Mutations in MMP9 and MMP13 Determine the Mode of Inheritance and the Clinical Spectrum of Metaphyseal Anadysplasia Ekkehart Lausch, Romy Keppler, Katja.

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Mutations in MMP9 and MMP13 Determine the Mode of Inheritance and the Clinical Spectrum of Metaphyseal Anadysplasia Ekkehart Lausch, Romy Keppler, Katja Hilbert, Valerie Cormier-Daire, Sarah Nikkel, Gen Nishimura, Sheila Unger, Jürgen Spranger, Andrea Superti-Furga, Bernhard Zabel The American Journal of Human Genetics Volume 85, Issue 2, Pages 168-178 (August 2009) DOI: 10.1016/j.ajhg.2009.06.014 Copyright © 2009 The American Society of Human Genetics Terms and Conditions

Figure 1 Clinical and Radiographic Presentation of MAD (A and B) Patient 1 (index patient IV.2 of family A) at the age of (A) 2 yrs, 3 mo and (B) 4 yrs, 3 mo. Radiographs demonstrate spontaneously regressive metaphyseal irregularities (arrows) of radius, ulna, tibia, and femur; the spine is normal. (C) The growth curve of patient 1 (red line) shows a transient growth deceleration maximal in the second year of life. (D) Genua vara and metaphyseal fraying in dominant (patient 5, index patient IV.2 of family B at the age of 1 yr, 8 mo) and recessive (patient 12, index patient II.2 of family E in Figure 2B at the age of 1 yr, 1 mo) MAD are indistinguishable. Patient numbers refer to Table 1 and Figure 2. The American Journal of Human Genetics 2009 85, 168-178DOI: (10.1016/j.ajhg.2009.06.014) Copyright © 2009 The American Society of Human Genetics Terms and Conditions

Figure 2 Pedigrees of MAD Families Abridged pedigrees of familes with (A) dominant and (B) recessive MAD are shown. Numbers of individuals correspond to Table 1; numbers in parentheses indicate individuals from whom no DNA was available. Symbol notations are as follows: squares, males; circles, females; open symbols, unaffected individuals; filled symbols, affected mutation carrier of the nucleotide and predicted amino acid change indicated on top; hatched symbols, likely affected individuals. Symbols with a slash through them indicate deceased individuals. Index patients are marked with a red arrow. The American Journal of Human Genetics 2009 85, 168-178DOI: (10.1016/j.ajhg.2009.06.014) Copyright © 2009 The American Society of Human Genetics Terms and Conditions

Figure 3 Concentration and Activity Levels of MMP9 and MMP13 in MAD Patients (A and B) MMP13 activity is not detectable in the serum (A), and MMP9 activity is reduced in the plasma (B), of patients with dominant MAD (MMP13 p.F55S, n = 3; MMP13 p.M72T, n = 3) in comparison to unaffected controls (n = 10). Specific MMP activity was quantified via a fluorogenic peptide substrate cleavage assay. Samples were measured in quadruplicates, and p values were obtained with a two-tailed Student's t test for the comparison of means (horizontal line). (C) Both proMMP13 and proMMP9 are strongly reduced in immunoblot analysis of whole-cell lysates from early-passage fibroblasts of patient 1 with dominant MAD (MMP13 p.F55S) as compared with an age-matched unaffected control; equal loading is demonstrated by reprobing with an antibody directed against β-actin. (D) The plasma of patients with recessive MAD lacks MMP9 activity (MMP9 c.21T>A, p.M1K, n = 2), and heterozygous carriers (n = 2) show a significant reduction in comparison to unaffected controls (n = 10). MMP9 activity was quantified via a fluorogenic peptide substrate cleavage assay. Samples were measured in triplicates and p values were obtained with a two-tailed Student's t test for the comparison of means (horizontal line). (E) No lower-sized bands indicative of initiation at downstream start codons are visible in immunoblot analysis of whole-cell lysates from fibroblasts transfected with minigene constructs bearing the recessive-MAD-associated mutation MMP9 c.21T>A, p.M1K. In wild-type constructs, proMMP9 is readily detectable with an antibody directed against the carboxy terminus. Transfection efficiency was similar, as monitored by fluorescence microscopy of EGFP expressed from an internal ribosome entry site downstream of MMP9; the promoter region includes 1644 bp (construct_1) and 762 bp (construct_2) upstream of the MMP9 start codon. The American Journal of Human Genetics 2009 85, 168-178DOI: (10.1016/j.ajhg.2009.06.014) Copyright © 2009 The American Society of Human Genetics Terms and Conditions

