Identification of a mutation causing deficient BMP1/mTLD proteolytic activity in autosomal recessive osteogenesis imperfecta

Abstract

Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C-terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI.

Figure 1

Clinical and radiological features of the patients studied in this report. A: Family pedigree showing parental consanguinity. The proband is indicated with an arrow. B: Severe bone deformities in both affected sibs. C–D: Panels illustrate severe kyphoscoliosis (C) and relatively large thorax, small abdomen and large umbilical hernia (arrow) in the proband (D). E: X-ray of the spine of the proband showing S-curve scoliosis of thoracic and lumbar spine and platyspondyly. F: X-ray of the left leg of the proband showing serpentine bowing and osteoporosis of tibia and fibula. G: X-ray of the right humerus showing healed fractures, angulations, and lack of bone modeling in the proband. H: X-ray of the pelvis and both femora of patient II-6 showing multiple healed fractures, with bowing, and lack of bone modeling.

Figure 2

Identification of a missense mutation in BMP1 in individuals with AR-OI. A: Exon–intron structure of the two alternative spliced isoforms (BMP1 and mTLD) transcribed from the BMP1 locus. B: Sequence analysis of genomic DNA from a control (ctrl), a heterozygous individual (ht), and one of the patients (p) showing the c.747C>G transversion in BMP1 exon 6 (red and squared nucleotide). C: Multiple sequence alignment of a fragment from the catalytic domain of different astacin-like metalloproteases from diverse organisms showing conservation of the F249 residue labeled in red. The lower cluster of sequences corresponds to protein members of the BMP1/tolloid-like subgroup of astacin-like metalloproteases both from vertebrate and invertebrate organisms. Aaa_Asta, astacin from the crayfish A. Astacus (P07584), Tpa_My01 myosinase from giant squid Todarodes pacificus (Q8IU46), Cel_Nas13 metalloproteinase from Caenorhabditis elegans, (Q20191), Qu_Cam-I from quail (AAA20842.1), Hu_Ovast, human ovastacin (Q6HA08), Hu_MEPα (Q16819) and Hu_MEPβ (Q16820), human meprins α and β, Hvu_Hmp1 (AAA92361.2) and Hvu_Hmp2 (AAD33860.1) from Hydra vulgaris, Spu_Span from sea urchin (P98068), Lpo_Astm from the horseshoe crab Limulus polyphemus (B4F320), Ola_Hce1 (NP_001188427.1) and Ola_Lce1 (NP_001098292.1) from medaka fish Oryzias latipes, Dr_tld, Drosophila tolloid (NP_524487.2), Ate_tll, tolloid-like peptidase from the spider Achaearanea tepidariorum (Q75UQ6), Ci_Bmp1 from Ciona intestinalis (NP_001071840.1), Da_Bmp1a from Danio rerio (NP_001035126.1), Xe_Bmp1 from Xenopus laevis (AAI70427.1), Mu_Bmp1 from Mus musculus (NP_033885.2), Hu_BMP1/mTLD, Hu_TLL1 and Hu_TLL2 human BMP1/TLD-like proteases (NP_001190.1/NP_006120.1, NP_036596.3 and NP_036597.1). For most of the proteins shown here, a wider sequence alignment of the catalytic domain and information about substrate specificity have been described previously [Bond and Beynon, 1995; Guevara et al., 2010].

Figure 3

Phe249Leu impairs type I procollagen C-propeptide protease activity of BMP1/mTLD. A: Diagram representing unprocessed type I procollagen and intermediate variants following removal of the N or C-terminal propeptides. LF antibodies are shown underneath their corresponding epitope. B: Western blot analysis of supernatants from fibroblasts derived from patients II-3 and II-6, their heterozygous sibling (II-2), and an unrelated normal control (C) incubated with the indicated LF antibodies to monitor proteolytic processing of proα1(I). For each sample the volume of supernatant loaded per total amount of protein in the cell extracts of the corresponding culture was the same (15 μl, 14 μl, 15 μl, and 18 μl were loaded for samples C, II-3, II-6, and II-2, respectively). Analysis of proα1(I) variants is shown in the upper panel and free PICP analysis with LF-42 is in the lower panel. Compared to controls, patients have lower levels of pNα(I) and free PICP, while pCα1(I) is accumulated. Slight impairment of PICP-processing is detected in the heterozygous individual II-2. Quantification of the pCα1(I)/pNα1(I) ratio in each lane using densitometry data from two independent LF-68 blots is shown in the graph on the right. C: Western blot analysis of supernatants run in parallel and probed with the indicated antibodies demonstrating the propeptide composition of each of the intermediately processed proα1(I) bands. D: Western blot analysis of supernatants from NIH3T3 cell cultures overexpressing wild type mTLD (mTLD-wt) or Phe249Leu mTLD (mTLD-mut). Cells infected with the empty retroviral vector (pBABE-puro) were used as control (Control). 30 μl of supernatant from each culture with the same number of cells were loaded in each lane. Both LF-68 and LF-42 antibodies detected a dramatic decrease in unprocessed proα1(I) that is converted into a smaller size C-terminally processed variant when mTLD-wt is overexpressed. Since LF-9 does not crossreact with the murine N-propeptide, we could not use this antibody, and hence, the processed variant detected by LF-68 is assumed to correspond to pNα1(I) because of its size and because it is not recognized by LF-42. The amount of mTLD-wt and mTLD-mut in the media of the corresponding culture was demonstrated by reprobing the upper LF-blots with anti-BMP1. E: Western blot of supernatants from a control individual (C) and patient II-3 fibroblast cultures infected with retroviral vectors as in panel D and probed against LF antibodies. Only expression of mTLD-wt strongly improves PICP-protease activity both in patient and control cells as demonstrated by the augmented pNα1(I)/pCα1(I) ratio and reduction of unprocessed proα1(I). Levels of mTLD variants in the media were monitored with anti-BMP1.