Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Abstract

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.

Figure 1.

ATP6i gene mutations. Electropherograms of the ATP6i gene in healthy donors (left) and osteopetrotic patients (right). In patient 1, boxes indicate the site of G deletion. In patients 2 to 4, arrows indicate the sites of base transversion or transition. Note that patients 1, 2, and 4 are homozygous, patient 3 is heterozygous.

Figure 2.

Restriction cuts sorted by MwoI enzyme. Analysis refers to patient 1. Top: Wild-type (left) and patient (right) sequences, spanning nucleotides 1073 to 1558/1557 of exons 10 to 12, and the positions of the gcnnnnn/nngc cuts by the MwoI restriction enzyme. Bottom: Control and patient fragments sorted by MwoI cuts resolved by ethidium-bromide 2.5% agarose gel electrophoresis and ultraviolet transillumination. Underlined, physiological cut sites by the MwoI enzyme; bold, additional cut-site in patient; g (bold and italics), G deleted in patient.

Figure 3.

Western blot analysis of a3 and TIRC7 proteins. Proteins were extracted from peripheral blood mononuclear cells of control and patient 1, as described in Materials and Methods, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting was performed using an antiserum against the C-terminal peptide of the a3 V-ATPase subunit, also recognizing the TIRC7 splice variant of the ATP6i gene (middle). Long-term film exposition allowed detection in these cells also of the modestly expressed a3 protein (top). Bottom: The constitutive protein actin used as an internal control.

Figure 4.

X-ray analysis. Radiographs of patient 1. A: Sclerosis of the base of the skull (arrow). Obliterated cavities and irregularly shaped extremities (arrows) in femurs and tibias (B) and in left forearm (C). Endobone appearance (arrow) in left hand phalanges (D). Similar patterns were noted in patients 2 to 4.

Figure 5.

Iliac crest bone biopsies. H&E routine staining of paraffin sections of an age-matched control (A, E) and of patients 1 (B, F), 3 (C, G), and 4 (D, H). Of note, irregular and massive primary trabeculae (b) surrounded by abnormal fibrous tissue (f), and layered by several osteoclasts (large arrows) are apparent in patients. In contrast, mature trabeculae (b) surrounded by normal hematopoietic bone marrow (bm) and no osteoclasts are observed in the control subject. Small arrows, Howship lacunae; arrowheads, unresorbed cartilage. Original magnifications: ×10 (A–D); ×40 (E–H).

Figure 6.

TRAP staining. Iliac crest bone biopsy paraffin section of patient 1 histochemically stained for the osteoclast-specific marker TRAP. Massive trabeculae (b) are layered by a prominent number of TRAP-positive osteoclasts (arrows) and surrounded by fibrous tissue (f). Original magnification, ×40.

Figure 7.

Immunohistochemical detection of the a3 V-ATPase subunit in bone biopsies. A: The bone biopsy of a patient affected by ATP6i-independent osteopetrosis was used as positive control for reaction to the anti-a3 antiserum. The arrow shows a group of osteoclasts labeled with brownish color because of positive reaction to the antiserum revealed by a horseradish-conjugated secondary antibody. B: Bone biopsy of patient 3 showing a3-negative osteoclasts (arrows) counterstained with hematoxylin. Original magnifications, ×40.

Figure 8.

In vitro bone marrow osteoclasts. Bone marrow was harvested from patient 1 and cultured in vitro by standard procedure (see Materials and Methods). A: Phase-contrast micrograph of a 1-day cultured osteoclast showing motile morphology and prominent lamellipodia (arrow). B: TRAP histochemical staining of 1-week culture showing several large, TRAP-positive, osteoclasts (arrows) against a background of TRAP-negative stromal cells. C: Bone section showing a small pit (arrow) excavated by an osteopetrotic osteoclast. Original magnifications: ×63 (A); ×25 (B and C).

Figure 9.

In vitro peripheral blood osteoclasts. Osteoclasts were generated in vitro from controls and patient 1 as described in Materials and Methods. A: Control culture showing several TRAP-positive osteoclasts. B: Patient’s culture showing similar number of osteoclasts with a strongly positive TRAP reaction. C: Patient’s culture at higher magnification showing strong TRAP positivity. D: Patient’s post-BMT osteoclasts showing less pronounced TRAP activity relative to the pre-BMT osteoclasts depicted in C. E: Pits (arrow) excavated by control osteoclasts showing regular morphology and intense toluidine-blue staining. F: Pits (arrow) excavated by the patient’s osteoclasts showing irregular edges and pale staining relative to control pits. Original magnifications: ×25 (A and B); ×40 (C–F).

Figure 10.

Cytoskeletal and adhesion properties of peripheral blood osteoclasts. Osteoclasts were generated in vitro from controls and patient 1 and processed for fluorescence microscopy as described in Materials and Methods. Actin rings (rhodamine-conjugated phalloidin staining) in control (A) and patient’s (B) osteoclasts. αVβ3 receptor in control (C) and patient’s (D) osteoclasts. c-Src (E) and PYK2 (F) in patient’s osteoclasts. No differences were noted between control and osteopetrotic controls. Original magnifications, ×40.