Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome - PubMed (original) (raw)

doi: 10.1016/j.ajhg.2008.09.005. Epub 2008 Oct 2.

Ingo Kurth, Fei Lan, Irene Meliciani, Wolfgang Wenzel, Soo Hyun Eom, Gil Bu Kang, Georg Rosenberger, Mustafa Tekin, Metin Ozata, David P Bick, Richard J Sherins, Steven L Walker, Yang Shi, James F Gusella, Lawrence C Layman

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Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome

Hyung-Goo Kim et al. Am J Hum Genet. 2008 Oct.

Abstract

CHARGE syndrome and Kallmann syndrome (KS) are two distinct developmental disorders sharing overlapping features of impaired olfaction and hypogonadism. KS is a genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia, and is most commonly due to KAL1 or FGFR1 mutations. CHARGE syndrome, a multisystem autosomal-dominant disorder, is caused by CHD7 mutations. We hypothesized that CHD7 would be involved in the pathogenesis of IHH and KS (IHH/KS) without the CHARGE phenotype and that IHH/KS represents a milder allelic variant of CHARGE syndrome. Mutation screening of the 37 protein-coding exons of CHD7 was performed in 101 IHH/KS patients without a CHARGE phenotype. In an additional 96 IHH/KS patients, exons 6-10, encoding the conserved chromodomains, were sequenced. RT-PCR, SIFT, protein-structure analysis, and in situ hybridization were performed for additional supportive evidence. Seven heterozygous mutations, two splice and five missense, which were absent in > or = 180 controls, were identified in three sporadic KS and four sporadic normosmic IHH patients. Three mutations affect chromodomains critical for proper CHD7 function in chromatin remodeling and transcriptional regulation, whereas the other four affect conserved residues, suggesting that they are deleterious. CHD7's role is further corroborated by specific expression in IHH/KS-relevant tissues and appropriate developmental expression. Sporadic CHD7 mutations occur in 6% of IHH/KS patients. CHD7 represents the first identified chromatin-remodeling protein with a role in human puberty and the second gene to cause both normosmic IHH and KS in humans. Our findings indicate that both normosmic IHH and KS are mild allelic variants of CHARGE syndrome and are caused by CHD7 mutations.

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Figures

Figure 1

Figure 1

CHD7 Domains and Positions of Mutations CHD7 structure with functional domains and the positions of five missense mutations and two splice-donor site mutations identified in IHH and KS patients are shown. The following abbreviations are used: chrom1, chromodomain 1; chrom2, chromodomain 2; SANT, SANT DNA binding domain; and LZD, leucine zipper domain. The function of CR1-CR3 and BRK domains are unknown. Three mutations affect chromodomains. Relative sizes and locations of domains are to scale.

Figure 2

Figure 2

CHD7 Exon Skipping in Two Patients (A) RT-PCR analysis of CHD7 exon-skipping event in a patient with IVS8+5G→A RT-PCR analysis confirms that the IVS8+5G→A mutation of CHD7 causes aberrant exon 8 skipping in an IHH patient. Splicing patterns are compared between a normal control and the mutant by cloned cDNA sequencing. An expected CHD7 product of 565 bp consisting of exons 4–10 is observed in the control (lane 2), whereas an abnormal product of 450 bp skipping exon 8 (115 bp) is observed in a patient with IVS8+5G→A (lane 3). Exon 9 nucleotide and out-of-frame AA sequence are depicted in blue. A 123 bp DNA marker is shown in the first lane. The aberrant exclusion of exon 8 (115 bp) in a patient with IVS8+5G→A is predicted to introduce a frameshift in the coding region and a subsequent premature termination codon at 16 AAs downstream from exon 8 skipping. In the truncated CHD7, 16 out-of-frame AA residues generated by the frameshift are depicted as a black bar at the end. The functional domains from N terminus to C terminus are depicted in color as follows: orange, chromodomains 1 and 2; navy blue, SNF2; sky blue, helicase; green, CR1, CR2, and CR3; deep green, BRK1 and BRK2; and red, leucine zipper domain. The SANT domain within CR3 was not depicted here. (B) RT-PCR analysis of CHD7 exon skipping event in a patient with IVS6+5G→C RT-PCR analysis shows that mutation IVS6+5G→C of CHD7 causes aberrant exon 6 skipping in a KS patient. Splicing patterns are compared between a normal control and the mutant by cloned cDNA sequencing. An expected CHD7 product of 576 bp consisting of exons 4–9 is observed in the control (lane 2), whereas an abnormal product of 510 bp skipping exon 6 (66 bp) is observed in a patient with IVS6+5G→C (lane 3). A 1 kb DNA marker is shown in the first lane. The abnormal exon 6 (66 bp) skipping in a patient with IVS6+5G→C causes a 66 bp in-frame deletion of 22 AAs. From chromodomain 1 comprising 66 AAs, 16 residues are deleted. Protein structure of the deleted region is depicted in yellow and chromodomains 1 and 2 in orange.

