Abstract The nucleotide sequence of the entire COL9A3 gene, coding for the human α3(IX) chain, was determined. The gene was approximately 23 kb in length and consisted of 32 exons. The polymerase chain reaction (PCR)-based procedure of conformation-sensitive gel electrophoresis (CSGE) was used to screen the gene for sequence variations and mutations in 83 unrelated patients with generalized primary osteoarthritis (OA), 31 with rheumatoid arthritis (RA), 171 with intervertebral disc disease (IDD), and 50 with various osteochondrodysplasias. The frequencies of certain sequence variations in healthy individuals were also determined. The COL9A3 gene was analyzed for mutations in two unrelated families with multiple epiphyseal dysplasia (MED). The analysis revealed a splice site mutation leading to skipping of exon 3 and an in-frame loss of 12 amino acid residues in the COL3 domain, the first diseasecausing mutation to be identified in the COL9A3 gene. Sequencing also indicated a 9 bp deletion in one allele in the second MED family that removed a Gly-Pro-Pro triplet. Surprisingly, the deletion did not co-segregate with the MED phenotype in the family. A similar 9 bp deletion, was also found in an unrelated family with no obvious phenotype, suggesting that the two 9 bp deletions represent neutral sequence variants. A construct with the deletion was then made in order to produce a recombinant protein, and the mutant type IX collagen was analyzed under reducing conditions by SDS-PAGE. The results indicated that the recombinant type IX collagen proteins consisted of three α chains, α1(IX), α2(JX), α3(IX), in a 1:1:1 ratio. To study the triple helix stability, pepsin treatment followed by SDS-PAGE was performed on normally folded and denatured recombinant type IX collagen samples. The results demonstrated that the recombinant type IX collagen containing the Gly-X-Y deletion in the a3(IX) chain is secreted as a correctly folded triple-helical molecule. CSGE analysis of exon 5 of the COL9A3 gene identified two nucleotide variations in the same codon, and thus three alleles: CGG (Arg), CAG (Gln), and TGG (Trp). The frequency of the Trp for Arg substitution, the Trp3 allele, was 0.244 among the probands with the IDD, while its overall frequency in the combined group of all non-IDD cases was 0.093. This difference was significant, with a p-value of 0.000013. The Trp3 allele increases the relative risk of IDD by a factor of 2.6 (95 percent confidence interval, 1.6 to 4.3). COL9A3 mutations are shown to be associated with mild cartilage and intervertebral disc diseases.

Abstract The role of sequence variations in genes encoding cartilage extracellular matrix (ECM) proteins were studied in osteoarthritis (OA) and multiple epiphyseal dysplasia (MED). The cartilage collagen genes COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, and COL11A2 were screened for sequence variations in 72 Finnish probands and one US family with primary early-onset hip and/or knee OA. Altogether 239 sequence variations were found, of which 16 were not present in the controls. Seven of the unique variations — four in COL11A1, two in COL11A2, and one in COL2A1 — were studied further, because they resulted in the substitution of conserved amino acids or were predicted to affect mRNA splicing. Association analysis was performed by genotyping 6–12 common polymorphisms from each gene in 72 OA patients and 103 controls; no common predisposing alleles were identified. The results, however, suggest that mutations in the minor cartilage collagen genes can be the cause of OA in a subgroup of OA patients. Two MED families with clinical and radiographic features suggestive of a collagen IX mutation were studied. Mutation screening of COL9A1, COL9A2, and COL9A3 yielded negative results. Instead, an R718W mutation in COMP was identified in both families. Clinical and radiographic overlap between patients with collagen IX mutations and patients with COMP mutations points to a common supramolecular complex pathogenesis. Clinical, radiological and molecular analyses of known MED genes were performed on a cohort of 29 consecutive MED patients. The DTDST mutation was identified in four patients (14%), the COMP mutation in three (10%), and the MATN3 mutation in three (10%). Two new distinct phenotypic entities were identified in patients in whom no mutation was found. The findings suggest that mutations in the above mentioned known MED genes are not the major cause of MED and are responsible for less than half of the cases. The existence of additional MED loci is supported by the exclusion of known loci and finding of the specific subgroups among these patients. The results suggest that genetic defects in ECM genes can predispose to OA and cause MED, even though the major genes involved in both disorders remain to be found.

