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Am J Med Genet A. Author manuscript; available in PMC Mar 8, 2010.
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PMCID: PMC2834558

Disruption of Chromodomain Helicase DNA Binding Protein 2 (CHD2) Causes Scoliosis


Herein we characterize an apparently balanced de novo translocation, t(X;15)(p22.2;q26.1)dn, in a female patient with scoliosis, hirsutism, learning problems, and developmental delay (DGAP025). Other clinical findings include a high-arched palate, 2–3 syndactyly of the toes, and mildly elevated serum testosterone. No known or predicted genes are disrupted by the Xp22.2 breakpoint. The 15q26.1 breakpoint disrupts chromodomain helicase DNA binding protein 2 (CHD2). Another member of the chromatin-remodeling gene family, CHD7, has been associated with a defined constellation of congenital anomalies known as coloboma, heart anomaly, choanal atresia, mental retardation, genital and ear anomalies syndrome (CHARGE) and idiopathic scoliosis. Monosomy of 15q26 also has been associated with a spectrum of congenital abnormalities and growth retardation that overlaps with those of DGAP025. To provide a biological correlate, we characterized a mutant mouse model with Chd2 disruption that is associated with embryonic and perinatal lethality. Expression analysis indicated that Chd2 is expressed in the heart, forebrain, extremities, facial and dorsal regions during specific times of embryonic development. Chd2+/m mice showed pronounced lordokyphosis, reduced body fat, postnatal runting, and growth retardation. These data suggest that haploinsufficiency for CHD2 could result in a complex of abnormal human phenotypes that includes scoliosis and possibly features similar to CHARGE syndrome.

Keywords: CHD2, chromodomain, helicase, congenital vertebral malformation, scoliosis, chromosomal translocation


Breakpoints analyzed in individuals with balanced chromosome rearrangements have led to the identification of various genes involved in human development [Ray et al., 1985; Blanquet et al., 1987; Turleau and de Grouchy, 1987; Ishikiriyama et al., 1989; Wallace et al., 1990; Zemni et al., 2000]. Identifying genes crucial in development through characterization of chromosomal rearrangements is the approach ongoing in the Developmental Genome Anatomy Project (DGAP; http://dgap.harvard.edu). Here we report our analysis of a 17-year-old Caucasian female (DGAP025) with multiple congenital abnormalities and t(X;15)(p22.2;q26.1)dn. Using high resolution FISH, we mapped the breakpoint on chromosome 15 within chromodomain helicase DNA binding protein 2 (CHD2), a member of theCHDfamily of genes. Mice in which a retroviral gene-trapping methodology truncates the murine ortholog within the DNA binding domain previously have been characterized to have growth and perinatal lethality in homozygous mutants, and glomerulopathy, other visceral organ pathology, and reduced survival in heterozygous mice [Marfella et al., 2006, 2007]. We independently characterized the same mouse model with a particular focus on skeletal analysis. In addition to finding embryonic lethality at E14.5 in homozygous mutants, we found runting, prominent vertebral anomalies and, occasionally, defective ocular development in heterozygous animals.

Chromodomain helicase DNA binding proteins were characterized as a distinct family of proteins in the late 1990s [Woodage et al., 1997]. CHD genes are evolutionarily conserved, and at least nine genes have been identified in humans ([Delmas et al., 1993; Woodage et al., 1997; Schuster and Stoger, 2002] and NCBI Build 36.2 (http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606)). The CHD gene family is defined by the presence of chromo (chromatin organization modifier) domains, an SNF2-related helicase/ATPase domain and distinct DNA binding domains [Woodage et al., 1997]. Chromodomain containing proteins can self-associate as well as interact with the heterochromatic regions at centromeres, telomeres, and polytene chromosomes [Delmas et al., 1993; Schuster and Stoger, 2002]. CHD proteins modulate transcription by virtue of their ability to remodel chromatin structure via their helicase activities and effect on histone deacetylation [Singh et al., 1991; Cowell and Austin, 1997].A wealth of data on the CHD family of proteins has come from studies showing CHD3 and CHD4 to be ATPases involved in chromatin remodeling [Cowell and Austin, 1997; Tong et al., 1998; Zhang et al., 1998; Brehm et al., 2000; Bowen et al., 2004]. The CHD3 and CHD4 proteins were also isolated as components of the nucleosome remodeling and histone deacetylation complex (NuRD) in HeLa cells [Targoff and Reichlin, 1985; Bowen et al., 2004]. The biological role of CHD2 is unknown. The chromosomal location of human CHD2 is 15q26.1, a region implicated in a rare genetic disorder that leads to growth retardation, cardiac defects, and early postnatal lethality [Wilson et al., 1985; Whiteford et al., 2000].

