Fracture healing, a critical and complex biological process, often presents challenges in clinical practice with the current standards failing to fully address the medical needs for rapid and effective recovery. In this work, a localized cold therapy is investigated as an alternative approach to expedite bone healing. We hypothesized that optimized cold application can enhance bone healing within a fracture model by inducing hypoxia, leading to accelerated angiogenesis along with improved osteogenesis. A short, localized cold exposure is directly applied to the fracture site over a 4-week period in a mouse fracture model, aiming to assess its impact on bone formation through mechanisms of angiogenesis and osteogenesis. Our results revealed a significantly greater volume of new bone tissue and enhanced vascularity at the fracture site in the cold-treated group compared with controls. Calcified tissue histology analysis showed that the accelerated callus maturation and development of the vascular network following cold exposure were associated with an activity increase of alkaline phosphatase and transient receptor potential vanilloid 1. These biological changes were accompanied by a hypoxic environment induced during cold therapy. The study provides compelling evidence supporting the efficacy of intermittent cold therapy in accelerating fracture healing. These promising results highlight the need for further research in larger-scale studies and diverse fracture models, underlining the potential of cold therapy as a novel, noninvasive treatment strategy in orthopedic care.

Introduction Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to test intervertebral disc (IVD) regeneration, application becomes costly when used as a screening method. The bovine IVD organ culture system offers an inexpensive alternative, however, in the current paradigm, the bony vertebrae is removed to allow for nutrient diffusion to disc cells. This provides limitations on the conditions and strategies one can employ in investigating IVD regeneration and mechanisms in degenerative disc disease (i.e., combination of axial and torsional loading). Although one method has been attempted to extend the survival of bovine vertebrae containing IVDs (vIVD) cell viability declined after two weeks in culture. Our goal was to develop and validate a long-term organ culture model with vertebral bone, which could be used subsequently for studying biological repair of disc degeneration and biomechanics. Material and Methods Preparation of vIVDs: Tails of 22- to 28-month-old steers were obtained from the local abattoir within 4h of slaughter. The largest IVDs ( n = 16) were prepared for organ culture by parallel cuts through the adjacent vertebral bodies at 1 cm from the endplates using an IsoMet®1000 precision sectioning saw (Buehler, Germany). vIVDs were split into two groups (PrimeGrowth or DMEM): eight were treated with PrimeGrowth Media kit (developed by Intervertech and licensed to Wisent Bioproducts) and eight with DMEM. The PrimeGrowth group was incubated for 1h in PrimeGrowth Isolation Medium (Cat# 319–511-EL) and the DMEM group for 1h in DMEM. After the isolation step, discs were washed 3 times in PrimeGrowth Neutralization Medium (Cat# 319–512-CL) while the other 8 IVDs were washed thrice in DMEM. The discs isolated with PrimeGrowth and DMEM were cultured for up to 5 months in sterile vented 60 ml Leakbuster™ Specimen Containers (Starplex) in PrimeGrowth Culture Medium (Cat# 319–510-CL) and DMEM with serum and antibiotics, respectively. Culture medium was replaced every three days with no mechanical load applied. Live/Dead Assay: vIVDs cultured for 1 or 5 months were dissected to separate NP, inner AF (iAF) and outer AF (oAF) regions. A 4 mm biopsy punch was used to prepare specimens for cell viability using a live/dead fluorescence assay (Live/Dead®, Invitrogen) and visualized by confocal microscopy. Glucose Diffusion: After one month of culture, vIVDs were incubated for 72h in diffusion medium containing PBS (1x), CaCl2 (1mM), MgCl2 (0.5mM), KCl2 (5mM), 0.1% BSA and 150µM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NDBG), a D-glucose fluorescent analogue. Discs were dissected and NP, iAF, oAF regions were incubated in guanidinium chloride extraction buffer. Extracts were measured for fluorescence. Results After 5 months of organ culture, vIVDs prepared with PrimeGrowth Media kit demonstrated ~95% cell viability in all regions of the disc. However, dramatic reductions (~90%) in vIVD viability were measured in DMEM-treated discs after 1 month. Interestingly, vIVD viability was related to the amount of 2-NDBG incorporated into the disc tissue. Conclusion We have developed a novel method for isolating IVDs with vertebral bone capable of long-term viability. This method may not only help in the discovery of novel therapeutics in disc regeneration, but could also advance our understanding on complex loading paradigms in disc degeneration.

