The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD)

Abstract

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.

Figure 1

GDD phenotype and mutations. A, Affected female from Japanese family at age 16 years; facial deformity characteristic of the GDD phenotype is shown. B, Three-dimensional image of computed tomography. The image shows jaw lesions occupying alveolar processes. Projections around teeth (asterisk) are metal artifacts caused by teeth fillings. C, D, Radiographs of upper and lower limbs showing diaphyseal cortical thickening of tubular bones (arrows). E, Physical map of the GDD critical region and genomic structure of GDD1. Exons (filled boxes) and UTRs (gray box) are shown. F, Sequence analysis showing heterozygous T-to-C and T-to-G changes in the codon for Cys³⁵⁶ in affected members of the Japanese and African American families, respectively. SSCP patterns show cosegregation of mutant alleles (mu) with the disease phenotype. Individuals available for analyses are indicated (arrows).

Figure 2

Characterization of the human GDD1 protein. A, The hydrophobic profile of the human GDD1 protein, which indicates that it contains eight potential transmembrane domains. B, Predicted membrane topology of the human GDD1 protein. The locations of the N-glycosylation sites (projections), the eight cysteine residues absolutely conserved in all species (C), the potential ER retention signal (gray box), and the mutated cysteine residue are shown. C, ClustalW multiple sequence alignment of GDD1 orthologs. Protein similarity between the GDD1 orthologs is relatively high, with the human protein 79%, 56%, 40%, and 41% identical to the mouse, zebrafish, fruit fly, and mosquito, respectively. The alignment shows that the eight cysteine residues in the putative extracellular loops are absolutely conserved in all species (highlighted in black). The predicted transmembrane domains (boxed), the putative N-glycosylation sites (red), and the potential ER retention signal (blue) are indicated. Identical (*), strictly conserved (:), and conserved (.) sites are indicated below the ClustalW alignment of GDD1 orthologs.

Figure 3

Expression analysis of GDD1 mRNA in human and mouse tissues. A, A human Multiple Tissue Northern Blot (Clontech), hybridized with a ³²P-labeled 581-bp fragment of hGDD1 cDNA (upper panel). The blot was rehybridized with ³²P-labeled β-actin cDNA as a loading control (lower panel). B, RT-PCR analysis for hGDD1 mRNA in human tissues, normal human osteoblasts, and normal human periodontal ligament cells. We used osteoblasts isolated from jawbone specimens (hOB1–4) by use of the explant culture method (Hahn et al. 1988), osteoblasts from femur (hOB5 [Cambrex]), and periodontal ligament cells (hPDL) isolated from extracted tooth by use of the explant culture method. These cells were maintained in α-minimum essential medium containing 10% fetal bovine serum, 50 μg/ml of L-ascorbic acid, 50 U/ml penicillin, and 50 μg/ml streptomycin, and used at passage 3–4 for extraction of total RNA. A 348-bp fragment of hGDD1 cDNA was amplified by use of primers from exons 9 and 12. PCR was performed by use of Platinum Taq (Invitrogen) at 95°C for 2 min, followed by 30 or 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. A 452-bp fragment of hGAPDH cDNA was amplified as an internal control. C, Relative expression of mGDD1 (normalized to mGAPDH) in mouse tissues. A real-time, two-step RT-PCR analysis was performed by use of SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7900HT system (Applied Biosystems). Total RNA was isolated from various tissues of mouse aged 8 wk and reverse transcribed into random-primed first-strand cDNA. Specific PCR primer sets for mGDD1 and mGAPDH were designed to avoid amplification of genomic DNA. The relative quantity was determined by the standard curve method, in which the standard curves were generated by 10-fold serial dilution of mouse skeletal muscle cDNA. The normalized values were derived from the ratio of the relative quantity of mGDD1 for each sample to that of mGAPDH for that sample. Data are presented as the means ± SD of three independent experiments.

Figure 4

Expression of the hGDD1 protein in COS-7 cells. A, Western blot analysis to confirm expression of the wild-type and mutant hGDD1 proteins. Whole-cell lysates from COS-7 cells transfected with each plasmid expression vector containing the wild-type (hGDD1-V5) or mutant (C356R or C356G) hGDD1 cDNA tagged with V5 epitope at its 3′ end were subjected to reducing SDS-PAGE, and western blot analysis was performed by use of anti-V5 antibody (Invitrogen). For control transfection, pcDNA6/lacZ-V5 (Invitrogen) was used to represent the expression of an unrelated exogenous protein. B, Cellular localization of the wild-type hGDD1 protein. V5-tagged hGDD1 protein (hGDD1-V5) formed a distinct reticular pattern around the nucleus (green). Costaining with antibody against the calreticulin ER-specific marker (red) showed colocalization with the hGDD1 protein (yellow). C, Immunofluorescent staining with anti-V5 antibody, which shows that the only cells overexpressing each mutant hGDD1 protein decrease cell adhesion and change the cell morphology to a round shape.