Xenopus Laevis has provided, during the last ten years, the basis of a growing body of physiological enquiries initiated by Jolly's research on reflex action. Its viability in the laboratory and amenability to operative procedure in particular, make it a peculiarly suitable object for investigation. It is regrettable therefore, that existing literature on the anatomy of Xenopus has been directed to elucidating those characteristics which are of especial interest to the Systematists and Morphologists rather than detailed information of a type which is essential to operative procedure. There is in particular no extant account of the endocrine system of Xenopus, although it is evident from superficial inspection that the suprarenal complex differs from that of the more familar Anura. The present enquiry concerns the Thyroid Gland, Pituitary and Epiphyseal Complexes with some observations concerning the possible occurrence of chromophil cells in the kidney of Xenopus laevis. The data have been placed on record specifically and constitution of these organs in physiological operations.

Abstract available through the Journal of Investigative Medicine.

No description available.

Philosophiae Doctor - PhD / Age estimation of the skeletal remains of children can be accomplished by examination of the ossification centres and the fusion of the epiphyseal plates of long bones. Dental age estimation is done by examining the eruption of the deciduous and permanent teeth. Both these methods are inaccurate and are subject to the nutritional status of the individual. A more accurate method of age estimation is by the examination of radiographic images of the developmental stages of the tooth crown and root formation. Two methods of dental age estimation used are those of Moorrees, Fanning and Hunt (1963) (MFH) and that of Demirjian, Goldstein and Tanner (1973) (DGT). These methods were tested on a sample of 913 Tygerberg dental patients; a random mixture of Caucasoid and Khoisanoid children. The MFH method under-estimated the ages of the sample by an average of 0.91 years and the DGT method over-estimated the ages by an average of 0.89 years. Samples of Indian and Negroid children from Kwa-Zulu Natal were tested in a similar manner and the results showed similar under and over-estimation of the ages by these methods. The Negroid children were labelled the Zulu sample. Correction factors were derived for the MFH and DGT methods of dental age estimation when used on Tygerberg, Indian and Zulu children. These correction factors were tested on the samples and found to improve the accuracy of the age estimation methods of MFH and DGT significantly.A second sample group of Tygerberg, Indian and Zulu children were then tested firstly using the standard method of MFH and DGT and the using the correction factors. The results showed that the correction factors improved the age estimation on these samples except in the case of the DGT method on Zulu children. A sample of Xhosa speaking children were added to the two Zulu samples and made an Nguni sample. The Tygerberg samples were combined as were the Indian samples to form data bases for the construction of dental age related tables for Tygerberg, Indian and Nguni children. These tables show that there are distinct differences in the ages at which the teeth develop in the different sample groups and that dental age related tables are necessary for children of different population origins. Statistical analysis of the age related tables from this study (Phillips Tables) show these tables are more accurate in the age estimation of South African children.

Magister Chirurgiae Dentium - MChD / Skeletal age assessment is not only an important aspect in orthodontic treatment planning, but is also widely used in forensic medicine and physi cal anthropology. Vari aus studi es have shown that chronological age may be at variance with an individual's biologic age. Current research would seem to indicate that the hand-wrist radiograph provides the most accurate method of assessing skeletal age for diagnostic purposes. In recent years the number of patients presenting with malocclusions of a skeletal nature at the University of the Western Cape has increased si gnifi cantly. If it i s accepted that treatment of jaw discrepancies associated with malocclusion is dependent on a large component of dentofacial orthopedics, then by implication it is necessary that a substanti al amount of faci al growth remai ns. The need has therefore arisen for the establishment of skeletal maturation trends in the Western Cape. Skeletal maturity was assessed from hand-wrist radiographs in a sample of 318 Western Cape chi 1dren aged 6 to 16 years for both sexes, utilising the bone specific Tanner-Whitehouse TW-2 scoring system. Data obtai ned from the present study showed a marked di fference in skel etal maturati on trends between femal es of the Western Cape to that of the British norm, while the males showed less divergence. Further, these findings show that in both sexes the epiphyseal bones matured in advance of the TW-standard. Carpal maturation, however, was delayed in the male when compared to the British standard, while that of the female conformed to that of the British standard.

Factors affecting morphometrics and epiphyseal closure are important in understanding regional variation and growth of white-tailed deer (Odocoileus virginianus). I compared body and antler growth from birth to 3 years of age in captive, first-generation, male white-tailed deer from three regions with varying soil quality and deer morphometrics. I also determined gender and age effects on epiphyseal closure timing in captive white-tailed deer. I found regional morphological variation present in first-generation male deer, which may be caused by regional genetic variation or lingering maternal effects. Determining cause of regional morphological variation will require data collection through a second-generation of males raised on the controlled diet. Epiphyseal closure timing was associated positively with age. Two of the four epiphyseal plates examined were affected by gender, with females closing prior to males. Morphometric and epiphyseal data confirm that age and gender affect epiphyseal closure timing in white-tailed deer.