OBJECTIVE Autosomal dominant hypocalcemia (ADH) is a rare disorder caused by activating mutations of the calcium-sensing receptor (CASR). The treatment of ADH patients with 1α-hydroxylated vitamin D derivatives can cause hypercalciuria leading to nephrocalcinosis. DESIGN AND METHODS We studied a girl who presented with hypoparathyroidism and asymptomatic hypocalcemia at age 2.5 years. Mutations of CASR were investigated by DNA sequencing. Functional analyses of mutant and WT CASRs were done in transiently transfected human embryonic kidney (HEK293) cells. RESULTS The proband and her father are heterozygous for an eight-nucleotide deletion c.2703_2710delCCTTGGAG in the CASR encoding the intracellular domain of the protein. Transient expression of CASR constructs in kidney cells in vitro suggested greater cell surface expression of the mutant receptor with a left-shifted extracellular calcium dose-response curve relative to that of the WT receptor consistent with gain of function. Initial treatment of the patient with calcitriol led to increased urinary calcium excretion. Evaluation for mosaicism in the paternal grandparents of the proband was negative. CONCLUSIONS We describe a novel naturally occurring deletion mutation within the CASR that apparently arose de novo in the father of the ADH proband. Functional analysis suggests that the cytoplasmic tail of the CASR contains determinants that regulate the attenuation of signal transduction. Early molecular analysis of the CASR gene in patients with isolated idiopathic hypoparathyroidism is recommended because of its relevance to clinical outcome and treatment choice. In ADH patients, calcium supplementation and low-dose cholecalciferol avoids hypocalcemic symptoms without compromising renal function.

Intervertebral disc (IVD) degeneration is a common cause of low back pain. Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to investigate IVD degeneration, they are technically challenging to prepare, expensive, present with limitations when performing biomechanical studies on the disc, and are impractical in large-scale screening of novel anabolic and scaffolding agents. An IVD organ culture system offers an inexpensive alternative. In the current paradigm, the bony endplates are removed to allow for nutrient diffusion and maintenance of disc cell viability. Although this is an excellent system for testing biologics, it results in concave cartilage endplates and, as such, requires special platens for loading purposes in a bioreactor as flat ones can overload the annular disc region leading to improper loading. Furthermore, the absence of bone makes it unsuitable for applying complex cyclic loading, a topic of interest in the study of chronic progressive degeneration, as multiaxial loading is more representative of daily forces encountered by the IVD. We have developed and validated a novel long-term IVD organ culture model that retains vertebral bone and is easy to prepare. Our model is ideal for testing potential drugs and alternate-based therapies, in addition to investigating the long-term effects of loading paradigms on disc degeneration and repair.

Introduction The intervertebral discs (IVDs) are the largest avascular tissues in the body characterized by an abundant extracellular matrix and low cell density, with innervation limited at the most peripheral annular layers. During IVD degeneration, the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), can stimulate nerve growth and hyperinnervation of the inner annulus fibrosus (AF) and nucleus pulposus (NP), which further could lead to back pain. Back pain can potentially be suppressed by BMP4 supplementation to decrease the density of innervation. Link N is a naturally occurring peptide that can stimulate BMP4 in human IVDs. The aim of the present study was to determine the effect of Link N on NGF, BDNF, and substance P (SP) in human AF cells stimulated with cytokines or punctured bovine discs and determine the possible mechanisms related to its effect. Material and Methods Human lumbar spines were obtained through Héma-Québec harvesting organ donation within 24 hour after death. Annulus fibrosus (AF) cells were isolated from AF region of human IVDs with Thompson grade 2 of degeneration, cultured in monolayers and stimulated with TNFα (100 ng/mL) and IL1β (10 ng/mL) in the presence or absence of Link N (1 µg/mL) for 48 hours. Total RNA was isolated and gene expression of neurotrophins, substance P, and their receptors was measured using RT PCR. AF cells isolated from human IVDs with Thompson grade 4 were cultured for 48 hours with or without Link N (1 µg/mL) and the NGF release in the media was measured by Western blotting. Coccygeal IVDs from the tails of adult bovine steers (20–25 months) were isolated with the cartilaginous endplates. After 24 hours preconditioning in complete DMEM, the IVDs were treated (control, capsaicin [1.5 µg/mL), punctured by 16 G needle, punctured by 16 G needle and incubated with Link N [10 µg/mL]) and the disc culture media was collected at different time points for analysis. SP in the media was concentrated by solid phase extraction and was assayed by Elisa. Results Proinflammatory cytokines IL1β and TNFα have been shown to trigger the expression of NGF and SP, as well as promoting the degradation of the extracellular matrix in human IVD cells. Our results demonstrate that gene expression of NGF, BDNF, TrkA, and TrkB significantly decreased in human disc cells stimulated with either IL-1β or TNFα supplemented with Link N when compared with the control. Additionally, Link N was also able to inhibit NGF release in the media when AF grade 4 was stimulated with IL-1β. Link N supplementation also led to a significant decrease in SP after 24 hour in injured discs, when compared with controls. Conclusion Link N can suppress the stimulation of NGF, BDNF, and their receptors TrkA and TrkB, respectively, as well as the neurotransmitter SP in degenerate discs. This suggests that Link N has the potential to inhibit pain induced by neuronal innervation caused by disc degeneration. Thus, administration of Link N has the potential to not only repair the discs in early stages of the disease but also suppress pain.