Recently, mutations and microdeletions inCHD7, a CHD family member, have been shown in more than 60% of cases of CHARGE syndrome (OMIM 214800), a complex and nonrandom constellation of multiple congenital anomalies including Coloboma, Heart defects, choanal Atresia, mental Retardation, Genital and Ear anomalies [Vissers et al., 2004; Jongmans et al., 2006; Lalani et al., 2006]. In addition, linkage analysis of familial idiopathic scoliosis has implicated CHD7, but the pathogenetic mutation(s) remain to be determined in the affected kindreds [Gao et al., 2007].

The association of scoliosis and other phenotypic problems with the disruption of CHD2 in our human patient, as well as the targeted disruption of its murine ortholog, suggests that this member of the CHD gene family also plays a significant role in development and growth of the spine.


Human Cell Line and Clinical Information

A lymphoblastoid cell line (NIGMS GM13992), established by Epstein–Barr virus transformation of peripheral blood lymphocytes from the patient (DGAP025), was obtained from the NIGMS Human Genetic Cell Repository at the Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). The clinical information for this patient was acquired by the Repository when the original blood sample was submitted. We attempted to obtain additional detailed clinical description and follow-up information with the assistance of the Repository, but were unsuccessful due to the long interval between its original submission and our subsequent studies.

Chromosome Preparations

Metaphase chromosomes were prepared using standard protocols. These chromosome spreads were used for GTG-banding, X-inactivation studies, and fluorescence in situ hybridization (FISH) [Ney et al., 1993]. FISH mapping of the chromosome breakpoints was carried out using bacterial artificial chromosome (BAC) and fosmid clones mapping to human chromosomes X and 15 (BACPAC Resource, CHORI, Oakland, CA) using methods previously described [Moore et al., 2004]. Clones were selected with the aid of the University of California Santa Cruz (UCSC) Genome Browser (May 2004 build; http://genome.ucsc.edu/cgi-bin/hggateway). BAC and fosmid DNA were prepared by strand displacement amplification using Phi29 DNA polymerase (GenomiPhi, GE Healthcare, Piscataway, NJ). DNA was directly labeled by nick translation using SpectrumGreen-dUTP or SpectrumRed-dUTP (Abbott Molecular/Vysis, Downers Grove, IL) and hybridized to metaphase chromosomes. Chromosomes were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) and at least 10 metaphases per probe were analyzed using a CytoVision/Olympus BX51 microscopy system (Applied Imaging, San Jose, CA and Optical Analysis Corp., Nashua, NH).

X-Inactivation Analysis

To assess the pattern of X-inactivation in DGAP025 lymphoblastoid cells, 5-bromo-2′-deoxyuridine (BrdU) replication timing studies were performed using standard protocols. Briefly, lymphoblastoid cells were grown in medium containing thymidine (0.3 mg/ml) and exposed to 30 µg/ml BrdU (Sigma, St. Louis, MO) for 6 hr prior to harvesting. Metaphases were denatured and dehydrated. Incorporated BrdU was then detected using fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal anti-BrdU antibody (Research Diagnostics, Flanders, NJ) according to the manufacturer’s directions; a chromosome 15 fosmid clone was used to differentiate between the normal and derivative X chromosomes.