Mammals are an extremely successful group of tetrapods, and the great diversity of limb morphologies and locomotor modes is underpinned by adaptations in their long bones. Elongation of long bones takes place at each end of the bone shaft, at the epiphyseal growth plate surface. Secondary ossification centres form bony epiphyses above these growth plates, eventually fusing with the plates and marking the end of bone elongation. The proximal growth plate surface in the humerus is not uniform, with varying distributions of peaks and grooves that appear to differ across Mammalia. Although the process of growth plate bone deposition is well understood, there has been limited research on the growth plate surface morphology and what factors may be influencing it, such as ecology, phylogeny, or ontogeny. Using synchrotron propagation phase-contrast X-ray microtomography to reconstruct 3D models of the growth plate surface of the proximal humerus. We applied a metric called ariaDNE to quantify and measure the curvatures of these surfaces. We studied several extant mammals and one fossil stem mammal. We found that there was a high diversity of growth plate morphologies across mammal species, but the overall morphology remained consistent within the same species. The morphology also appears to shift throughout different ontogenetic stages, with a trend towards higher complexity in later developmental stages. The fossil specimen, G. planiceps, has a growth plate morphology that is remarkably similar to the short-beaked echidna (Tachyglossus aculeatus). Preliminary statistical tests suggest that growth plate morphology may be influenced by phylogeny and locomotory mode, although our small dataset has likely introduced sampling bias and the sample size should be expanded to find significant patterns. Growth plate surface morphology should be further explored due to its potential palaeobiological applications for the interpretation of ecology and evolution of extinct taxa.

Mutations in genes of importance for cartilage development may lead to skeletal malformations, chondroskeletal dysfunction and increased susceptibility to degenerative joint disease. Characterization of these mutations and identification of molecular pathways for the corresponding gene products have contributed to our understanding of mechanisms regulating skeletal patterning, endochondral ossification and joint formation. A five generation family segregating autosomal dominant osteochondritis dissecans (OCD) was identified. Affected family members presented with OCD in knees, hips and elbows, short stature, and early osteoarthritis. A genome wide scan and a multipoint linkage analysis identified aggrecan (ACAN) as a prime candidate gene. DNA sequence analysis of the ACAN-gene revealed heterozygosity for a missense mutation (c.6907G>A) in affected subjects, resulting in a p.V2303M substitution in the aggrecan G3 domain C-type lectin. This domain is important for the interaction with other proteins in the cartilage extracellular matrix. To determine the effect of the V2303M substitution on secretion and interaction, we performed binding studies with recombinant mutated and wild type G3 proteins. We found decreased affinity or complete loss of interaction between V2303M aggrecan and fibulin1, fibulin2 and tenascin-R. Analysis of articular cartilage from an affected family member confirmed that V2303M aggrecan is produced and present. In search for gene mutations associated with multiple epiphyseal dysplasia (MED) we considered the ACAN-gene a likely candidate. The ACAN-gene was analysed in 39 individuals with MED and screened negative for mutations in six previously known MED genes. Sequence analysis revealed a heterozygous missense mutation (c.1448G>T) in one adult male and compound heterozygous missense mutations (c.1366T>C and c.836G>A) in a five year old boy with healthy parents, each of them carrier for one of the mutations. A large family segregating autosomal dominant brachymesophalangia and OCD in finger joints was characterised. The clinical presentation in six affected family members was consistent with the diagnosis Brachydactyly type A1, in this family characterized by shortening of the middle phalanges, short ulnar styloid process, flattening of the metacarpal heads and mild osteoarthritis. The condition may be caused by mutations in the Indian hedgehog gene (IHH) or a yet unidentified gene on chromosome 5p13. Sequence analysis of the IHH-gene in affected individuals revealed a novel C to T transition (c.472C>T) leading to a p.158Arg>Cys substitution. Residue 158 in IHH is highly conserved throughout evolution and molecular structure modelling of IHH suggests that the R158C substitution leads to a conformational change at the site of interaction with the IHH-receptor. This supports that the substitution causes Brachydactyly type A1 in this family. In summary, we report on the clinical, radiological and molecular genetic characteristics of the three skeletal disorders OCD, MED and BDA1. Our results provide a novel molecular mechanism in the pathophysiology of familial osteochondritis dissecans confirming the importance of aggrecan C-type lectin for cartilage function. We also show that ACAN-gene mutations may be associated with MED extending the spectrum of skeletal dysplasias associated with the aggrecan gene. Finally, we report on a novel missense mutation in a conserved region of the IHH-gene associated with BDA1.