Introduction Intervertebral disc (IVD) calcification frequently occurs with age as demonstrated by radiographic analysis of elderly spines and is enhanced in patients with scoliosis. In one study by Chanchairujira K. et al (Spine 2004;230:499–503), the degree of disc degeneration as measured by disc space height was significantly correlated with IVD calcification. Several systemic disorders such as hyperparathyroidism and chondrocalcinosis, which are related to calcium handling, arthritis, trauma, vertebral fusion, and infection, have been associated with IVD calcification. Our preliminary data suggest that ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR) are increased in NP and AF cells of human Thompson graded discs, however, a role in disc degeneration remains unknown. Material and Methods Cells: Human NP and AF cells were incubated in culture media containing various concentrations of calcium (1.0, 1.5, 2.5, and 5.0 mM) a CaSR agonist (5 µM), or IL-1β (10 ng/mL) for 7 days. Lysates were extracted and the expression of aggrecan and type II collagen (Col II) were measured by Western blotting. IVDs: Caudal IVDs from the tails of 20- to 24-month-old steers were isolated and the vertebral bone was removed. IVDs were cultured for 4 weeks in culture medium containing calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist (5 µM). NP and AF tissues were subjected to guanidium extraction and Western blotting was performed to determine the expression of aggrecan and Col II. Histological sections were prepared to determine degree of mineralization by von Kossa staining and expression of alkaline phosphatase. Results The expression of aggrecan and Col II decreased dose-dependently in both NP and AF cells following incubation with calcium or the CaSR agonist. A similar phenomenon was observed in the expression of aggrecan and Col II in IVDs following calcium incubation. In addition to decreases in Col II and aggrecan, increase in mineralization and expression of alkaline phosphatase was observed in calcium-incubated IVDs. Conclusion Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of CaSR may be a contributing factor in IVD degeneration.

Introduction Although the disc has limited endogenous repair activity, induced repair of disc tissue may be possible by the intradiscal injection of growth factors to stimulate the production of disc matrix. We previously demonstrated that Link N (DHLSDNYTLDHDRAIH), a naturally occurring peptide generated by the N-terminal proteolytic fragmentation of link protein during tissue turnover, can act as a growth factor in the disc. It can stimulate matrix production in vitro, in vivo, and in intact ex vivo human intervertebral discs (IVDs). We have recently discovered that AF cells have the ability to proteolytically process Link N resulting in a fragment spanning amino acid residues 1 to 8 (US patent 61870394)—short Link N (sLink N). Our in vitro data indicates that the biologically active sequence is preserved within this fragment and, thus, sLink N could represent a potential stable growth factor able to stimulate disc repair. Separately, we developed a long-term organ culture model with vertebral bone. The purpose of the present study was to evaluate the effect of sLink N and compare its efficacy to Link N in this novel organ culture model of early disc degeneration. Material and Methods Caudal IVDs from the tails of 20 to 24-month old steers were isolated with adjacent vertebral bone. After 7 days of preconditioning in culture, degeneration was induced in IVDs by a single injection of 50 µg trypsin into the NP. After 7 days after induced-degeneration, the trypsin-treated discs were injected with either sLink N or Link N (100 µg/disc, n = 6 discs/group). Four of the trypsin-treated degenerate discs were injected with PBS alone to serve as a control for degeneration while four discs served as nondegeneration controls. At 2, 4, and 8 weeks posttreatment, two discs from each treatment and control groups were processed for biochemical analyses. Proteoglycan (predominantly aggrecan) synthesis in the NP was monitored as sulfated glycosaminoglycan using the 1,9-dimethylmethylene blue dye-binding assay, and Western blotting was performed to determine the expression of aggrecan and type II collagen in the tissue. Results Without intervention, at all-time points, the GAG content in degenerate discs dropped to approximately 50% of that in nondegenerate controls. In contrast, sLink N or Link N, significantly increased the GAG content of the discs compared with the GAG content in degeneration control discs. However, sLink N was more potent at inducing proteoglycan and type II content than Link N. Conclusion The results revealed that sLink N or Link N have the ability to restore tissue content and that sLink N is more potent than Link N in an early state of the disease. These results have implications in relation to using either sLink N or Link N when regenerating a functional NP in the degenerated IVD or in repairing cartilage.

