift mutations, which most likely lead to haploinsufficiency. Phenotypically, the mutation-positive group was more likely to exhibit cardiovascular malformations (54 of 59 in the mutation-positive group vs. 30 of 42 in the mutation-negative group; P=.014), coloboma of the eye (55 of 62 in the mutation-positive group vs. 30 of 43 in the mutation-negative group; P=.022), and facial asymmetry, often caused by seventh cranial nerve abnormalities (36 of 56 in the mutation-positive group vs. 13 of 39 in the mutation-negative group; P=.004). Mouse embryo whole-mount and section in situ hybridization showed the expression of Chd7 in the outflow tract of the heart, optic vesicle, facio-acoustic preganglion complex, brain, olfactory pit, and mandibular component of the first branchial arch. Microarray gene-expression analysis showed a signature pattern of gene-expression differences that distinguished the individuals with CHARGE syndrome with CHD7 mutation from the controls. We conclude that cardiovascular malformations, coloboma, and facial asymmetry are common findings in CHARGE syndrome caused by CHD7 mutation.

Figure 1

Individuals with CHARGE syndrome who have CHD7 mutations. A and a, A 17-year-old girl with retinal coloboma, characteristic ear malformation, hearing loss, partial atrioventricular canal defect, growth retardation, and delayed puberty who has a frameshift mutation in exon 37. B and b, An 11-year-old boy with colobomas of the iris and the macula, choanal stenosis, characteristic ear malformation, bilateral Mondini malformation, profound hearing loss, submucous cleft palate, bilateral facial palsy, undescended testes, and pulmonary atresia with ventricular septal defect who has a de novo frameshift mutation in exon 18. C and c, A 3-year-old boy with bilateral iris coloboma, choanal stenosis, ear malformation, hearing loss, facial palsy, and abnormal cochlea but without cardiovascular malformation who has a nonsense mutation in exon 13. D and d, A 12-year-old boy with coloboma involving the optic nerve, choanal atresia, characteristic ear malformation, profound hearing loss, abnormal inner ear, patent ductus arteriosus, and genital abnormalities who has a frameshift mutation in exon 2. Note that the individuals in panels B and D illustrate the characteristic facial appearance of CHARGE syndrome.

Figure 2

Microarray gene-expression profile and the resulting cluster analysis. Patterns of gene expression were assessed on Affymetrix U133Plus2.0 arrays (see “Material and Methods” section for details). After identification of a subset of transcripts with apparently significant differences in expression between the individuals with CHARGE syndrome with CHD7 mutations and the controls, the combined data were used to produce a hierarchical cluster analysis. Expression values shown were standardized using the gene-specific mean and SD of log₂ expression. Note that the profile of CHA230 (who had no detectable CHD7 mutation) is highly similar to those of individuals with CHD7 mutations.

Figure 3

Genomic and protein map of CHD7, indicating the spectrum of mutations in CHARGE syndrome. On the protein map, blackened circles signify nonsense mutations, blackened diamonds signify frameshift mutations, unblackened squares signify missense mutations, and the unblackened circle specifies a mutation with an in-frame deletion of 3 aa. On the genomic sequence, triangles represent splice-site mutations.

Figure 4

Five pedigrees with CHARGE syndrome illustrating four familial cases and an affected pair of MZ twins. CHD7 mutations were identified in pedigrees CHA76, CHA166, and CHA172. Germline mosaicism is suggested in CHA76, whereas CHA166 indicates autosomal dominant transmission of the missense mutation in this mildly affected family. CHA172 shows a nonsense mutation in MZ twins. Mutations in CHD7 were not identified in families CHA192 and CHA88. Probands are indicated by arrows.

Figure 5

Mouse embryo whole-mount and section in situ hybridization showing the expression of Chd7. A, Whole-mount in situ hybridization of the embryo at 10.5 dpc, demonstrating expression of Chd7 in the craniofacial region. B, Expression of Chd7 observed in cardiac outflow tract (OFT). LA = left atrium; RA = right atrium. C, Sagittal section of embryo at 10.5 dpc, showing expression in the facio-acoustic (VII–VIII) preganglion complex (FA), hindbrain (HB), forebrain (FB), mandibular component of the first branchial arch (BA), and otic vesicle (OV). D and F, Sagittal section of embryo with expression in optic stalk/optic vesicle (OPV) and olfactory pit (OP). E and G, Magnified images of panel C, indicating expression of Chd7 in facio-acoustic preganglion complex and otic vesicle, with hematoxylin and eosin stain. H, Sagittal section of the embryo, demonstrating expression in truncus arteriosus (TA) at 10.5 dpc.