Chronic Joint Pain and Dysfunction via the MMP13 Gene
- Summary and Pricing
- Clinical Features and Genetics
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A clinical study involving 13 patients diagnosed with MAD (belonging to five families) showed that all individuals harbored causative sequence variants in the MMP13 gene (Lausch et al. 2009).
Genetic bone disorders are characterized by defective growth and modeling of the spine, long bones (e.g., femur, tibia, or both), and cartilage, as well as joint pain. These disorders include several skeletal diseases such as spondyloepimetaphyseal dysplasias (SEMDs), metaphyseal anadysplasia (MAD), and osteoarthritis (OA). SEMDs generally feature moderate to severe metaphyseal changes, mild epiphyseal alterations, rhizomelic shortening of the lower limbs, pear-shaped vertebrae during childhood, and deformities in the genu varum or genu valgum (knock knee) secondary to bowing of the legs, resulting in short-trunk disproportionate dwarfism (Wynne-Davies and Hall 1982; Gertner et al. 1997; Isidor et al. 2013). The defects in modeling often improve during adolescence, although the affected individual usually remains shorter than age-matched children. The results of biochemical tests for skeletal homeostasis are usually normal, whereas radiologic and histopathologic assessment often indicates a primary abnormality involving growth plate development. MAD pertains to the early-onset regression category of metaphyseal dysplasia (Sobreira et al. 2014). A MAD diagnosis is often established during the first few months after birth when the long bones show irregular distal metaphyses. Individuals with MAD also present with hypoplastic femoral necks, with the edges of the metaphyses positioned in a vertical fashion (Verloes et al. 1990). The femoral shaft of individuals with MAD is generally bowed (MacDermot et al. 1991). Similar to SEMDs, MAD anomalies dissipate when a child reaches the age of two years old. MAD is also characterized by slight shortness and varus deformities involving the lower limbs, but stature remains unaffected. OA is a debilitating joint disorder that involves the mechanical disruption of the cartilage matrix, changes in bone mass, and localized inflammation (Mitchell et al. 1996). Inflammatory cytokines such as IL-1, IL-6, and TNF-alpha are expressed at the articular joint, causing inflammation and in turn stimulating the production of various matrix metalloproteinases (MMPs) (Goldring et al. 2011). Radiologic examination is considered the most reliable diagnostic and monitoring test for SEMDs, MAD, and OA.
Chronic degenerative joint pain and dysfunction such as that observed in SMED, MAD, and OA is an autosomal dominant disorder caused by pathogenic sequence variants in the matrix metalloproteinase 13 (MMP13) gene. The MMP13 gene has been localized to chromosome 11q22.2 and consists of 10 exons that encode for a 471-amino acid polypeptide containing a sequence motif specific to the collagenase subfamily (Tardif et al. 1997).
The MMP13 gene is also known as collagenase-3 (CLG3), based on its identification as the third member of the collagenase family (Freije et al. 1994). MMP13 is commonly expressed by chondrocytes and is capable of degrading a wide range of collegenous and noncollagenous extracellular matrix macromolecules such as types 1-4 and 9-11 collagens, gelatin, fibrinogen, and laminin (Mitchell et al. 1996). Unlike most human MMPs, its expression is restricted in normal tissues and is often upregulated in pathologic conditions (Reboul et al. 1996; Goldring et al. 2011). Detection of MMP-13 is therefore often observed in situations where rapid collagen remodeling is required, which include fetal bone development and postnatal bone development (Dancevic and McCulloch 2014). In chronic disorders, MMP-13 is generally expressed at sites of excessive degradation of the extracellular matrix, which include the cartilage of patients with osteoarthritis, rheumatoid arthritis, and cancer (Freije et al. 1994; Mitchell et al. 1996; Reboul et al. 1996).
To date, a total of five pathogenic missense sequence variants have been reported to cause chronic degenerative joint pain and dysfunction (Kennedy et al. 2005; Lausch et al. 2009; Bonafe et al. 2014; Li et al. 2014). A small study involving two brothers diagnosed with MAD showed that both individuals possessed pathogenic sequence variants in the MMP13 gene (Li et al. 2015). In a study involving a three-generation family consisting of 12 family members, 5 affected individuals were determined to harbor causative sequence variants in the MMP13 gene (Bonafe et al. 2014). In a larger 4-generation family study comprising 32 family members (13 affected and 11 unaffected kin, and 8 unaffected spouses), 13 individuals carried pathogenic sequence variants in the MMP13 gene (Kennedy et al. 2005).
Full gene sequencing of all 10 coding exons of the MMP13 gene is performed. The full coding region of each exon plus ~20 bp of flanking non-coding DNA on either side are sequenced. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.
Indications for Test
The ideal candidate for MMP13 testing should have been previously diagnosed with SMED, MAD, or OA by using radiologic examination.
