Geleophysic dysplasia (from the Greek geleos
, “happy”, and physis
, “nature”; OMIM 231050) is a rare autosomal recessive disorder characterized by short stature, small hands and feet with broad proximal phalanges, cone-shaped epiphyses, delayed bone age and shortened tubular bones1
(). Other features include a ‘happy’ face with upturned corners of the mouth, hepatomegaly, restricted joint mobility, skin thickening and muscle hypertrophy. Affected individuals present with progressive cardiac disease with dilation and thickening of the pulmonary, aortic or mitral valves, often leading to death before 5 years of age. Tracheal stenosis responsible for severe respiratory problems is frequent. Geleophysic dysplasia biopsy material has shown abundant lysosome-like vacuoles in hepatocytes, fibroblasts and macrophages suggestive of a storage disorder2,3
(Supplementary Fig. 1
online). However, biochemical analyses have not detected an enzymatic deficiency or precisely characterized the accumulated material.
Figure 1 Clinical and radiological manifestations of geleophysic dysplasia in individual 3. (a) Note the facial features, including round face, long philtrum, thin upper lip and very short hands and feet (individual 3 at age 9 years). (b) Hand X-rays of individual (more ...)
Homozygosity mapping in four consanguineous geleophysic dysplasia families of French Polynesian, Moroccan, Algerian and Pakistani origins (families 1–4; ) showed linkage of the underlying gene to chromosome 9q34.2–q34.3 in a 619-kb interval (Zmax
= 4.52 at θ
= 0 at the gt-AL590710 locus). A recombination event in family 4 defined the proximal boundary of the region (gt-AL593848), and a second recombinant in the same family defined the distal boundary (gt-AL593186). Because geleophysic dysplasia belongs to the group of acromelic dysplasias that also includes the autosomal recessive form of Weill-Marchesani syndrome caused by ADAMTS10
mutations, we considered ADAMTSL2, the product of the ADAMTSL2
(ADAMTS-like 2) gene, as a likely candidate among the seven genes located within the critical interval (). ADAMTSL2 belongs to a large superfamily containing 19 ADAMTS proteases and at least five ADAMTS-like proteins. ADAMTS proteases are secreted enzymes with a conserved organization that includes a metalloprotease domain and an ancillary domain containing one or more thrombospondin type 1 repeats (TSR). Some ADAMTS proteases participate in extracellular matrix (ECM) turnover in arthritis, and others are involved in procollagen and von Willebrand factor maturation or in angiogenesis4
. The ADAMTS-like subfamily comprises proteins homologous to the ADAMTS ancillary domains but lacking the protease domain and hence lacking catalytic activity. ADAMTSL-1 and ADAMTSL-3 proteins are closely related secreted glycoproteins5,6
, whereas ADAMTSL-2 has a different domain structure7
. Their functions are unknown.
Figure 2 Genetic mapping of the locus involved in geleophysic dysplasia. (a) Pedigrees of geleophysic dysplasia families. (b) The region of homozygosity is located between gt-AL593848 and gt-AL593186; seven genes are located in this region. (c) Exon and intron (more ...)
The 18 coding exons of ADAMTSL2
encode a 951-residue protein composed of a signal peptide, a TSR, a cysteine-rich module, a spacer module, an N-glycan–rich module, six additional TSRs and a PLAC module7
. Direct sequence analysis of the coding region of ADAMTSL2
in families 1–4 and in two additional affected individuals detected four distinct missense mutations and a nonsense mutation ( and ). The missense mutations consistently involved residues that are conserved across species and across the ADAMTSL family members. The mutations clustered in the regions encoding the cysteine-rich domain (three mutations) and TSR6 (two mutations) ( and ). We identified the same mutation in the two families from North Africa. All mutations cosegregated with the disease and were absent in chromosomes from ethnicity-matched controls. We did not find any mutations in family 4 (RNA was not available for this family).
ADAMTSL2 mutations identified in individuals with geleophysic dysplasia
To correlate the expression pattern of ADAMTSL2 with the clinical manifestations of geleophysic dysplasia, we performed in situ hybridization experiments on tissue derived from a human fetus at 35 weeks of gestation. In agreement with the clinical manifestations observed in individuals with geleophysic dysplasia, we found ADAMTSL2 mRNA expression in heart, skin and pulmonary arteries (). In the heart, we detected ADAMTSL2 mRNA in cardiomyocytes (). We also observed strong expression in skin epidermis, dermal blood vessels () and in the tracheal wall (). ADAMTSL2 mRNA was present in developing skeletal muscle (), and we observed strong expression in the pulmonary arteries and developing bronchioles of the lung (). Because geleophysic dysplasia is a chondrodysplasia, we also performed in situ hybridization of the proximal femoral growth plate. On longitudinal sections, we found a high level of ADAMTSL2 mRNA expression in chondrocyte columns in the hypertrophic and reserve zones ().
Figure 3 In situ hybridization analysis of ADAMTSL2 mRNA expression in a human fetus at 35 weeks of gestation. The purple staining indicates sites of RNA hybridization. (a) Transverse section through the heart showing specific signal in cardiomyocytes. (b) In (more ...)
We tested the functional consequences of the geleophysic dysplasia mutations using myc-tagged wild-type and mutant ADAMTSL2 (R113H, P147L and G811R) in parallel transfections of HEK293F cells. Protein blot analyses after 48 h of transfection confirmed that wild-type ADAMTSL2 was secreted into the medium7
. Although there was not a statistically significant alteration in cellular levels of each mutant protein, we found significantly reduced secretion of each mutant protein compared to wild-type protein (). Thus, the mutant proteins are likely to be synthesized, but it is possible that they are misfolded, which may interfere with their efficient secretion. One can also consider an increased turnover or an altered function of secreted mutant proteins.