Somatostatin (SST) is a peptide hormone that acts through a family of heptahelical receptors belonging to the G-protein coupled receptor (GPCR) superfamily. There are five known SST receptor subtypes termed SSTR1-5 and all couple to G alpha(i/o) G-proteins. It has been previously demonstrated that these receptors can form both homo- and heterodimers within their family or with other GPCR family members. Although agonist was demonstrated as a factor in modulating certain dimeric pairs, the molecular mechanism(s) underlying this regulation remains undetermined. Here, we demonstrate the coupling of G-protein as a contributing factor in the homo- and heterodimerisation of human (h) SSTR2 and SSTR5. When cells stably expressing hSSTR2 are pretreated with pertussis toxin (PTX), dissociation of hSSTR2 dimers occurs. Interestingly, although dimerisation of hSSTR5 was unaffected following PTX treatment, heterodimerisation between hSSTR2 and hSSTR5 is potentiated in the absence of receptor-stimulation. These results demonstrate the importance of G-protein in the maintenance and regulation of hSSTR dimers.

The biological effects of somatostatin (SST) were first encountered unexpectedly in the late 1960s in two unrelated studies, one by Krulich et al. (1968) who reported on a growth hormone (GH)-releasing inhibitory substance from hypothalamic extracts, and the other, by Hellman and Lernmark (1969), on the presence of a potent insulin inhibitory factor from the extracts of pigeon pancreatic islets. However, the inhibitory substance was not officially identified until 1973 by Guillemin’s group (Brazeau et al. 1973). In both synthetic and naturally occurring forms, this tetradecapeptide, originally coined as somatotropin release-inhibitory factor (SRIF, SST-14) was shown by Brazeau et al. to be the substance controlling hypothalamic GH release. This single achievement not only pioneered SST research but was also duly recognized, as Guillemin shared the 1977 Nobel Prize in Medicine. The following years bequeathed an exponential increase in SST-related studies. It soon became clear that SST-synthesis was not restricted to the hypothalamus. Its production is widely distributed throughout the central nervous system (CNS), peripheral neurons, the gastrointestinal tract, and the pancreatic islets of Langerhans (Luft et al. 1974; Arimura et al. 1975; Dubois 1975; Hokfelt et al. 1975; Orci et al. 1975; Pelletier et al. 1975; Polak et al. 1975; Patel and Reichlin 1978). In fact, SST-like immunoreactivity can be found throughout various tissues of vertebrates and invertebrates, including the plant kingdom (Patel 1992; Tostivint et al. 2004). Given its broad anatomical distribution, it is no wonder that SST produces a wide spectrum of biological effects. Generally regarded as an inhibitory factor, SST can function either locally on neighboring cells or distantly through the circulation, to regulate such physiological processes as glandular secretion, neurotransmission, smooth muscle contractility, nutrient absorption, and cell division (Reichlin 1983a, b; Patel 1992, 1999; Patel et al. 2001; Barnett 2003).

