Journal of Human Genetics (2014) 59, 631–637;

Functional characterisation of a novel mutation affecting the catalytic domain of MMP2 in siblings with multicentric osteolysis, nodulosis and arthropathy.

Jacopo Azzollini1, Davide Rovina1, Cristina Gervasini1, Ilaria Parenti1, Alessia Fratoni1, Maria Vittoria Cubellis2, Amilcare Cerri3, Luca Pietrogrande4 and Lidia Larizza1,5

1Medical Genetics, Department of Health Sciences, University of Milan, Milan, Italy; 2Department of Biology, University ‘Federico II’, Naples, Italy; 3Dermatologic Unit, Departmentof Health Sciences, San Paolo Hospital, University of Milan, Milan, Italy; 4Operative Unit of Orthopaedics and Traumatology, Department of Health Sciences, San Paolo Hospital,University of Milan, Milan, Italy and 5Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Milan, Italy.



Multicentric osteolysis, nodulosis and arthropathy (MONA) is a rare autosomal recessive disorder. To date, 13 mutations of the matrix metalloproteinase 2 (MMP2) gene have been detected in 26 patients with MONA and other osteolytic syndromes. Here, we describe the molecular and functional analysis of a novel MMP2 mutation in two adult Italian siblings with MONA. Both siblings displayed palmar-plantar subcutaneous nodules, tendon retractions, limb arthropathies, osteolysis in the toes and pigmented fibrous skin lesions. Molecular analysis identified a homozygous MMP2 missense mutation in exon 8 c.1228G4C (p.G410R), not detected in 260 controls and predicted by several bioinformatic tools to be pathogenic. By protein modelling, the mutant residue was predicted to affect the main chain conformation of the catalytic domain. Gelatin zymography, the gold standard test for MMP2 function, of serum-free conditioned medium from G410R-MMP2-expressing human embryonic kidney (HEK) cells, showed a complete loss of gelatinolytic activity. The novel mutation is located in the catalytic domain, as are 3 (p.E404K, p.V400del and p.G406D) of the other 13 MMP2 mutations described to date; however, p.G410R underlies a phenotype that is only partially overlapping that of other MMP2 exon 8 mutation carriers. Our results further delineate the complexity of genotype–phenotype correlations in MONA, broaden the repertoire of reported MMP2 mutation and enhance the comprehension of the protein motifs crucial for MMP2 catalytic activity.

PMID: 25273674



Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases with important functions in many pathophysiological processes, including organ growth, wound healing, bone remodeling, ischemic response, tumor invasion and metastasis. Mutations affecting MMP genes are also responsible for Mendelian osteolytic disorders: alterations of the MMP2 gene underlie Multicentric Osteolysis, Nodulosis, and Arthropathy (MONA)/Torg Syndrome (OMIM #259600), while mutations of MMP14 were found in individuals affected by Winchester syndrome (OMIM #277950). The MONA/Torg syndrome, subject of our study, is an extremely rare autosomal recessive condition, characterised by prominent involvement of the bones, including diffuse osteoporosis, osteolysis and arthropathies with progressive multiple joint contractures. The patients’ phenotype is broad and highly variable and might feature subcutaneous fibrotic nodules, cutaneous pigmented lesions, corneal opacities, cardiac defects, growth impairment and facial dysmorphisms.

We described two Italian siblings, born to consanguineous parents, displaying infancy-onset progressive diffuse reduction of bone mineral density, followed by bone resorption at hands and feet, progressive flexion contractures at the upper limbs with multiple arthropathies, painful subcutaneous nodules and cutaneous alterations (hyperpigmented fibrotic striae, and achromic plaques). The elder sister also presented signs not detected in her brother, such as dorsal scoliosis, relapsing corneal pterygium and a first-degree atrioventricular block without evident structural heart defects.

As sibs overall clinical characteristics were consistent, albeit not completely overlapping, with the features observed in Torg-MONA patients, we performed a mutation screening of the MMP2 gene, which allowed the identification of the novel homozygous mutation c.1228G>C (p.G410R) affecting exon 8, within a sequence encoding the protein catalytic domain.

