Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development

Abstract

The generation and homeostasis of bone tissue throughout development and maturity is controlled by the carefully balanced processes of bone formation and resorption. Disruption of this balance can give rise to a broad range of skeletal pathologies. Lethal osteosclerotic bone dysplasia (or, Raine syndrome) is an autosomal recessive disorder characterized by generalized osteosclerosis with periosteal bone formation and a distinctive facial phenotype. Affected individuals survive only days or weeks. We have identified and defined a chromosome 7 uniparental isodisomy and a 7p telomeric microdeletion in an affected subject. The extent of the deleted region at the 7p telomere was established by genotyping microsatellite markers across the telomeric region. The region is delimited by marker D7S2563 and contains five transcriptional units. Sequence analysis of FAM20C, located within the deleted region, in six additional affected subjects revealed four homozygous mutations and two compound heterozygotes. The identified mutations include four nonsynonymous base changes, all affecting evolutionarily conserved residues, and four splice-site changes that are predicted to have a detrimental effect on splicing. FAM20C is a member of the FAM20 family of secreted proteins, and its mouse orthologue (DMP4) has demonstrated calcium-binding properties; we also show by in situ hybridization its expression profile in mineralizing tissues during development. This study defines the causative role of FAM20C in this lethal osteosclerotic disorder and its crucial role in normal bone development.

Figure 1.

a, G banding of the pseudodicentric chromosome 7. b, FISH staining with probes directed against chromosome 7 centromere (pink) and chromosome 7p telomere (blue). The upper panel displays a normal karyotype from the affected child’s father, and the lower panel is from the affected child, with an absence of signal from the chromosome 7pter probe. c, Schematic representation, from contig NT_079592 (GenBank), of the terminal region of chromosome 7p, including the location of the deleted 7pter FISH probe. Marker D7S2563 delimits the deleted region, which contains five potential transcriptional units.

Figure 2.

Sequence chromatograms, predicted effects, and locations of the mutations identified in the six additional affected individuals. Mutations are present in affected individuals as homozygotes, apart from two compound heterozygotes (c914+5G→C, c1401-1G→A, designated by a number sign [#], and 1094G→A, c1322-2A→G, designated by an asterisk [*]). Statistical analysis of each of the splice-site variants is displayed above the chromatograms (blue path). Also shown is a schematic representation of multiple-species sequence alignment of the FAM20C protein sequences affected by respective variants (red path).

Figure 3.

a, In situ hybridization of the mouse FAM20C orthologue (DMP4) on E15 mouse, with light microscopy and dark field images showing the developing vertebral column with expression on the mineralizing surface of somites (red arrow) and limb girdle (blue arrow). b, Light microscopy and dark field images showing expression of FAM20C (DMP4) in osteoblasts (yellow arrow), odontoblasts (red arrow), and ameloblasts (blue arrow) of the alveolar bone in a P1 mouse. c, PCR analysis of Fam20C (DMP4) mRNA from multiple tissues from a P5 SD rat (1 = spleen, 2 = skeletal muscle, 3 = bone, and 4 = tooth germ) (left). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal control. Right, With northern-blot analysis, 20 μg of total RNA isolated from P5 rats (1 = spleen, 2 = skeletal muscles, 3 = bones, and 4 = tooth germs), was electrophoresed, blotted, and probed for FAM20C (DMP4). The blot was stripped and reprobed for GAPDH.