Generation of Chd2 Mutant Mice

We generated Chd2-deficient mice using the BayGenomics genetrap embryonic stem cell (ES) cell resource [Stryke et al., 2003]. Chd2 trapped ES cells were obtained from BayGenomics and analyzed by PCR to confirm Chd2 disruption using primers specific for Chd2 and the gene-trap sequences. The following primers were used for genotype analysis of mutant and wild type mice: TR3, 5′-GTG AGC GAG TAA CAA CCC GTC-3′; TR2, 5′-AGC TGT TGG GAG GGT CAC TTT ATG-3′; TR1, 5′-ACC TGG CTC CTA TGG GAT AG-3′; GSP1, 5′-TGT GTG TCA GCA ATG CAG GA -3′; GSP2, 5′-TGC ATA ACC ATT CCG GGT GTG-3′. Sequencing of the PCR product indicated that the gene trap was integrated within intron 27 (1,563 base pairs from the beginning of the intron) of Chd2. Blastocyst injections from the validated ES cells were performed using the microinjection services at the University of Massachusetts Medical School, Worcester, MA. Resulting chimeras were bred to C57BL6/J mice to obtain founder Chd+/m mice (henceforth designated as Chd2+/m). These heterozygotes were then inter-crossed to yield 22 litters of 109 pups at F2 for phenotype analysis. Genotyping of the Chd2m allele was performed by Southern blot assays (data not shown) and PCR using the primers described above.

Expression Analysis of Chd2 During Mouse Development

Embryos obtained from timed matings from wild-type females and Chd2+/m males were fixed with 1% paraformaldehyde and stained in a solution (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.1 M phosphate buffer, pH 7.3) containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Genotypes of embryos were determined from genomic DNA isolated from yolk sacs.

Imaging and 3D Reconstruction of Mutant Mice

High-resolution CT (Computed Tomography) images were acquired with a MicroCAT™ II (Siemens Medical Solutions Molecular Imaging, LLC, Knoxville, TN) instrument [Paulus et al., 2000; Wall et al., 2006]. The scanner is equipped with a 20–80 kVp microfocus X-ray source and has a 90 mm × 60 mm field of view. Each CT dataset was composed of 360 1-degree projections acquired over 8 min using a 310 msec exposure for each. Images were reconstructed using a modified version of the Feldkamp algorithm [Gregor et al., 2002] on a 512×512×768 matrix with an isotropic voxel size of 77 µm. Micro CT data were visualized using the Amira 3-D image analysis software package (Amira, Version 3.1: Mercury Computer Systems, Chelmsford, MA).


Karyotypic and Phenotypic Description of DGAP025

DGAP025 is a 17-year-old Hispanic Caucasian female who presented with scoliosis, developmental delay, high arched palate, 2–3 syndactyly of the toes, learning problems, height <30th centile and occipital frontal circumference (OFC) <25th centile (Table I). In addition, the patient has a masculinized face, hirsutism, and excessive hair on her extremities. Her voice was characterized as low and somewhat masculine, her thorax shows secondary sexual development, her serum testosterone was 59 ng/dl (normal range for females: 10–55 ng/dl). Information allowing assignment to a specific Tanner Stage was not available. Her face (including the eyes and ears) was otherwise unremarkable (inner canthal distance, 3 cm; palpebral fissure distance, 2.5 cm; and ears, 6 cm).

Phenotypic Features Associated With Abnormalities of 15q26

The NIGMS Human Genetic Cell Repository karyotype, 46,X,t(X;15)(p22;q26.1)dn, for DGAP025 was confirmed prior to any FISH-mapping studies. The ideogram depicting the normal and derivative chromosomes X and 15 is shown in Figure 1A.

FIG. 1
Cytogenetic analysis of DGAP025

X-Inactivation Analysis

The X-inactivation pattern was determined using an EBV-transformed lymphoblastoid cell line. The der(X) was identified using simultaneous FISH with a chromosome 15 fosmid clone. Results confirmed the expected inactivation pattern, in which the normal X was late replicating, in each of 50 metaphases analyzed (data not shown). This result indicates that the normal X was inactivated and that the etiology of this subject’s phenotype was not subsequent to allelic imbalance due to inappropriate inactivation of autosomal DNA on the der(X) or lack of inactivation of X chromosomal DNA on the der(15).

Breakpoint Mapping of the X Chromosome

To determine the site of breakage on the X chromosome, we performed FISH analysis with an ordered series of BAC and fosmid clones mapped to Xp22. The Xp22 breakpoint interval was narrowed to a region of about 1.46 kb using overlapping fosmid clones, G248P86980D11 and G248P80445G9 (Fig. 1B).