Figure 3

Figure 3

Protein Sequence Alignment and Structure Modeling of CHD7 (A) Multiple protein-sequence alignment of CHD7 with its orthologs. The positions of residues affected by missense mutations in IHH/KS patients are marked by asterisks and bold letters in available CHD7 animal orthologs. H55 and S834 are evolutionarily fully conserved, whereas A2789 and P2880 are highly conserved. K2948 shows relative conservation. Human CHD7 N-terminal residues 1–1423 are missing in the predicted rat Chd7 protein (NP_001101376). Blue shading represents the invariant residues that match the consensus exactly, and pink shading shows partial matching. (B) CHD7 structure modeling. (B1 and B2) Shown are alternate views of the model of the 300 AA C-terminal region of CHD7 based on the 3DJURY model, which results from the alignment of this region with that of mdia1 gbd-fh3 in complex with rhoc (1z2c/B). Regions of the model that were predicted solely on the basis of homology in 3DJURY are shown in green, but additional information was used to substantiate the model. There is an additional long consecutive region where EMBOSS found substantial sequence similarity to the CHD7 sequence—these regions are marked in dark blue. Regions that, in addition to the alignment, agree in their secondary structure with the consensus prediction of PHYRE are shown in light brown. Finally, we found a region that, in addition to the alignment and secondary structure, exhibits a motif commensurate with the leucine zipper (bright blue). Sites and side chains of the three mutations Lys2948Glu, Pro2880Leu, and Ala2789Thr are shown in red for better visibility. All mutation sites lie in highly flexible loop regions. (B1) and (B2) show the same model rotated 180° around the vertical axis though the center of the molecule. (B3) Shown is the model of the 155 AA region around Ser834Phe (AA 795–950). The side chain of Phe834 is highlighted in dark blue for better visibility. In addition to the secondary and tertiary structure, side chains of the AA in a 6 Å radius of Phe834 are shown in the standard color coding (carbon green, oxygen red, sulfur yellow). Of particular importance is the strong stacking interaction of Phe834 (blue) with the ring of Tyr881 (green) directly underneath.

Figure 4

Figure 4

Chd7 Expression during Murine Development The following abbreviations are used: OP, olfactory placode; GCL, granule cell layer of the cerebellum; OE, olfactory epithelium; aPG, anterior pituitary gland; pPG, posterior pituitary gland; SC, spinal cord; CA1, CA1 region of the hippocampus; CA3, CA3 region of the hippocampus; DG, dentate gyrus; CO, cochlea; E, eye; and NP, nasal pit. (A) DIG-labeled whole-mount in situ hybridization using a Chd7 antisense probe shows high expression in the olfactory placode at E10.5. (B and C) Strong labeling of neuroepithelial structures is seen at E11, shown in higher magnification in (C). (D and E) Expression of Chd7 in the olfactory epithelium, developing cortex, cochlea, spinal cord, and anterior pituitary gland at E14. [35S]-UTP-labeled in situ hybridizations are shown in (E). Inset shows magnification of the pituitary gland. (F–I) Expression of Chd7 in the adult brain: Singular DIG-positive cells are found scattered within hypothalamic nuclei of the preoptic area (F). One cluster of cells within the medial preoptic area consistently stained positively for Chd7 (G). DIG-labeled in situ hybridizations on 12 μm cryosections show expression in the cerebellum (H). The inset in (H) shows an overview of the adult brain with a [35S]-UTP labeled Chd7 antisense probe. The hippocampal region also stains positive for Chd7 (I).

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