Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia

Abstract

Metatropic dysplasia is a clinical heterogeneous skeletal dysplasia characterized by short extremities, a short trunk with progressive kyphoscoliosis, and craniofacial abnormalities that include a prominent forehead, midface hypoplasia, and a squared-off jaw. Dominant mutations in the gene encoding TRPV4, a calcium permeable ion channel, were identified all 10 of a series of metatropic dysplasia cases, ranging in severity from mild to perinatal lethal. These data demonstrate that the lethal form of the disorder is dominantly inherited and suggest locus homogeneity in the disease. Electrophysiological studies demonstrated that the mutations activate the channel, indicating that the mechanism of disease may result from increased calcium in chondrocytes. Histological studies in two cases of lethal metatropic dysplasia revealed markedly disrupted endochondral ossification, with reduced numbers of hypertrophic chondrocytes and presence of islands of cartilage within the zone of primary mineralization. These data suggest that altered chondrocyte differentiation in the growth plate leads to the clinical findings in metatropic dysplasia.

FIG. 1

Radiographs of nonlethal metatropic dysplasia cases. Case numbers are shown below each radiograph. Arrows identify some of the characteristic findings in metatropic dysplasia including marked platyspondyly with dense, wafer-like vertebral bodies (A–D) and metaphyseal abnormalities of the metacarpals (I).

FIG. 2

Two cases of neonatal lethal metatropic dysplasia. (A, B) Both infants have short limbs and small chest cavities. (C, D) Note the nodular appearance of the right ear of R09-35 in B. The left ear was similarly enlarged.

FIG. 3

Model of the TRPV4 molecule showing the locations of the implied sequence changes in the protein relative to the recognized protein domains.

FIG. 4

Basal currents through TRPV4 in metatropic dysplasia (A–C) and effects of stimulation with 2 μM 4α-PDD in WT TRPV4 (E), I331F (F), P799L (G) on the increase in the intracellular Ca²⁺ concentration [Ca²⁺]_i. A voltage step protocol was used activating TRPV4 channels consisting of 40 mV steps from −80 to +200 mV. Note that the constitutive channel activity of the mutant channels was higher than that of the wild-type channel (histogram in panel D). Panels E–G show the Ca²⁺ imaging data for activation of TRPV4 with 2 μM 4α-PDD. Note the increased basal Ca²⁺ level before the agonist stimulation. H: Histogram showing average increases in fluorescence ratio in response to 4α-PDD. Error bars represent means ±SE. Asterisk (*) indicates significant differences when compared with cells expressing wild-type TRPV4 (one-sided Student’s t-test, P <0.01).

FIG. 5

Morphology of the bones in metatropic dysplasia cases R08-023 and R09-035. A: Gross appearance of sectioned lower limb and histology of case R09-035. The femur and tibia are shortened with very little diaphyseal bone (arrow). There is extensive nodular overgrowth of disordered cartilage in the patella and at the ends of both long bones, which contain brown appearing bone marrow and bone. P, patella; DF, distal femur; PF, proximal femur; T, tibia. B: Low magnification of an H&E stained, paraffin-embedded whole section cut through the knee joint of case R09-035 and mounted on a 7.5 cm × 5 cm glass slide showing lack of growth plates, the nodular nature of the cartilaginous proliferation, and cortical bone (arrow) of the short femoral diaphyses. P, patella; F, femur; T, tibia; BM, bone marrow. C: Section showing the growth plate in case R09-035 illustrating the short hypertrophic zone and cartilage within the primary spongiosa (arrow). D: Growth plate from case R08-023 showing irregular primary spongiosa with trapped cartilage nodules (arrowhead) and a poorly organized hypertrophic zone (thin arrow). PS, primary spongiosa; H, hypertrophic zone. E: Nodules of cartilage (arrowheads) and woven bone (arrows) in case R09-035 arising in islands of mesenchymal cells. M, mesenchymal cell. F: H&E stained diaphyseal cortical bone from case R09-035 showing islands of immature woven bone (arrows) surrounded by normal lamellar bone.

FIG. 6

Tracheal cartilage in case R09-035. A: The cartilage in the trachea at the level of the thyroid gland is thickened by irregular nodules of cartilage separated by strands of mesenchymal cells. The adjacent tracheal mucosa (arrow) is unremarkable. B: Trabeculae of immature woven bone (arrows) are present within the foci of mesenchymal cell proliferation.