Breakpoint Mapping of Chromosome 15

FISH performed with BAC clones RP11-52D3 and RP11-577O14 localized the breakpoint within an interval of ~98 kb. Further refinement of the chromosome 15 breakpoint using fosmids identified an ~416 bp interval within fosmid G248P80128E6 (Fig. 1C,D) flanked by fosmids G248P83477D10 and G248P81760B10. It also is possible that the breakpoint is located in one of the flanking fosmid clones because fluorescent signal generated by such a highly asymmetrically distributed FISH probe might be below the image capture system’s detection threshold for one of the derivatives. In our experience, the breakpoint is usually with 10 kb of the clone’s “flanking” end in such cases.

Only a single known gene, CHD2, maps within this interval, and the breakpoint region defined by FISH mapping localized it within intron 18 (Fig. 1D). Based on this FISH analysis, the chromosome 15 breakpoint was refined to 15q26.1.

Expression of Chd2 During Murine Development

To understand the relationship between the expression of chromodomain helicase DNA binding protein 2 and the abnormal phenotypes observed in both DGAP025 and our mutant mouse model, we performed expression analysis using embryos obtained from timed matings between wild-type female mice and Chd2 heterozygous males. The genetrap vector present in the Chd2-targeted ES cell clone contains a promoter-less β-galactosidase-neomycin fusion gene that allows expression analysis of the trapped Chd2 gene (Fig. 2). The genotype of parents and offspring were determined by Southern blot analysis (not shown) and PCR (Fig. 2).

FIG. 2
A: Schematic representation of wild-type and trapped Chd2 alleles. B: Genotype analysis of mutant and wild-type mouse embryonic fibroblasts are shown above. The relative positions of PCR primers used in the genotype analysis are indicated.

Chd2 expression was limited to the para-aortic splanchnopleural mesoderm (P-Sp) region consisting of heart precursors in E10.5 embryos (Fig. 3A). At 10.5 d.p.c., expression was confined to the bulbus cordis and the common atrial chamber, predominantly areas that ultimately would develop into the right atrium and ventricle. One day later, new X-gal staining highlighted the forebrain and eye. Interestingly, strong X-gal staining appeared in the extremities, as well as the dorsal and facial regions in E15.5 embryos.

FIG. 3
Expression analysis of Chd2 and phenotypic characterization of Chd2+/m and Chd2m/mmice

Phenotypic Characterization of Chd2 Mutant Mice

The germline disruption of genes with essential functions in embryogenesis usually leads to either embryonic lethality or developmental defects. Genotype analysis of tail DNA from offspring of F1 heterozygous intercrosses indicated that Chd2 nullizygous mutants fail to survive. As shown in Table II, offspring from the 22 different F1 intercrosses did not yield any homozygous mutant offspring at weaning. These data also indicate that embryonic lethality is likely in a subset of heterozygous pups because the number of heterozygotes obtained was less than the expected 2:1 ratio of the total offspring. To assess developmental defects that arise due to Chd2 deficiency, we initiated timed matings and found that the lethality of the embryos began to occur around E14.5 as the number of null offspring was decreased at E14.5 (Table II). Neonatal mutants also appeared pale, runted and tended to wean later than their wild-type littermates. A small fraction (2/15 mutants) exhibited defective eye formation and eye migration defects (Fig. 3B). The adult heterozygous offspring did not show any overt abnormalities except for apparent growth retardation. Most significantly, by 4 months, pronounced lordokyphosis was readily apparent (Fig. 4). In addition, the heterozygotes had a runt phenotype in which the subcutaneous fatty tissues were absent or extremely hypoplastic (Fig. 3D and Fig. 4D). Careful review of the distal extremities in heterozygous animals (n=12) did not reveal any evidence of syndactyly or other gross digital abnormalities; filling defects in the distal vasculature, however, were noted and currently are under study (Fig. 2C). Finally, cystic endometrial hyperplasia was noted in three of eight female heterozygotes (data not shown). Unlike the murine phenotype described in a previous report [Marfella et al., 2006] we did not detect any cardiac anomaly apart from mild to moderate atrial enlargement in a subset of the Chd2+/m and Chd2m/m neonates (data not shown); nor were the stigmata of cardiac failure (viz., hepatic centrilobular necrosis and hemosiderin-laden alveolar macrophages) present in the neonates. Renal histopathology also was not observed in the neonates, but~47% (15/32) of adults exhibited glomerulonephropathy (data not shown). The reasons for the phenotypic differences between mice characterized herein and those by Marfella et al. [2006] remain to be determined.