Figure 4 Functional Analysis of Disease-Associated MMP13 Mutations in HEK Cells (A) Schematic diagram of the genomic structure of the MMP13 gene, illustrating the position of MAD-associated mutations and protein domains. MMP13 encodes a 471 amino acid (aa) preproprotein including a 19 aa signal sequence (S) removed upon secretion, a 84 aa prodomain (Pro) maintaining latency of the proenzyme, and a 166 aa catalytic domain (Cat) connected by an 11 aa hinge peptide (H) to the 191 aa hemopexin-like domain (Hpx). The contribution of MMP13 exons to each protein domain is indicated by color. Within the catalytic domain, a conserved motif contains three histidine residues that ligate the active site Zn2+. The prodomain regulates enzymatic activity through the molecular interaction between the thiol group of cysteine 77 with the zinc ion in the catalytic center, modeled in the ribbon diagram of proMMP13 structure. In addition to the zinc-binding amino acids C77 (the “switching” cysteine), H203, H207, and H213, the atomic structure and relative positions of disease-associated F55 and M72 are shown. For simplicity, only the Zn2+ of the catalytic core and the central Ca2+ of the hemopexin-like domain are represented, as red and green spheres, respectively. (B) All dominant-MAD-associated mutations cause a strong reduction of the proMMP13-specific signal in immunoblot analysis of whole-cell lysates from human embryonic kidney cells transfected with expression constructs bearing disease-associated MMP13 mutations; there is no apparent difference between the individual dominant-MAD- and SEMDMO-associated mutations. Protein stability is normal for the mutation p.H213N associated with recessive MAD and the missense mutation p.E204A ablating enzymatic activity. Transfection efficiency and mRNA stability was similar for all constructs, as analyzed in parallel by quantitative reverse transcriptase-PCR (not shown); equal protein loading is documented by reprobing the blot with an antibody directed against β-actin. (C) Collagenase activity for native collagen type II is reduced to background levels by all disease-associated MMP13 missense mutations, similar to the catalytically inactive mutant p.E204A. Conditioned supernatants of transfected cells were activated with 1 mM APMA for 1 hr. No collagenolysis was detected without activation. Relative collagenase activity was normalized to recombinant MMP13 and total protein content. Bars represent the mean of triplicates, and error bars indicate the SDM. The American Journal of Human Genetics 2009 85, 168-178DOI: (10.1016/j.ajhg.2009.06.014) Copyright © 2009 The American Society of Human Genetics Terms and Conditions

Figure 5 Transcatalytic Intracellular Degradation of MMP9 by Autoactivated MMP13 (A) Dominant-MAD-associated MMP13 mutations cause a strong reduction of both MMP13- and MMP9-specific signals in immunoblot analysis of whole-cell lysates from human embryonic kidney cells cotransfected with expression constructs bearing disease-associated MMP13 mutations and wild-type MMP9; loss-of-function mutations do not affect the stability of either protein. (B) Dominant MMP13 mutations lead to autoactivation and intracellular degradation of both MMP13 and MMP9; MMP stability is restored by additional inactivation of the catalytic center through the missense mutation p.E204A. Constructs bearing compound mutations were analyzed by immunoblotting. (C) Transcatalysis reduces enzymatic activity of secreted MMPs. Dominant MMP13 mutations cause a strong decrease of gelatinase activity in conditioned supernatants of cotransfected cells, whereas MMP9 activity is not altered by the recessive-MAD-associated mutant p.H213N. Bars represent the mean of triplicates, and error bars indicate the SDM. (D) Additional ablation of enzymatic activity in dominant MMP13 mutants by site-directed mutagenesis brings secreted gelatinase activity back to levels measured in MMP9 wild-type transfectants. Conditioned supernatants were activated with 1 mM APMA for 16 hr. Bars represent the mean of triplicates, and error bars indicate the SDM. The American Journal of Human Genetics 2009 85, 168-178DOI: (10.1016/j.ajhg.2009.06.014) Copyright © 2009 The American Society of Human Genetics Terms and Conditions