Figure 4 Functional consequences of ADAMTSL2 mutations. (a) Characterization of wild-type and mutant ADAMTSL2 proteins. Conditioned medium (top) from transfected HEK293F cells or cell lysate (bottom) was normalized to either secreted IgG (medium) or actin (cell (more ...)
To define the molecular pathway in which ADAMTSL2 might participate, we used full-length ADAMTSL2 lacking its signal peptide as bait to screen for putative ADAMTSL2-binding proteins in a yeast two-hybrid screen of a human muscle cDNA library. Among several positive clones, one clone contained a 783-bp insert that corresponded to residues 673–933 of human latent TGF-β–binding protein 1 (LTBP-1). We verified the interaction of LTBP-1S, the dominant and more widely distributed form, with ADAMTSL2 using immunoprecipitation ().
LTBP-1 has a major role in the storage of latent TGF-β in the ECM and regulates its availability. TGF-β is secreted from cells either as a dimeric small latent complex (SLC) comprising noncovalently bound latency-associated propeptide and mature TGF-β and/or as a large latent complex (LLC) comprising SLC bound to LTBP-1, LTBP-3 or LTBP-4 through a TGF-β binding motif8
. LTBPs are structurally related to fibrillins9
, and LTBP-1 is an associated component of tissue microfibrils10
. The activation of the TGF-β–SMAD signaling pathway is tightly regulated through various ECM proteins involved in the release of LLC from microfibrils and ECM and in the release of TGF-β from latency-associated propeptide11,12
To determine the functional significance of this interaction, we quantified active and total TGF-β in the cultured medium of fibroblasts from individuals with geleophysic dysplasia and age- and passage-matched control skin fibroblasts by ELISA. There was a tenfold higher amount of total TGF-β in the cultured medium of geleophysic dysplasia fibroblasts than in cultured medium from control fibroblasts (P < 0.0003) (). Whereas active TGF-β represented 85% and 92% of total TGF-β in the medium of individuals 6 and 2, respectively, active TGF-β represented only 7% of total TGF-β in control medium (). Consistent with this observation, protein blot analysis of geleophysic dysplasia cell lysates showed fivefold greater amounts of phosphorylated SMAD2/3 (phospho-SMAD2/3) than control cell lysates (). Moreover, immunostaining with antibodies to phospho-SMAD2 (anti-phospho-SMAD2) showed an increase in nuclear-localized phospho-SMAD2/3 in two geleophysic dysplasia skin fibroblasts ().
Figure 5 Analysis of TGF-β signalling pathway in geleophysic dysplasia fibroblasts. (a) Quantification of total (gray bars) and active (black bars) TGF-β in the conditioned medium of fibroblasts from individuals with geleophysic dysplasia and control (more ...)
TGF-β is a growth factor that regulates cell proliferation, migration, differentiation and survival in a context-dependent fashion and whose activity is tightly regulated through the ECM. TGF-β signaling is crucial in various developmental and homeostatic processes. Enhanced TGF-β signaling has been shown to be a major pathophysiologic factor in Marfan syndrome and related disorders, including Loeys-Dietz syndrome and Camurati-Engelmann disease, all characterized by tall stature and thin habitus13–15
. In Marfan syndrome, the diminished ability of microfibrils to bind the LLC presumably results in elevated active TGF-β. Enhanced TGF-β signaling has been demonstrated in various tissues (lung, heart and aorta) in mouse models of Marfan syndrome16,17
. In Loeys-Dietz syndrome (which is caused by TGFBR1/R2
loss-of-function mutations), the paradoxical increase of TGF-β signaling has been demonstrated only in the aortic wall14
. In addition, the observation of more diffuse and severe arterial disease in Loeys-Dietz syndrome suggests that the disease mechanisms in FBN1
mutations are different18
. Similarly, the increased bone density observed only in Camurati-Engelmann disease (due to mutations in the latency-associated peptide of TGF-β) supports bone-specific consequences of enhanced TGF-β signaling in this disorder18
. In the context of geleophysic dysplasia, the observation of increased TGF-β signaling in fibroblasts from individuals with geleophysic dysplasia further illustrates the ECM dependence and complexity of the TGF-β–SMAD signaling pathway and demonstrates the existence of new physiological mechanisms regulating TGF-β action. Further-more, the findings of ADAMTS10
mutations in Weill-Marchesani syndrome, a condition related to geleophysic dysplasia, suggests that ADAMTSL2 is a component of a key regulatory network in the ECM that also contains these two proteins. Future studies will clarify the nature of the interactions between LTPB-1, ADAMTS10, FBN1 and ADAMTSL2 and how the lack of functional ADAMTSL2 results in the release of TGF-β. One can hypothesize that ADAMTSL2 acts as a cofactor that enhances or stabilizes binding of LLC to fibrillins. The tissue-specific consequences of ADAMTSL2
mutations also raise the intriguing possibility that other ADAMTS-like proteins may regulate the bioavailability of TGF-β in a site-specific manner dictated by their individual expression profiles. Ongoing studies should contribute to further understanding of the regulatory networks in the ECM and the context-dependent mechanisms leading to the activation of TGF-β signaling.