FIG. 4
Chd2+/m mice develop lordokyphosis as they age
Embryonic and Neonatal Lethality of Chd2 Mutant Mice*


We report on an X;autosome translocation, t(X;15)(p22.2;q26.1)dn, in a 17-year-old female with scoliosis, hirsutism, learning problems and developmental delay. Upon initial clinical examination, this particular constellation of malformations and developmental abnormalities did not correspond to any known syndrome.

Although we cannot completely exclude the possibility of disruption of an unannotated gene at Xp22.2, our results suggest that the chromosome 15 breakpoint is likely the pathogenetically relevant breakpoint. In addition, chromosome rearrangements as far as 1 Mb away from the transcription and promotor region have been shown to affect gene expression [Velagaleti et al., 2005]. Accordingly, a position effect altering expression of a gene near the chromosome X breakpoint is possible, but data from other examples suggest that the distance is usually under 200 kb [Bedell et al., 1996; Kleinjan and van Heyningen, 1998, 2005]. Some of the genes in the vicinity of the chromosome X breakpoint and their respective distances relative to the breakpoint are AP1S2—16 kb, U2AF1RS2—48 kb, CA5B—86 kb, and GRPR—81 kb. The nearest of these, adaptor-related protein complex 1 (AP1S2, σ2 subunit), localizes to the cytoplasmic face of coated vesicles of the Golgi complex, where it mediates clathrin recruitment [Takatsu et al., 1998]. U2AF1RS2 (U2AF1L2, U2 small nuclear ribonucleoprotein auxiliary factor, small subunit 2), encodes a protein related to an essential splicing factor [Kitagawa et al., 1995].CA5B(carbonic anhydrase VB, mitochondrial) is a member of the zinc metalloenzyme family that catalyze the reversible hydration of carbon dioxide [Fujikawa-Adachi et al., 1999]. The most distant, GRPR (gastrin-releasing peptide receptor), regulates numerous functions of the gastrointestinal and central nervous systems, including release of gastro-intestinal hormones, smooth muscle cell contraction, and epithelial cell proliferation [Spindel et al., 1990]. Of the genes near the X chromosome breakpoint, only GRPR has been implicated to play a role in human development. Specifically, an individual with autism, a phenotype unlike that of DGAP025, was found to have a balanced translocation between GRPR on Xp22.12 and 8q22.1 [Ishikawa-Brush et al., 1997].

In addition to DGAP025, at least eight other patients are reported in the literature with some overlapping phenotype and similar chromosomal breakpoints or deletions involving 15q25–26 (Table I) [de Jong et al., 1989; Rosenberg et al., 1992; Chen et al., 1998; Schlembach et al., 2001; Klaassens et al., 2005]. Six of these eight patients had craniofacial dysmorphism and limb abnormalities with 15q26 chromosomal breakpoints. The growth retardation in DGAP025 and in other cases reported in the literature (Table I) also is consistent with the location of a key developmental regulator gene at 15q26. The most interesting candidate gene in the region of overlapping deletion is the one directly disrupted by the chromosomal rearrangement in DGAP025, namely CHD2 [Woodage et al., 1997].

Recently, causative mutations and deletions of CHD7, another CHD family member, have been identified in roughly 60% of individuals with CHARGE syndrome [Vissers et al., 2004; Lalani et al., 2006]. Expression analysis of murine embryos and neonates demonstrates increased CHD7 expression in tissues corresponding to the adult structures affected in CHARGE syndrome [Bosman et al., 2005; Lalani et al., 2006; Sanlaville et al., 2006]. Multiple ENU-induced mutations in Chd7 result in a variety of partially or nearly fully truncated polypeptide products and a range of defects with reduced penetrance that include cleft palate, choanal atresia, cardiac septal defects, vulvar and clitoral defects, keratoconjuctivitis sicca, and perinatal death [Bosman et al., 2005]. In the human, CHD7 polymorphisms have recently been associated a familial susceptibility to idiopathic scoliosis, particularly those manifesting during the accelerated growth of adolescence [Gao et al., 2007]. In contrast to the murine mutations, the mechanistic significance of the newly identified human CHD7 polymorphisms remains to be elucidated. Finally, Gao et al. [2007] have suggested that CHD7 may have been disrupted by position effect over 9.9 Mb in an individual with idiopathic scoliosis and pericentric chromosomal inversion between 8p23.2 and 8q11.21 [Bashiardes et al., 2004].