Bioinformatic analysis predicted a damaging effect of the mutation on the protein function, as well as on its structure and stability. In order to confirm the expected functional derangement, following molecular cloning and transfection of wild type and G410R-mutant MMP2 into HEK cells, we performed gelatin zymography, the gold standard test for the assessment of MMP2 catalytic function, on the conditioned media containing either the normal or the altered protein. This assay, in combination with the MMP2 western blot analysis, allowed us to demonstrate that the mutation does not impair significantly the protein synthesis, but determines a complete loss of its enzymatic activity.

Previous studies reported only other 13 different MMP2 mutations, spread throughout the sequence of the gene: of note, although no mutational hotspot could be evidenced, 9 out of 14 mutations are clustered within the catalytic domain of the protein, with 4 amino acid changes sited in exon 8 (Fig. 1a).The other identified mutations appear to be equally distributed in all the protein domains, including prodomain, fibronectin II-like and hemopexin-like domains (Fig. 1b). With respect to the geographical distribution of patients, although MONA is considered a panethnic condition, most of the mutations have been observed in individuals from Mediterranean or nearby countries (Saudi Arabia); a founder effect, though, cannot yet be ascertained, due to the exiguous number of MMP2-mutated individuals identified so far (Fig. 1c). The mutations are private and, with only one exception, are in homozygous state; indeed consanguinity has been recorded in all families, except for a family from Korea and one from the US.

As highlighted in Figure 1a, the functional assessment, showing a complete loss of activity, was reported for 6 different mutations: 2 missense affecting residue 101, known to be crucial for the activation of the proenzyme, 3 nonsense/frameshift, localized in the catalytic, fibronectin II-like and hemopexin-like domains, and 1 missense mutation, affecting, likewise our mutation, exon 8.

The definition of genotype-phenotype correlations in MONA is thus challenging, because all functionally tested mutations seem to abolish MMP2 proteolytic activity, making it difficult to link the underlying molecular mechanism to the high variability of patients’ clinical features. Based on previous observations, some authors suggested that carriers of homozygous mutations within the MMP2 catalytic domain may display a distinct phenotype characterised by the absence of subcutaneous nodules, which, however, represent a prominent feature of our patients. Furthermore, we noticed that no congenital heart malformation has been found in patients with missense mutations/small in-frame deletions localised within the catalytic domain; this finding, though, needs further evidences to be confirmed. Of note, we also attested a certain degree of intrafamilial variability, as some relevant clinical features were identified in only one of the two siblings.

In conclusion, our study demonstrated the crucial functional role of R410 aminoacid residue in the protein enzymatic activity and expanded the restricted MMP2 mutation repertoire. We also highlighted the complexity of genotype–phenotype correlations in MONA, as well as the variable intrafamilial expressivity, thus further broadening and refining the clinical definition of the syndrome.


Figure 1. Overview of the reported MMP2 mutations. (a) Schematic view of the MMP2 gene (Genbankrefseq accession numbers: NG_008989, NM_004530) showing the position of all 14 reported mutations, including seven missense plus one in-frame deletion (blue frame), five truncating (red frame) and one splice site (pink frame); G410R is shown in red characters; mutations within dashed diagonal frames have been functionally tested. Exons are coloured according to the encoded protein domains as depicted. (b) X-ray crystallographic-derived structure of the pro-MMP-2 (Protein DataBank ID-1ck7) molecule showing, clockwise the fibronectin II-like, the hemopexin-like, the prodomain and the catalytic domain (1). (c) World map showing the geographical distribution of the 14 identified MMP2 mutations; attached frames indicate the two mutations found in a compound heterozygous patient.



  1. Morgunova, E., Tuuttila, A., Bergmann, U., Isupov, M., Lindqvist, Y., Schneider, G. et al. Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed. Science 284, 1667-1670 (1999).



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