Antonella Forlino

New perspectives on osteogenesis imperfecta

A new paradigm has emerged for osteogenesis imperfecta as a collagen-related disorder. The more prevalent autosomal dominant forms of osteogenesis imperfecta are caused by primary defects in type I collagen, whereas autosomal recessive forms are caused by deficiency of proteins which interact with type I procollagen for post-translational modification and/or folding. Factors that contribute to the mechanism of dominant osteogenesis imperfecta include intracellular stress, disruption of interactions between collagen and noncollagenous proteins, compromised matrix structure, abnormal cell–cell and cell–matrix interactions and tissue mineralization. Recessive osteogenesis imperfecta is caused by deficiency of any of the three components of the collagen prolyl 3-hydroxylation complex. Absence of 3-hydroxylation is associated with increased modification of the collagen helix, consistent with delayed collagen folding. Other causes of recessive osteogenesis imperfecta include deficiency of the collagen chaperones FKBP10 or Serpin H1. Murine models are crucial to uncovering the common pathways in dominant and recessive osteogenesis imperfecta bone dysplasia. Clinical management of osteogenesis imperfecta is multidisciplinary, encompassing substantial progress in physical rehabilitation and surgical procedures, management of hearing, dental and pulmonary abnormalities, as well as drugs, such as bisphosphonates and recombinant human growth hormone. Novel treatments using cell therapy or new drug regimens hold promise for the future.

Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome

BACKGROUND Corticotropin-independent Cushings syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushings syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates.

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength.

The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties.

Use of the Cre/lox Recombination System to Develop a Non-lethal Knock-in Murine Model for Osteogenesis Imperfecta with an α1(I) G349C Substitution VARIABILITY IN PHENOTYPE IN BrtlIV MICE

We utilized the Cre/lox recombination system to develop the first knock-in murine model for osteogenesis imperfecta (OI). The moderately severe OI phenotype was obtained from an α1(I) Gly349 → Cys substitution in type I collagen, reproducing the mutation in a type IV OI child. We introduced four single nucleotide (nt) changes into murine col1a1 exon 23: the disease causing G→T transversion (nt 1546), an adjacent G→T change (nt 1551) to generate a GUC ribozyme cleavage site, and two transversions (nt 1567 C→A and nt 1569 C→G) to cause a Leu → Met substitution. We also introduced a 3.2-kilobase pair transcription/translation stop cassette in intron 22, flanked by directly repeating loxrecombination sites. After homologous recombination in ES cells, two male chimeras were obtained. Chimeras were mated with transgenic females expressing Cre recombinase to remove the stop cassette from a portion of the progenys cells. To generate mice with full expression of the Gly349 → Cys mutation, these offspring were then mated with wild-type females. Skeletal staining and bone histology of the F2 revealed a classical OI phenotype with deformity, fragility, osteoporosis and disorganized trabecular structure. We designate these mice BrtlIV (Brittle IV). BrtlIV mice have phenotypic variability ranging from perinatal lethality to long term survival with reproductive success. The phenotypic variability is not associated with differences in expression levels of the mutant allele in total RNA derived from tissue extracts. Expression of the mutant protein is also equivalent in different phenotypes. Thus, these mice are an excellent model for delineation of the modifying factors postulated to affect human OI phenotypes. In addition, we generated knock-in mice carrying an “intronic” inclusion by mating chimeras with wild-type females. Alternative splicing involving the stop cassette results in retention of non-collagenous sequences. These mice reproduce the lethal phenotype of similar human mutations and are designated BrtlII.

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

PRKACB and Carney Complex

The authors report that a gain of function in the catalytic subunit beta of the cyclic AMP–dependent protein kinase (protein kinase A), resulting from the presence of four copies of PRKACB (instead of the normal two), may lead to a Carney complex phenotype.

