NMIHBA results from hypomorphic PRUNE1 variants that lack short-chain exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

Generation of Fbn1 conditional null mice implicates the extracellular microfibrils in osteoprogenitor recruitment.

Loss-of-function experiments in mice have yielded invaluable mechanistic insights into the pathogenesis of Marfan syndrome (MFS) and implicitly, into the multiple roles fibrillin-1 microfibrils play in the developing and adult organism. Unfortunately, neonatal death from aortic complications of mice lacking fibrillin-1 (Fbn1(-/-) mice) has limited the scope of these studies. Here, we report the creation of a conditional mutant allele (Fbn1(fneo) ) that contains loxP sites bordering exon1 of Fbn1 and an frt-flanked neo expression cassette downstream of it. Fbn1(fneo/+) mice were crossed with FLPeR mice and the resulting Fbn1(Lox/+) progeny were crossed with Fbn1(+/-) ;CMV-Cre mice to generate Fbn1(CMV-/-) mice, which were found to phenocopy the vascular abnormalities of Fbn1(-/-) mice. Furthermore, mating Fbn1(Lox/+) mice with Prx1-Cre or Osx-Cre mice revealed an unappreciated role of fibrillin-1 microfibrils in restricting osteoprogenitor cell recruitment. Fbn1(Lox/+) mice are, therefore, an informative genetic resource to further dissect MFS pathogenesis and the role of extracellular fibrillin-1 assemblies in organ development and homeostasis.

Differential effects of alendronate and losartan therapy on osteopenia and aortic aneurysm in mice with severe Marfan syndrome.

Reduced bone mineral density (osteopenia) is a poorly characterized manifestation of pediatric and adult patients afflicted with Marfan syndrome (MFS), a multisystem disorder caused by structural or quantitative defects in fibrillin-1 that perturb tissue integrity and TGFß bioavailability. Here we report that mice with progressively severe MFS (Fbn1(mgR/mgR) mice) develop osteopenia associated with normal osteoblast differentiation and bone formation. In vivo and ex vivo experiments, respectively, revealed that adult Fbn1(mgR/mgR) mice respond more strongly to locally induced osteolysis and that Fbn1(mgR/mgR) osteoblasts stimulate pre-osteoclast differentiation more than wild-type cells. Greater osteoclastogenic potential of mutant osteoblasts was largely attributed to Rankl up-regulation secondary to improper TGFß activation and signaling. Losartan treatment, which lowers TGFß signaling and restores aortic wall integrity in mice with mild MFS, did not mitigate bone loss in Fbn1(mgR/mgR) mice even though it ameliorated vascular disease. Conversely, alendronate treatment, which restricts osteoclast activity, improved bone quality but not aneurysm progression in Fbn1(mgR/mgR) mice. Taken together, our findings shed new light on the pathogenesis of osteopenia in MFS, in addition to arguing for a multifaceted treatment strategy in this congenital disorder of the connective tissue.

Extracellular microfibrils control osteoblast-supported osteoclastogenesis by restricting TGF{beta} stimulation of RANKL production.

Mutations in fibrillin-1 or fibrillin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that fibrillin-1 and fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGFß and bone morphogenetic protein signals. Here, we describe in vivo and ex vivo experiments that implicate the fibrillins as negative regulators of bone resorption. Adult Fbn2(-/-) mice display a greater than normal osteolytic response to locally implanted lipopolysaccharide-coated titanium particles. Although isolated cultures of Fbn2(-/-) preosteoclasts exhibited normal differentiation and activity, these features were substantially augmented when mutant or wild-type preosteoclasts were co-cultured with Fbn2(-/-) but not wild-type osteoblasts. Greater osteoclastogenic potential of Fbn2(-/-) osteoblasts was largely accounted for by up-regulation of the Rankl gene secondary to heightened TGFß activity. This conclusion was based on the findings that blockade of TGFß signaling blunts Rankl up-regulation in Fbn2(-/-) osteoblasts and bones and that systemic TGFß antagonism improves locally induced osteolysis in Fbn2(-/-) mice. Abnormally high Rankl expression secondary to elevated TGFß activity was also noted in cultured osteoblasts from Fbn1(-/-) mice. Collectively our data demonstrated that extracellular microfibrils balance local catabolic and anabolic signals during bone remodeling in addition to implying distinct mechanisms of bone loss in Marfan syndrome and congenital contractural arachnodactyly.

Functional biology of the Steel syndrome founder allele and evidence for clan genomics derivation of COL27A1 pathogenic alleles worldwide.