CHD2 is a 38 exon, ~122 kb member of the CHD gene family. CHD2, like CHD7, is a protein with two chromatin organization modifier (chromo) domains, a SNF2-related helicase/ATPase domain and a DNA-binding domain [Woodage et al., 1997]. CHD proteins alter gene expression by modifying nucleosome binding and remodeling, which presumably modulates access of the transcriptional machinery to the DNA template.

Prior to this study, no association between CHD2 defects and a human phenotype has yet been proven prior to this study, although CHD2 recently was one of several genes within 15q26.1 implicated in congenital diaphragmatic hernia [Klaassens et al., 2005]. Specifically, monosomy 15q26.1–15q26.2 occurs recurrently in individuals with congenital diaphragmatic hernia [Slavotinek et al., 2006]. In the study by Slavotinek and co-authors, only one Mexican-American patient of more than 100 ethnically diverse individuals with congenital diaphragmatic hernia had a missense change (C5128T, corresponding to R1710W), which was not found in 100 ethnically matched normal controls [Slavotinek et al., 2006]. Of note, this particular sequence variation was not associated with any other phenotypic abnormalities and, consequently, C5128T was judged not to be pathogenetic for congenital diaphragmatic hernia. The lack of clinically evident diaphragmatic herniation in DGAP025 and our mutant mouse model is consistent with lack of a critical role for CHD2 in development of the diaphragm, but raises the possibility that another gene in the critical deletion interval might be causative. Recently, it has been shown that mice lacking NR2F2, which maps to 15q26.2 approximately 3.3 Mb from CHD2, are born with Bochdalek-type congenital diaphragmatic hernias [You et al., 2005].

Disruption of CHD2 could contribute to the phenotype in DGAP025 either through formation of a fusion product with a gene on the X chromosome or by CHD2 truncation, causing haploinsufficiency or perhaps a dominant negative effect. In CHARGE syndrome associated with CHD7 mutations, nearly three-quarters (47/64) of mutations in a single series were predicted to result in premature termination of the protein, which was interpreted as supporting the haploinsufficiency model [Lalani et al., 2006]. The genomic orientation of the genes surrounding the chromosomal breakpoints in DGAP025 is such that a 5′-CHD2-AP1S2-3′ fusion would be feasible if the transcription excluded the first exon of AP1S2 (exon jumping). The orientation of the other nearby genes would not support the formation of additional fusion products. Alternatively, the hypothetical truncation product would retain two chromo domains and the SNF2 domain, but omit the C-terminal helicase domain. Nonetheless, we searched for either a fusion transcript between the 5′ of CHD2 and the 3′ of a gene on the X chromosome or a truncated CHD2 transcript using 3′ RACE PCR experiments (data not shown) using the EBV-transformed lymphoblast cell line and found neither. The absence of such aberrant transcripts suggests that they may not contribute to the DGAP025 phenotype, although it is possible that such mRNA species were expressed either at different times or in different cell types during the development of DGAP025. In the mouse model, insertion of the retroviral gene-trap results in truncation of Chd2 in the DNA binding domain and, presumably, abrogation of the DNA binding domain’s function [Marfella et al., 2007]. Although the extent of truncation differs between DGAP025 and the mouse model, the similarity in these mutations suggests that the mechanism responsible for the resulting phenotypes also maybe shared. Consequently, our analysis of the t(X;15)(p22.2;q26.1)dn in DGAP025 supports the view that haploinsufficiency for CHD2 is the most likely explanation for the observed phenotypic features.