XX males SRY negative: a confirmed cause of infertility

Background SOX9 is a widely expressed transcription factor playing several relevant functions during development and essential for testes differentiation. It is considered to be the direct target gene of the protein encoded by SRY and its overexpression in an XX murine gonad can lead to male development in the absence of Sry. Recently, a family was reported with a 178 kb duplication in the gene desert region ending about 500 kb upstream of SOX9 in which 46,XY duplicated persons were completely normal and fertile whereas the 46,XX ones were males who came to clinical attention because of infertility. Methods and results We report a family with two azoospermic brothers, both 46,XX, SRY negative, having a 96 kb triplication 500 kb upstream of SOX9. Both subjects have been analyzed trough oligonucleotide array-CGH and the triplication was confirmed and characterised through qPCR, defining the minimal region of amplification upstream of SOX9 associated with 46,XX infertile males, SRY negative. Conclusions Our results confirm that even in absence of SRY, complete male differentiation may occur, possibly driven by overexpression of SOX9 in the gonadal ridge, as a consequence of the amplification of a gene desert region. We hypothesize that this region contains gonadal specific long-range regulation elements whose alteration may impair the normal sex development. Our data show that normal XX males, with alteration in copy number or, possibly, in the critical sequence upstream to SOX9 are a new category of infertility inherited in a dominant way with expression limited to the XX background.

Current and emerging treatments for the management of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is the most common bone genetic disorder and it is characterized by bone brittleness and various degrees of growth disorder. Clinical severity varies widely; nowadays eight types are distinguished and two new forms have been recently described although not yet classified. The approach to such a variable and heterogeneous disease should be global and therefore multidisciplinary. For simplicity, the objectives of treatment can be reduced to three typical situations: the lethal perinatal form (type II), in which the problem is survival at birth; the severe and moderate forms (types III–IX), in which the objective is ‘autonomy’; and the mild form (type I), in which the aim is to reach ‘normal life’. Three types of treatment are available: non-surgical management (physical therapy, rehabilitation, bracing and splinting), surgical management (intramedullary rod positioning, spinal and basilar impression surgery) and medical-pharmacological management (drugs to increase the strength of bone and decrease the number of fractures as bisphosphonates or growth hormone, depending on the type of OI). Suggestions and guidelines for a therapeutic approach are indicated and updated with the most recent findings in OI diagnosis and treatment.

Cellular mechanism of decreased bone in Brtl mouse model of OI: imbalance of decreased osteoblast function and increased osteoclasts and their precursors.

The Brtl mouse, a knock‐in model for moderately severe osteogenesis imperfecta (OI), has a G349C substitution in half of type I collagen α1(I) chains. We studied the cellular contribution to Brtl bone properties. Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40–45%. Brtl ObS/BS is comparable to wildtype, and Brtl and wildtype marrow generate equivalent number of colony‐forming units (CFUs) at both ages. However, OcS/BS is increased in Brtl at both ages (36–45%), as are TRACP+ cell numbers (57–47%). After puberty, Brtl ObS/BS decreases comparably to wildtype mice, but osteoblast matrix production (MAR) decreases to one half of wildtype values. In contrast, Brtl OcS falls only moderately (∼16%), and Brtl TRACP staining remains significantly elevated compared with wildtype. Consequently, Brtl BFR decreases from normal at 2 mo to one half of wildtype values at 6 mo. Immunohistochemistry and real‐time RT‐PCR show increased RANK, RANKL, and osteoprotegerin (OPG) levels in Brtl, although a normal RANKL/OPG ratio is maintained. TRACP+ precursors are markedly elevated in Brtl marrow cultures and form more osteoclasts, suggesting that osteoclast increases arise from more RANK‐expressing precursors. We conclude that osteoblasts and osteoclasts are unsynchronized in Brtl bone. This cellular imbalance results in declining BFR as Brtl ages, consistent with reduced femoral geometry. The disparity in cellular number and function results from poorly functioning osteoblasts in addition to increased RANK‐expressing precursors that respond to normal RANKL/OPG ratios to generate more bone‐resorbing osteoclasts. Interruption of the stimulus that increases osteoclast precursors may lead to novel OI therapies.