Somatostatin (SST) analogs have been successfully used in the medical treatment of acromegaly, caused by GH hypersecreting pituitary adenomas. Patients on SST analogs rarely develop tachyphylaxis despite years of continuous administration. It has been recently proposed that a functional association between SST receptor (SSTR) subtypes 2 and 5 exists to account for this behavior; however, a physical interaction has yet to be identified. Using both coimmunoprecipitation and photobleaching fluorescence resonance energy transfer microscopy techniques, we determined that SSTR2 and SSTR5 heterodimerize. Surprisingly, selective activation of SSTR2 and not SSTR5, or their costimulation, modulates the association. The SSTR2-selective agonist L-779,976 is more efficacious at inhibiting adenylate cyclase, activating ERK1/2, and inducing the cyclin-dependent kinase inhibitor p27(Kip1) in cells expressing both SSTR2 and SSTR5 compared with SSTR2 alone. Furthermore, cell growth inhibition by L-779,976 treatment was markedly extended in coexpressing cells. Trafficking of SSTR2 is also affected upon heterodimerization, an attribute corresponding to modifications in beta-arrestin association kinetics. Activation of SSTR2 results in the recruitment and stable association of beta-arrestin, followed by receptor internalization and intracellular receptor pooling. In contrast, heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with beta-arrestin. Given that SST analogs show preferential binding to SSTR2, these data provide a mechanism for their effectiveness in controlling pituitary tumors and the absence of tolerance seen in patients undergoing long-term administration.

The Fell-Muir Award requires the recipient to deliver a lecture and a review manuscript which provides a personal overview of significant scientific developments in the field of matrix biology over the period of the recipient's career. In this context, this review considers the collagen family of structural proteins and the advances in biochemical, molecular biological and genetic techniques which led to the elucidation of the structure, synthesis and function of this important group of extracellular matrix constituents. Particular attention is focussed on early research on the identification and assembly of the soluble precursors of collagen types I and II, and the identification of the precursor of basement membrane collagen type IV. In subsequent studies investigating the maintenance of the chick chondrocyte phenotype in culture, the influence of the extracellular milieu was found to influence markedly both cell morphology and collagen gene expression. These studies led to the discovery of collagen type X whose expression is restricted to hypertrophic chondrocytes at sites of endochondral ossification. Such research provided a prelude to investigations of mammalian endochondral ossification which is known to be aberrant in a variety of human chondrodysplasias and is reactivated in bone fracture repair and in osteoarthritis. The cloning of bovine and then human collagen type X genes facilitated studies in relevant human diseases and contributed to the discovery of mutations in the COL10A1 gene in families with metaphyseal chondrodysplasia type Schmid. Clustering of mutations in the C-terminal domain of the type X collagen molecule has now been widely documented and investigations of the pathogenic mechanisms in animal models are beginning to suggest the prospect of novel treatment strategies.

The Fell–Muir Award requires the recipient to deliver a lecture and a review manuscript which provides a personal overview of significant scientific developments in the field of matrix biology over the period of the recipient's career. In this context, this review considers the collagen family of structural proteins and the advances in biochemical, molecular biological and genetic techniques which led to the elucidation of the structure, synthesis and function of this important group of extracellular matrix constituents. Particular attention is focussed on early research on the identification and assembly of the soluble precursors of collagen types I and II, and the identification of the precursor of basement membrane collagen type IV. In subsequent studies investigating the maintenance of the chick chondrocyte phenotype in culture, the influence of the extracellular milieu was found to influence markedly both cell morphology and collagen gene expression. These studies led to the discovery of collagen type X whose expression is restricted to hypertrophic chondrocytes at sites of endochondral ossification. Such research provided a prelude to investigations of mammalian endochondral ossification which is known to be aberrant in a variety of human chondrodysplasias and is reactivated in bone fracture repair and in osteoarthritis. The cloning of bovine and then human collagen type X genes facilitated studies in relevant human diseases and contributed to the discovery of mutations in the COL10A1 gene in families with metaphyseal chondrodysplasia type Schmid. Clustering of mutations in the C-terminal domain of the type X collagen molecule has now been widely documented and investigations of the pathogenic mechanisms in animal models are beginning to suggest the prospect of novel treatment strategies.

We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L-363,301, c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11] (numbering as in the native hormone somatostatin-14). The d-Trp in position 8 was replaced with (2R,3S)- and (2R,3R)-beta-MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d-Trp upon binding to human somatostatin receptors hsst1-5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3. Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.

In this chapter, we discuss the current understanding of the family of matrix macromolecules that are classified as collagens. Reference is made to reviews and recent papers to provide the reader with a way into the relevant literature rather than an exhaustive survey of the bibliography.