Mutation of both Chd2 alleles in the mouse results in embryonic and perinatal lethality, clearly indicating that this chromodomain helicase DNA binding protein family member is important during development. Like CHD7, the pattern of embryonic expression (eye, heart, face, and forebrain) correlates with most of the affected organs and tissues defining the coloboma, heart anomaly, choanal atresia, retardation, genital and ear anomalies syndrome (CHARGE). The concurrence of cardiac Chd2 expression and embryonic lethality of Chd2 nullizygosity suggests that lethality may be due to a failure to achieve a critical milestone in cardio-vascular or hematopoietic development. Later in postnatal life, the most striking anomaly noted in Chd2 heterozyogotes was marked lordokyphosis. CHD2 disruption in the human (DGAP025) also was associated with clinically significant scoliosis. Of note, Doyle and Blake [Doyle and Blake, 2005] report that nearly two-thirds (19/31) of individuals with a clinical diagnosis of CHARGE syndrome have scoliosis that was moderate to severe in 40% (8/19) of cases. Interestingly, they suggest that CHARGE syndrome with scoliosis may represent a specific subtype because, on average, the diagnosis of CHARGE syndrome was delayed by over 2 years (6.3 years of age compared to 3.7 years of age) in cases without scoliosis. In addition, scoliosis was associated with growth hormone therapy. The location of the anomalous vertebral development and growth may correlate with the dorsal expression of Chd2 in the mouse embryo at 15.5 d.p.c. Although syndactyly was not observed in the mouse model, Chd2 expression was also prominent in growing limbs, and a mechanism similar to that responsible for the vertebral anomaly could be postulated for anomalous digital development in DGAP025.

Other phenotypic features in DGAP025, and possibly in those individuals with monosomy for regions of 15q (Table I), also can be correlated with the heterozygous mouse. Intrauterine growth retardation (IUGR) is a common characteristic of all previously reported patients with monosomy 15q26 (Table I and [Kristoffersson et al., 1987; Formiga et al., 1988; Roback et al., 1991]). DGAP025 showed a decreased occipital-frontal circumference (OFC) and height, whereas the Chd2+/m mouse showed a runt phenotype with reduced body fat. Reduced fetal growth might be attributable to dysregulation of a key regulator of early fetal development. Haploin-sufficiency of insulin-like growth factor 1 receptor (IGF1R) has been proposed as a possible factor for growth retardation [Nagai et al., 2002]. IGF1R, however, is unlikely to be the responsible gene in DGAP025 as it maps telomeric to CHD2 in 15q26.3. The relationship, if any, between the other phenotypic features of DGAP025 (viz., stigmata of hyperandrogenism, high arched palate, and learning problems) and the embryonic murine pattern of expression (facial area and forebrain) remains to be determined. Of note, both DGAP025 and Chd2+/m females show phenotypic features (hyperandrogenism and endometrial hyperplasia, respectively) that could be attributed to an abnormal anovulatory endocrinological state. Elevation of serum testosterone levels, however, could not be confirmed in Chd2+/m female mice (data not shown). This potential difference in phenotypic expression between mice and human, however, may be a reflection of basic differences in their respective reproductive physiology. It is also possible that further investigation of CHD2 in DGAP025 and our mouse model may expand the definition of CHARGE syndrome.

In conclusion, we mapped a developmentally critical CHD gene to 15q26.1 in the human, a region that has been implicated in multiple congenital anomalies, and we report similar features in a Chd2 mutant mouse model. Taken together with known disruptions and mutations in another chromatin remodeling gene family member (CHD7), these data suggest a potential role for CHD2 in embryonic development, possibly through alteration of chromatin structure and subsequent gene expression. This first demonstration of CHD2 mutation leading to abnormal development of the spine and other organs, in both man and mouse, provides the basis for future studies to explore further growth and development, as well as to study further the origins of the CHARGE syndrome. Identification of similar individuals with breakpoints, deletions, or mutations involving CHD2 and phenotypes similar to those observed in DGAP025 and our mouse model will clarify further the contributions of CHD2 and chromatin regulation to embryonic development.


We are indebted to Robert E. Eisenman for technical assistance and to Amy Bosco, Heather L. Ferguson, and Chantal Kelly for their expertise as genetic counselors for the Developmental Genome Anatomy Project. We are most grateful to Dr. Cynthia Morton for the helpful conversations during this project and for reviewing this manuscript. We would also like to thank kindly Dr. Jay C. Leonard of the NIGMS Cell Repository, Coriell Cell Repositories, Coriell Institute for Medical Research for his assistance with and support of the Developmental Genome Anatomy Project. The authors wish to acknowledge the following support of the research by the National Institutes of Health (T32 HL007627 to S.K.; GM061354 to A.H.L., and B.J.Q.) and University of Tennessee seed funds (to S.V.).


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