Previously we reported the identification of a homozygous COL27A1 (c.2089G>C; p.Gly697Arg) missense variant and proposed it as a founder allele in Puerto Rico segregating with Steel syndrome (STLS, MIM #615155); a rare osteochondrodysplasia characterized by short stature, congenital bilateral hip dysplasia, carpal coalitions, and scoliosis. We now report segregation of this variant in five probands from the initial clinical report defining the syndrome and an additional family of Puerto Rican descent with multiple affected adult individuals. We modeled the orthologous variant in murine Col27a1 and found it recapitulates some of the major Steel syndrome associated skeletal features including reduced body length, scoliosis, and a more rounded skull shape. Characterization of the in vivo murine model shows abnormal collagen deposition in the extracellular matrix and disorganization of the proliferative zone of the growth plate. We report additional COL27A1 pathogenic variant alleles identified in unrelated consanguineous Turkish kindreds suggesting Clan Genomics and identity-by-descent homozygosity contributing to disease in this population. The hypothesis that carrier states for this autosomal recessive osteochondrodysplasia may contribute to common complex traits is further explored in a large clinical population cohort. Our findings augment our understanding of COL27A1 biology and its role in skeletal development; and expand the functional allelic architecture in this gene underlying both rare and common disease phenotypes.

Fibrillin-rich microfibrils - structural and instructive determinants of mammalian development and physiology.

Fibrillin-rich microfibrils have emerged recently as an informative model system in which to study fundamental questions related to extracellular matrix biology and connective tissue pathophysiology. As a result, these studies have yielded novel clinical concepts and promising therapeutic strategies. These achievements have been based on the realization from studies of genetically engineered mice that mutations in fibrillin-rich microfibrils impair both the structural integrity of connective tissues and signaling events by TGF-beta/BMP superfamily members. In this view, fibrillin-rich microfibrils represent architectural assemblies that specify the concentration and timely release of local effectors of morphogenesis and tissue remodeling, in addition to conferring structural integrity to individual organ systems. This review summarizes the evidence supporting our current understanding of the structural and instructive roles that fibrillin-rich microfibrils play during embryonic development and in human diseases.

Caffey disease: new perspectives on old questions.

The autosomal dominant form of Caffey disease is a largely self-limiting infantile bone disorder characterized by acute inflammation of soft tissues and localized thickening of the underlying bone cortex. It is caused by a recurrent arginine-to-cysteine substitution (R836C) in the α1(I) chain of type I collagen. However, the functional link between this mutation and the underlying pathogenetic mechanisms still remains elusive. Importantly, it remains to be established as to how a point-mutation in type I collagen leads to a cascade of inflammatory events and spatio-temporally limited hyperostotic bone lesions, and how structural and inflammatory components contribute to the different organ-specific manifestations in Caffey disease. In this review we attempt to shed light on these questions based on the current understanding of other mutations in type I collagen, their role in perturbing collagen biogenesis, and consequent effects on cell-cell and cell-matrix interactions.

Drug-based therapies for vascular disease in Marfan syndrome: from mouse models to human patients.

Marfan syndrome is a congenital disorder of the connective tissue with a long history of clinical and basic science breakthroughs that have forged our understanding of vascular-disease pathogenesis. The biomedical importance of Marfan syndrome was recently underscored by the discovery that the underlying genetic lesion impairs both tissue integrity and transforming growth factor-beta regulation of cell behavior. This discovery has led to the successful implementation of the first pharmacological intervention in a connective-tissue disorder otherwise incurable by either gene-based or stem cell-based therapeutic strategies. More generally, information gathered from the study of Marfan syndrome pathogenesis has the potential to improve the clinical management of common acquired disorders of connective-tissue degeneration.

Extracellular regulation of transforming growth factor beta and bone morphogenetic protein signaling in bone.

Systemic and local factors regulate the activity of osteoblasts and osteoclasts during bone growth and remodeling by modulating a complex array of intracellular signaling events. Recent genetic evidence implicates extracellular fibrillin assemblies (microfibrils and elastic fibers) in imparting contextual specificity to endogenous transforming growth factor-beta and bone morphogenetic protein ligands in the forming and mature skeleton. The evidence is based on the characterization of the cellular and molecular mechanisms responsible for the unique bone manifestations that characterize mouse models of Marfan syndrome and congenital contractural arachnodactyly. Collectively, the studies indicate that fibrillin assemblies play a key role both in establishing morphogen gradients within the developing limbs and in restricting growth factors activity in remodeling bones. The latter finding is likely to improve the design of more effective therapeutic interventions in osteoporosis and of bioengineering formulations for the repair of bone fractures.

Fibrillin-1 and -2 differentially modulate endogenous TGF-ß and BMP bioavailability during bone formation.

Extracellular regulation of signaling by transforming growth factor (TGF)-ß family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-ß and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2-null (Fbn2(-/-)) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2(-/-) phenotype is accounted for by improper activation of latent TGF-ß that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1(-/-) mice exhibit improper latent TGF-ß activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-ß and BMP signaling.