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis1,2,3. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4–13). These dominant forms of MED (EDM1–3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro14,15. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene16 (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1–3 genes17 provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24–p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous disorder with marked clinical and radiographic variability. Traditionally, the mild "Ribbing" and severe "Fairbank" types have been used to define a broad phenotypic spectrum. Mutations in the gene encoding cartilage oligomeric-matrix protein have been shown to result in several types of MED, whereas mutations in the gene encoding the alpha2 chain of type IX collagen (COL9A2) have so far been found only in two families with the Fairbank type of MED. Type IX collagen is a heterotrimer of pro-alpha chains derived from three distinct genes-COL9A1, COL9A2, and COL9A3. In this article, we describe two families with distinctive oligo-epiphyseal forms of MED, which are heterozygous for different mutations in the COL9A2 exon 3/intron 3 splice-donor site. Both of these mutations result in the skipping of exon 3 from COL9A2 mRNA, but the position of the mutation in the splice-donor site determines the stability of the mRNA produced from the mutant COL9A2 allele.

To the Editor: We have previously reported the clinical and radiographic features of affected individuals from a large kindred who have an autosomal dominant form of bilateral dysplasia of the hip joints with severe secondary osteoarthrosis (Cilliers and Beighton Cilliers and Beighton, 1990Cilliers HJ Beighton P Beukes familial hip dysplasia: an autosomal dominant entity.Am J Med Genet. 1990; 36: 386-390Crossref PubMed Scopus (28) Google Scholar). This family came to the attention of one of us (H.C.) because of the number of patients with the family name, Beukes, who presented to the Department of Orthopaedic Surgery, University of Orange Free State, South Africa, for prosthetic hip-joint replacement as a consequence of bilateral premature degenerative osteoarthropathy. Genealogical studies subsequently revealed that all the affected individuals were members of an extended family that could be traced back to a single Dutch immigrant to South Africa who arrived in 1685 (Cilliers and Beighton Cilliers and Beighton, 1990Cilliers HJ Beighton P Beukes familial hip dysplasia: an autosomal dominant entity.Am J Med Genet. 1990; 36: 386-390Crossref PubMed Scopus (28) Google Scholar). Our continued investigation of this family has now traced 55 individuals in eight generations who, on the basis of either their medical histories or clinical and radiographic presentation of the disorder, appear to have inherited the disorder. The disorder clearly has an autosomal dominant mode of inheritance, but there is some evidence of nonpenetrance in that apparently unaffected individuals have had affected offspring. The clinical and radiographic manifestations have been described in detail elsewhere (Cilliers and Beighton Cilliers and Beighton, 1990Cilliers HJ Beighton P Beukes familial hip dysplasia: an autosomal dominant entity.Am J Med Genet. 1990; 36: 386-390Crossref PubMed Scopus (28) Google Scholar). In brief, the presenting symptom is hip-joint discomfort, which usually develops during childhood at age <2 years but may develop either later in childhood or, as in one instance, as late as the age of 35 years. After onset of symptoms, the hip joints deteriorate progressively, gait is disturbed, and, by early adulthood, affected persons are crippled by degenerative arthropathy. The earliest radiological changes are broadening of the femoral necks, late appearance of the secondary ossification centers of the femoral head, and an irregular appearance of the proximal epiphyseal line of the femur. By mid childhood, the femoral heads are flat (coxa plana), with broadening of the femoral necks, adaptation of the acetabulum to the malformed femoral head, superolateral displacement of the femoral head, and an irregular appearance of the greater trochanteric epiphyses. By adulthood, these features are more pronounced, and there is superior migration and superolateral displacement of the femoral head and overgrowth of the greater trochanter in a superomedial direction. In the later stages, coxa vara is a prominent finding. Signs of degenerative osteoarthrosis (periarticular cysts, periarticular sclerosis, and narrowing of the joint space) are evident in early to mid childhood and are progressive. Apart from the hip problems, the