Genomic view on candidate genes and BP sequences. (A) PTHLH on chromosome (Chr.) 12p11.2 and KCNB2 on Chr. 8q13. Endonucleases (SfiI, PmeI, PstI) for Southern blotting (* indicate restriction sites; probes indicated as arrows) narrowed down the BP regions; fragments are shown in kilobases (kb). The Chr. 12p11.2 BP was at bp 28.102.311 and the Chr. 8q13 BP in KCNB2 intron 2 at bp 73.779.815 (UCSC Genome Browser Mar. 2006). Southern blotting: Chr.12 probes 1, 2 (left) and Chr. 8 probes 1, 2 (right). Non-affected persons (III:3, IV:3) showed the expected restriction fragments (refer to top). In BDE affected patients (III:2, IV:2, V:1) unexpected bands displayed the affected allele. (B) BP sequences on the derivative chromosomes der(8) and der(12). Der(8) BP is 86.128 kb upstream of PTHLH and 167.635 kb downstream of KCNB2, disrupted by the translocation in intron 2. The der(8) BP is surrounded by AP-1 (TGAGTCA) and bidirectional EBS binding motifs. At der(12) BP a 5-bp micro-duplication generated the core consensus sequence for EBS (GGA^A/_T); KCNB2 exon 3 (Ex.) is 233.327 kb upstream of der(12) BP.

Electrophoretic mobility shift analysis (EMSA) and Chromatin immunoprecipitations (ChIP) at der(8) BP and wt(12). (A) EMSA of the wild-type (wt) and translocation BPs far upstream of PTHLH. Recombinant human (rh) AP-1 (c-Jun) and rhC-ets-1 were incubated with 3′-biotin-labeled oligonucleotides representing der(8) BP and wt(8) and wt(12). rhAP-1 bound der(8) BP (lane 5) and wt(12) (lane 8), but not wt(8) (lane 11). (B) rhC-ets-1 bound der(8) BP (lane 5) and wt(8) (lane 11), but not wt(12) (lane 8). Competition with 400-fold excess unlabeled oligo (lanes 3, 6, 9, 12) prevented binding in all cases; AP-1 or C-ets-1 consensus oligos as controls (lane 2); probes without protein (lanes 1, 4, 7, 10). (C) ChIP to determine chromatin stage at der(8) BP. The regulatory histone modifications H3K4me1 and H3K4me3 enrichments in fibroblasts of a BDE patient between der(8) BP affected and wt(12) unaffected allele were 2-fold higher at der(8) BP. Epigenetic modifications occurred only at the translocation BP. (D) The transcription factors AP-1 (c-Jun) and C-ets-1 in vivo binding at der(8) BP and wt(12) allele in chondrogenically differentiated fibroblasts of a BDE patient. AP-1 and C-ets-1 were significantly enriched at der(8) BP, AP-1 was also found at wt(12) (n = 3, **P < 0.01, *P < 0.05).

PTHLH promoter characterization and PTHLH reporter gene assays in chondrocyte cell lines. (A) 5′ RACE PCR of PTHLH-transcripts in ATDC5 and C28/I2 cells. Two TATA boxes (P1, P3), one GC-rich promoter (P2), the UCSC mammalian conservation with approximate genomic kilobases distances and the PTHLH exons 1–5 (ex.) are shown. Identical chondrogenic transcription start site (TSS, bp 28014179) were revealed in ATDC5 and C28/I2 cells downstream of GC-rich promoter (bottom). Two further PTHLH transcripts start at bp 28016183 (see NCBI RefSeq). The red box represents 3080 bp of the PTHLH promoter used to generate the reporter construct. (B) PTHLH-luciferase reporter gene assays with EBS (C-ets-1 core consensus sequence), wt(12) (AP-1 motif), der(8) BP (C-ets-1 and AP-1 motif) or a three time repeats (×3) inserted upstream of the 3080 bp-PTHLH promoter (scheme right). Only the wt(12) × 3 permitted strong stimulation of PTHLH promoter activity and the presence of the C-ets-1 motifs in der(8) BP × 3 prevented activation by endogenous factors (n = 3, **P < 0.01, *P < 0.05).

BDE characteristics and cytogenetic karyotyping. (A) The hands of patient III:2 and index patient IV:2 displayed type E brachydactyly with shortened metacarpals, particularly 3, 4 and 5. Amputation of distal phalanges in digit 3, patient III:2. (B) Family tree; solid symbols are affected. (C) Metaphase FISH with BACs spanning breakpoints (BPs) revealed t(8;12)(q13;p11.2) translocation; BAC RP11-1151B7 for chromosome 12 (green, FITC) and BAC RP11-313C15 for chromosome 8 (red, TMR), present on the normal and derivative chromosomes, der(12) and der(8).

Analysis of the putative cause of the translocation by asymmetrical and symmetrical topoisomerase (TOPII a, s) and translin binding. Wild-type sequences are from RefSeq (NCBI) with homology matching bp (*); BPs as bold lines. TOPII asymmetric (a) consensus sequence: 5′-^A/_GN^c/_TNNCNNG^C/_T^NG^G/_TTN^C/_TN-3′; symmetric (s): 5′-^A/_GNNNN^C/_T^A/_GN^A/_G^C/_T^^A/_TVB^T/_A^A/_G^C/_TN^C/_T^A/_GNNNN^C/_T-3′. Two different translin recognition motifs: 5′-ATGCAG-0–4 bp-GCCC^A/_T^G/_C^G/_C^A/_T-3′ and 5′-GCNC^A/_T^G/_CCT-0–2 bp-GCCC^A/_T^G/_C^G/_C^A/_T-3′. ↑↓ Potential TOPII restriction sites, * verified +2 (guanine)/+4 (thymine) TOPII at a position behind the restriction site (72).

Gene expression in BDE and unaffected fibroblasts undergoing chondrogenic induction in pelleted micromass cultures. (A) The cartilage markers Aggrecan (AGC), COL2A1, COL10A1 and IHH were upregulated in the non-affected control individuals (NON-AFF) and in the BDE affected patient (AFF) and verified the chondrogenic differentiation. (B–D) PTHLH, ADAMTS-7 and ADAMTS-12 expressions were similar in differentiated NON-AFF, but significantly downregulated in differentiated BDE cultures (independent differentiations, n = 4, **P < 0.01, *P < 0.05).