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.

In dieser Dissertation wurde eine isolierte Brachydaktylie vom Typ E (BDE) untersucht. Grundlage war eine Familie mit autosomal-dominanten Erbgang BDE. Der genetische Hintergrund ist eine balancierte Translokation t(8;12)(q13;p11.2). Der Bruchpunkt auf derivativem Chromosom der(8) liegt 86 kb strangaufwärts des chondrogenetisch essentiellen Kandidatengens PTHLH (Parathyroid hormone like hormone). PTHLH ist für die Differenzierungsrate von proliferativen Chondrozyten verantwortlich. Positiv oder negativ reguliertes Pthlh führen zu einer Dysbalance mit Brachydaktylie-ähnlichen Phänotypen in murinen Tiermodellen. Der Leserahmen des Kaliumkanals KCNB2 auf Chromosom 8 wurde durch die Translokation in Intron 2 getrennt. Chrondrogenetische KCNB2 Funktionen konnten durch in situ Hybridisierungen ausgeschlossen werden. Der Translokationsbruchpunkt auf der(8) liegt in einer in Mammalia hochkonservierten Region und beeinhaltet ein Bindungsmotiv für AP1 Transkriptionsfaktoren. Durch die Translokation befindet sich in unmittelbarer Nähe eine Kernkonsensussequenz für ETS Transkriptionsfaktoren. AP1 und ETS Transkriptionsfaktoren interagieren und wurden auf eine potentielle PTHLH Regulation untersucht. Epigenetische Histonmodifizierungen, charakteristisch für cis-regulatorische Elemente, sowie Reportergenassays mit AP1 und ETS1 Bindungsmotiven zeigten einen Bezug zur PTHLH Regulation. Bindungsassays mit AP1 und ETS1 Transkriptionsfaktoren an den Bruchpunktsequenzen, sowie funktionelle in vitro Experimente mit Chondrozyten verifizierten die Hypothese, dass der Translokationsbruchpunkt strangaufwärts von PTHLH regulatorische Eigenschaften besitzt. Die AP1 und ETS1 Transkriptionsfaktoren regulierten PTHLH positiv in ATDC5 und C28/I2 Chondrozyten. In chondrogeninduzierten Patientenfibroblasten war die PTHLH Expression inhibiert. Die molekulare Pathogenese der BDE wurde durch die bisher unbekannte chondrogene PTHLH Fehlregulation dargestellt. / We studied a 3-generation family with Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation. We identified PTHLH (Parathyroid hormone like hormone) on chromosome 12p11.2 and the ionchannel KCNB2 on chromosome 8q13 as candidate genes. KCNB2 was disrupted in intron 2, while the chromosome 12 breakpoint is localized 86 kb upstream of PTHLH; only the latter gene is involved in chondrogenesis. The 12p11.2 breakpoint is conserved and features an AP1 binding site 86 kb upstream of PTHLH. Due to the translocation, an ETS binding site from 8q13 resided near the AP1 site. Since both transcription factors interact, we tested if AP1 and ETS1 can activate PTHLH in ATDC5 and C28/I2 chondrocytes. We used the breakpoint sequences of the derivative chromosomes 8 and 12 and the nonaffected chromosome 8 and 12 allele sequences in reporter-gene assays. Reporter-gene constructs containing the der(8) breakpoint revealed activation in murine and human chondrocytes. The enrichment of histone modifications, implicating cis-regulatory effects were investigated in the breakpoint area. We found the enriched histone H3K4me1 modification at the chromosome 12 breakpoint position in murine and human chondrocytes, while affected fibroblasts showed higher H3K4me1 enrichment at the der(8) breakpoint compared to wt(12) allele. Furthermore, the breakpoint sequence bound to AP1 and C-ets-1 in EMSA. Western blotting after PMA-stimulated AP1 and ETS1 activation and overexpression of different AP1 and ETS1 combinations showed activated PTHrP expression in chondrocytes. In chondrogenic induced BDE fibroblasts PTHLH was inhibited, while IHH was upregulated. We suggest that PTHLH was dysregulated by the translocation in BDE chondrocytes. This could lead to BDE. We highlight the impact to characterize genomic breakpoints in detail and demonstrate a novel AP1- and ETS1-directed chondrogenic PTHLH regulation in wild-type chondrocytes and dysregulation in the pathogenesis of BDE.