general health of affected individuals is good, their height is normal, and, other than in one instance in which a young adult had severe kyphoscoliosis that necessitated spinal fusion, involvement of the vertebral bodies and other joints is minimal. The radiographic findings, the absence of involvement of the vertebral bodies and joints other than the hip, and the normal stature of affected individuals has led to the conclusion that this disorder is distinct from other autosomal dominant forms of chondrodysplasia, in which premature degenerative osteoarthropathy of the hip joint is a major complication. It therefore seemed appropriate that this condition be categorized as a familial hip dysplasia, and it was called “Beukes hip dysplasia” (BHD [MIM 142669]), on the basis of the name of the affected family (Cilliers and Beighton Cilliers and Beighton, 1990Cilliers HJ Beighton P Beukes familial hip dysplasia: an autosomal dominant entity.Am J Med Genet. 1990; 36: 386-390Crossref PubMed Scopus (28) Google Scholar). Mutations in the genes encoding components of the extracellular matrix of cartilage have been identified in families with forms of chondrodysplasia with secondary osteoarthritis (see Kuivaniemi et al. Kuivaniemi et al., 1997Kuivaniemi H Tromp G Prockop DJ Mutations in fibrillar collagens (types I, II, III and XI), fibril-associated collagen (type IX) and network-forming collagen (type X) cause a spectrum of diseases of bone, cartilage and blood vessels.Hum Mutat. 1997; 9: 300-315Crossref PubMed Scopus (261) Google Scholar; Briggs et al. Briggs et al., 1998Briggs MD Mortier GR Cole WG King LM Golik SS Bonaventure J Nuytinck L et al.Diverse mutations in the gene for cartilage oligomeric matrix protein in the pseudoachondroplasia-multiple epiphyseal dysplasia disease spectrum.Am J Hum Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, and references therein). We commenced our studies to locate the gene responsible for BHD, by performing analyses of linkage to polymorphic markers within or near cartilage candidate genes. We found, however, no evidence of linkage to COL2A1 (Beighton et al. Beighton et al., 1994Beighton P Cilliers HJ Ramesar R Autosomal dominant (Beukes) premature degenerative osteoarthropathy of the hip joint unlinked to COL2A1.Am J Med Genet. 1994; 53: 348-351Crossref PubMed Scopus (8) Google Scholar), COL9A1, COL9A2, COL11A1, COL11A2, COL10A1, CRTL-1, CRTM, AGC1, or COMP (Al-Ali et al. Al-Ali et al., 1994Al-Ali M Beighton P Read A Warman M Donn R Boot-Handford R Wallis GA Exclusion of candidate genes in two families with inherited forms of osteoarthritis.Int J Exp Pathol. 1994; 75: A71PubMed Google Scholar; G. Wallis, P. Roby, and S. Eyre, unpublished data). We therefore performed a genomewide screen with a panel of 290 markers with an average spacing of 11 cM (Davies et al. Davies et al., 1994Davies JL Kawaguchi Y Bennett ST Copeman JB Cordell HJ Pritchard LE Reed PW et al.Genome-wide search for human type 1 diabetes susceptibility genes.Nature. 1994; 371: 130-136Crossref PubMed Scopus (1247) Google Scholar). For this purpose, genotype data were obtained from 32 individuals from the BHD kindred, including 15 affected individuals, 11 unaffected related individuals, and 6 unrelated spouses. The affected status of the 15 individuals who were genotyped had been established on the basis of their clinical and radiological presentation of the disorder. The BHD pedigree shown in figure 1 has been condensed and includes only those individuals who were genotyped and those who were required for the linkage analysis. Despite the fact that the pedigree has been condensed, the relationships between the members of the pedigree have been retained. Genotyping was done with an ABI 373 sequencer and GENESCAN 1.2.2-1 and GENOTYPER 1.1.1 software. Two-point LOD score (Z) values were computed by the LINKAGE package (Lathrop and Lalouel Lathrop and Lalouel, 1984Lathrop GM Lalouel JM Easy calculations of LOD scores and genetic risks on small computers.Am J Hum Genet. 