Identification of biallelic LRRK1 mutations in osteosclerotic metaphyseal dysplasia and evidence for locus heterogeneity

Abstract

Background: Osteosclerotic metaphyseal dysplasia (OSMD) is a unique form of osteopetrosis characterised by severe osteosclerosis localised to the bone ends. The mode of inheritance is autosomal recessive. Its genetic basis is not known.

Objective: To identify the disease gene for OSMD.

Methods and results: By whole exome sequencing in a boy with OSMD, we identified a homozygous 7 bp deletion (c.5938_5944delGAGTGGT) in the LRRK1 gene. His skeletal phenotype recapitulated that seen in the Lrrk1-deficient mouse. The shared skeletal hallmarks included severe sclerosis in the undermodelled metaphyses and epiphyseal margins of the tubular bones, costal ends, vertebral endplates and margins of the flat bones. The deletion is predicted to result in an elongated LRRK1 protein (p.E1980Afs*66) that lacks a part of its WD40 domains. In vitro functional studies using osteoclasts from Lrrk1-deficient mice showed that the deletion was a loss of function mutation. Genetic analysis of LRRK1 in two unrelated patients with OSMD suggested that OSMD is a genetically heterogeneous condition.

Conclusions: This is the first study to identify the causative gene of OSMD. Our study provides evidence that LRRK1 plays a critical role in the regulation of bone mass in humans.

Keywords: LRRK1 mutation; Lrrk1 deficient mouse; Osteosclerotic metaphyseal dysplasia; Whole exome sequencing.

Figure 1

LRRK1 mutation in patient 1. (A) Electropherograms in family 1. Homozygous deletion (c.5938-5944delGAGTGGT) in the patient (P1). The parents are heterozygous for the deletion. (B) LRRK1 protein structure and location of the mutation. The mutation causes a frame-shift within the seventh WD40 domain in the C-terminus of the LRRK1 protein. The additional 66 amino acids in the mutated protein are shown by a green box and green letters.

Figure 2

Radiographs of patient 1 at age 14 months. (A) Skull anteroposterior (A-P). Normal skull. (B) Lateral spine. No platyspondyly. Ribs are broad. (C) Left hand A-P. Broad and sclerotic metaphyses in the distal radius and ulna. Sclerotic metaphyses of metacarpals and phalanges. (D) Pelvis A-P. Marginal sclerosis of the ilia. Broad and sclerotic metaphyses of the proximal femur.

Figure 3

Radiographs of the Lrrk1 knock-out (KO) mouse at 8 weeks compared with the wild-type (WT). (A) Skull. There is no difference in the bone density of the skull and teeth. (B) The height of the vertebral bodies of the KO mouse is decreased with marginal sclerosis and the ribs are broad and sclerotic. (C) Lower leg. In the KO mouse, the metaphyses of the long tubular bones are severely sclerotic and undermodelled. There is also shortening of the long bones. (D) Feet. In the KO mouse, the metaphyses of the short tubular bones are sclerotic and undermodelled, while the bone density of the diaphyses is decreased.

Figure 4

Overexpression of the LRRK1 mutant protein does not rescue bone resorption defect of Lrrk1-deficient osteoclasts. (A) Comparable expression of FLAG-tagged wild-type (LRRK1) and mutant (LRRK1mt: p.E1980Afs*66) human LRRK1 proteins. 293T cells were infected with lentiviral particles and cultured in fresh medium for 12–36 h after infection. Total cellular extracts were subjected to western blot using anti-FLAG antibody. (B) Efficient transduction of lentiviral particle in osteoclast precursors. Primary monocytes derived from the spleen of Lrrk1 knockout mice were seeded in 48-well plates containing bone slices and infected with lentiviral particles. The transduction efficiency is monitored by green fluorescent protein (GFP) expression in the cells that grow at the edges of bone slices 48 h after transduction. (C) Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and resorptive pit formation on bone slices (×100). Left panel: lentivirus transduced monocytes were differentiated on bone slices for 6 days, followed by TRAP staining. Right panel: transduced monocytes in another 48-well plate were differentiated on bone slices for 10 days. The cells were removed and pits were stained with haematoxylin. (D) Quantitative data of TRAP-positive multinuclear cells (left) and pit formation (right) from three independent experiments, with three bone slices each time (N=9). Data are the means±SEM and analysed using Student’s t test. *p<0.01.