Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia

Abstract

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we describe a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)(2)D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16-month-old brother and his 12.5-year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development.

Figure 1

Panel A: Pedigree of the kindred with autosomal recessive hypophosphatemia (ARHP). The index case is indicated by the arrow; solid black symbols depict affected individuals; half-filled symbols represent unaffected, obligate carriers, and healthy siblings shown to be carriers of the identified DMP-1 mutation; stippled boxes represent healthy family members, who were not investigated; /, deceased family members. Panel B: Age at the time of laboratory measurements of affected and unaffected individuals. *Age- and sex-specific reference ranges are given in parenthesis just below the each individual's values for PO_4,, ALP and TMP/GFR. Conversion factors: Ca, 1 mg/dl = 0.25 mmol/L; PO₄, 1 mg/dl = 0.323 mmol/L. Panel C: Confirmation of the identified single nucleotide deletion (c.485delT) by digestion of PCR-amplified genomic DNA (1619 bp) with the endonuclease EcoNI. The PCR product derived from the wild-type allele yields two bands of 397 and 1222 bp, respectively, while the product carrying the identified mutation is not reduced in size by digestion with EcoNI.

Figure 2

Nucleotide sequence analysis of DNA from a healthy control (upper), from the unaffected family member #8 carrying a heterozygous DMP-1 mutation in exon 6 (c.485Tdel), and from the affected family member #5 carrying a homozygous mutation.

Figure 3

Radiograph of the lower extremities of patient #6 at the age of 16 months before treatment (lower and upper left panel), which showed metaphyseal fraying, cupping and bowing. At the age of 9.5 years, after 2 years of treatment with oral phosphate and 1,25(OH)₂D, the radiographs continued to show some metaphyseal fraying and severe bowing of the femur as well as osteosclerosis of medial aspect of femur pelvic bones (right panel).

Figure 4

Radiographs of the left hand of patient #6 at the age of 16 months (Panel A) and at the age of 9.5 years (Panel B). The metatarsals and phalangial bones were short and broad, the distal phalanges were dysplastic, and ulna and fibula were broad (Panel B) and showed an increased thickness of the growth plates, some metaphyseal fraying, and bowing (Panel A). Patient #5 (Panel C) showed at the age of 11 years, broad bones and bowing of radius.

Figure 5

Panoramic radiographs of the three affected individuals (left panel), which show in comparison to the findings in an age-matched healthy controls (right panel), enlarged pulps, decreased dentin and enamel layers (*), as well as the absence of the lower third molar (**) in individual #2.

Figure 6

Retroalveolar radiographs of the two affected individuals #2 and #5 at the ages of 20 and 11 years, respectively (upper left and right panel). The films showed, in comparison to the findings in a healthy heterozygous carrier of the mutation (#1; lower left panel) and an 11 years old age-matched healthy controls (lower right panel), hypomineralization, enlarged pulps and decreased layers of dentin and enamel; patient #1 had one remaining deciduous tooth due to the absence of the root of the permanent tooth (*)

Figure 7

Panel A: Schematic presentation of the gene encoding DMP-1 showing the approximate locations of the DMP-1 mutation identified in this report (c.485delT) and of previously defined mutations (9, 19). Coding exons are shown as black boxes and non-coding exonic regions as white boxes. The approximate locations of the oligonucleotide primers (F, forward and R, reverse) that were used for DNA amplification are indicated. Panel B: Schematic representation of the wild-type DMP-1 protein, which undergoes extensive post-translational modifications (glycosylation and phosphorylation) resulting in a full-length 94 kDa protein that is cleaved into a 37 kDa (stippled bar) and a 57 kDa fragment (black bar) by subtilisin-like proprotein convertases (SPC); the locations of Nuclear Export Signal (NES) and of Nuclear Localization Signals-1, -2 and -3 (NLS-1, -2 and -3) are indicated above the schematic protein molecule; arrows points to the location of the identified mutation and of SPC cleavage site. Panel C: Partial amino acid sequences of wild-type and mutant DMP-1 proteins, number above indicates the amino acid residue; the novel unrelated amino acid sequence derived from the mutant allele that comprises 53 residues is shown in italics; the site for SPC cleavage in the wild-type protein is shown in underlined italics.