1984; 36: 460-465PubMed Google Scholar), for various recombination fraction (θ) values, with penetrance values of 90%, 95%, and 100%, and a disease frequency of .0001. One marker on chromosome 4, D4S408, had a maximum Z (Zmax) value of 3.58 at a maximum θ (θmax) of .00, at 90% penetrance (see table 1). Further analysis with markers from this region, with penetrance values of 85%, 90%, and 95%, gave a two-point Zmax value of 5.73 for marker D4S2924, at θ=.00 and at a penetrance of 90% (see table 1). Haplotypes were constructed with the map order D4S1607–1.5 cM–D4S2951–1 cM–D4S1554–4 cM–D4S408–3.2 cM–D4S2924–0.5 cM–D4S171–0.5 cM–D4S1540–3 cM–D4S3051–1 cM–D4S426–1.3 cM–D4S2930. The order of the markers was derived from on-line genetic mapping data at the Center for Medical Genetics, Marshfield Medical Research Foundation Website. As judged on the basis of an examination of the marker haplotypes segregating with BHD (fig. 1), the closest recombinants involving affected family members were at D4S1554 proximally and D4S3051 distally, which limits the BHD gene locus to an interval of ∼11 cM. Three apparently clinically unaffected individuals (VII-4, age 49 years; VII-7, age 33 years; and VII-9, age 33 years) were found to have inherited the disease-linked haplotype. Individual VII-4 transmitted the disorder and the disease-linked haplotype to her two affected offspring, demonstrating that she is a nonpenetrant carrier of the mutated gene. However, to date, her only potential clinical symptom of the disorder has been hip-joint pain during pregnancy. The remaining two individuals have not reported any symptoms of the disorder. Attempts are currently underway to obtain recent radiographs of these three individuals, to determine whether they have any radiological evidence of the disorder.Table 1Two-Point Z Values, between BHD and Chromosome 4q35 MarkersZat θ =MarkerZmax (θmax).00.01.05.10.20.30.40D4S16071.54 (.18)−11.96−2.19.371.231.531.14.48D4S29511.46 (.17)−12.20−1.32.631.261.431.07.53D4S15542.00 (.10)−1.421.271.892.01.751.25.63D4S4083.58 (.00)3.583.553.373.052.231.30.39D4S29245.73 (.00)5.735.685.404.963.872.581.17D4S1714.84 (.00)4.844.794.554.153.162.01.82D4S15405.11 (.00)5.115.034.674.173.061.90.78D4S3051.18 (.33)−3.14−2.35−1.01−.43.04.17.14D4S426.03 (.40)−7.10−2.63−1.18−.60−.150.03D4S2930.18 (.29)−12.36−3.64−1.44−.55.07.18.11Note.—All values are calculated under the assumption of 90% penetrance and a disease-allele frequency of .0001. Open table in a new tab Note.—All values are calculated under the assumption of 90% penetrance and a disease-allele frequency of .0001. Multipoint analysis was done with the LINKAGE 5.1 LINKMAP program and the marker order given above. Multipoint analysis with the complete set of markers spanning the disease interval was not possible, because of both the high number of alleles per marker and the large number of individuals in the pedigree, so sequential three-point analyses were done. The combined results are shown in figure 2. The multipoint location score for the chromosome 4 markers was 30.05 (equivalent to Z=6.5), and the likely location of the BHD gene was confirmed to be the 11-cM interval between D4S1554 and D4S3051. Currently, within the linked region there are no known or obvious potential candidate genes for the disease, and no other forms of familial osteochondrodysplasia are known to map to this region. Physical mapping data for this region include a single YAC contig, WC4.7, to which a number of expressed sequence tags (ESTs) have been mapped (Whitehead Institute/MIT Genome Sequencing Project). Our finding that BHD does not map to any of the loci that have previously been identified for other forms of autosomal dominant chondrodysplasia with associated osteoarthropathy (notably, spondyloepiphyseal dysplasia [MIM 184100 and MIM 183900], multiple epiphyseal dysplasia [MIM 226900], and pseudoachondroplasia [MIM 177170]) supports the clinical and radiographic data suggesting that this disorder is a distinct form of familial hip dysplasia. Identification of the BHD gene within the linked region on 4q35 could have implications for the investigation of other, more common forms of idiopathic hip osteoarthritis. We sincerely thank the family members who participated in this study, as well as the many genetics nurses who were involved in the collection of the DNA. We thank Mike Briggs and Mike Dixon for helpful discussions. This work was supported by grants from the Royal Society (United Kingdom), the Arthritis Research Campaign (United Kingdom), the U.C.T. Staff Research Fund, and the Mauerberger Foundation, S.A.

TypeX collagen isa homotrimer ofml(X) chainsencodedbytheCOLlOAIgene. It issynthesised specifically andtransiently byhypertrophic chondrocytes atsitesof endochondral ossification. Pointmutationsanddeletions intheregionofthe COLlOAlgene encoding theal(X)carboxyl-terminal (NC1)domainhavepreviously beenidentified insubjects with metaphysealchondrodysplasia type Schmid(MCDS).To determine whether mutationsinotherregionsofthegene causedMCDS or comparable phenotypes, we usedPCR followed bySSCPtoanalyse thecodingandpromoterregions ofthe COLlOAlgene, as wellas theintron/exon boundaries offivefurther subjects with MCDS, one subject withatypical MCDS, and ninesubjects withotherformsof metaphyseal chondrodysplasia.