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- Genetic Counselor Team - email@example.com
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- Bonafé L, Liang J, Gorna MW, Zhang Q, Ha-Vinh R, Campos-Xavier AB, Unger S, Beckmann JS, Le Béchec A, Stevenson B, Giedion A, Liu X, Superti-Furga G, Wang W, Spahr A, Superti-Furga A. 2014. MMP13 mutations are the cause of recessive metaphyseal dysplasia, Spahr type. American Journal of Medical Genetics A. 164A(5): 1175-1179. PubMed ID: 24648384
- Dancevic CM, McCulloch DR. 2014. Current and emerging therapeutic strategies for preventing inflammation and aggrecanase-mediated cartilage destruction in arthritis. Arthritis Research and Therapy. 16(5): 429. PubMed ID: 25606593
- Freije JM, Díez-Itza I, Balbín M, Sánchez LM, Blasco R, Tolivia J, López-Otín C. 1994. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. Journal of Biological Chemistry. 269(24): 16766-16763. PubMed ID: 8207000
- Gertner JM, Whyte MP, Dixon PH, Pang JT, Trump D, Pearce SH, Wooding C, Thakker RV. 1997. Linkage studies of a Missouri kindred with autosomal dominant spondyloepimetaphyseal dysplasia (SEMD) indicate genetic heterogeneity. Journal of Bone and Mineral Research. 12(8): 1204-1209. PubMed ID: 9258750
- Goldring MB, Otero M, Plumb DA, Dragomir C, Favero M, El Hachem K, Hashimoto K, Roach HI, Olivotto E, Borzì RM, Marcu KB. 2011. Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. European Cells and Materials. 21: 202-220. PubMed ID: 21351054
- Goldring MB, Otero M, Plumb DA, Dragomir C, Favero M, El Hachem K, Hashimoto K, Roach HI, Olivotto E, Borzì RM, Marcu KB. 2011. Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. Europeran Cells and Materials. 21:202-220.
- Isidor B, Geffroy L, de Courtivron B, Le Caignec C, Thiel CT, Mortier G, Cormier-Daire V, David A, Toutain A. 2013. A new form of severe spondyloepimetaphyseal dysplasia: Clinical and radiological characterization. American Journal of Medical Genetics A. 161A(10): 2645-26451. PubMed ID: 23956136
- Kennedy AM, Inada M, Krane SM, Christie PT, Harding B, López-Otín C, Sánchez LM, Pannett AA, Dearlove A, Hartley C, Byrne MH, Reed AA, Nesbit MA, Whyte MP, Thakker RV. 2005. MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (SEMD(MO). Journal of Clinical Investigations. 115(10): 2832-2842. PubMed ID: 16167086
- Lausch E, Keppler R, Hilbert K, Cormier-Daire V, Nikkei S, Nishimura G, Unger S, Spranger J, Superti-Furga A, Bernhard Z. 2009. Mutations in MMP9 and MMP13 determine the mode of inheritance and the clinical spectrum of metaphyseal anadysplasia. The American Journal of Human Genetics. 85: 168-178. PubMed ID: 19615667
- Lausch E, Keppler R, Hilbert K, Cormier-Daire V, Nikkel S, Nishimura G, Unger S, Spranger J, Superti-Furga A, Zabel B. 2009. Mutations in MMP9 and MMP13 determine the mode of inheritance and the clinical spectrum of metaphyseal anadysplasia. American Journal of Human Genetics. 85(2): 168-178.
- Li D, Weber DR, Deardorff MA, Hakonarson H, Levine MA. 2015. Exome sequencing reveals a nonsense mutation in MMP13 as a new cause of autosomal recessive metaphyseal anadysplasia. European Journal of Human Genetics. 23(2): 264-266. PubMed ID: 24781753
- MacDermot KD, Winter RM, Wigglesworth JS, Strobel S. 1991. Short stature/short limb skeletal dysplasia with severe combined immunodeficiency and bowing of the femora: report of two patients and review. Journal of Medical Genetics. 28(1): 10-17. PubMed ID: 1999827
- Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, Geoghegan KF, Hambor JE. 1996. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. Journal of Clinical Investigations. 97(3): 761-768. PubMed ID: 8609233
- Mitchell PG1, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, Geoghegan KF, Hambor JE. 1996. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. Journal of Clinical Investigations. 97(3): 761-768.
- Reboul P, Pelletier JP, Tardif G, Cloutier JM, Martel-Pelletier J. 1996. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. Journal of Clinical Investigations. 97(9): 2011-2019. PubMed ID: 8621789
- Sobreira N, Modaff P, Steel G, You J, Nanda S, Hoover-Fong J, Valle D, Pauli RM. 2015. An anadysplasia-like, spontaneously remitting spondylometaphyseal dysplasia secondary to lamin B receptor (LBR) gene mutations: further definition of the phenotypic heterogeneity of LBR-bone dysplasias. American Journal of Medical Genetics A. 167A(1): 159-163. PubMed ID: 25348816
- Tardif G, Pelletier JP, Dupuis M, Hambor JE, Martel-Pelletier J. Cloning, sequencing and characterization of the 5 PubMed ID: 9173871
- Verloes A, Van Maldergem L, de Marneffe P, Dufier JL, Maroteaux P. 1990. Microspherophakia-metaphyseal dysplasia: a PubMed ID: 2395168
- Wynne-Davies R, Hall C. 1982. Two clinical variants of spondylo-epiphysial dysplasia congenita. Journal of Bone and Joint Surgery British Volume. 64(4): 435-441. PubMed ID: 6807992
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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