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Case Reports
. 2005 Apr;76(4):652-62.
doi: 10.1086/429252. Epub 2005 Feb 22.

Position Effects Due to Chromosome Breakpoints That Map Approximately 900 Kb Upstream and Approximately 1.3 Mb Downstream of SOX9 in Two Patients With Campomelic Dysplasia

Free PMC article
Case Reports

Position Effects Due to Chromosome Breakpoints That Map Approximately 900 Kb Upstream and Approximately 1.3 Mb Downstream of SOX9 in Two Patients With Campomelic Dysplasia

Gopalrao V N Velagaleti et al. Am J Hum Genet. .
Free PMC article


Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.


Figure  1
Figure 1
Photographs of the proband (A, B, and C), her father (C and D), and her brother (C, E, and F). Note the Robin sequence.
Figure  2
Figure 2
Mapping of chromosome breakpoints in patient 1. A, Metaphase chromosomes after FISH with BAC clones RP11-879D6 (green) and RP11-261A13 (red) showed that RP11-879D6 spans the breakpoint. B, DNA sequencing of both breakpoints of the derivative chromosomes 4 (green) and 17 (yellow) identified a 13-bp deletion on 4q28.3 generated by the translocation breakpoint. The breakpoint on chromosome 4 maps within a mariner transposon-like element HSMAR2. The trinucleotide CAC in bold represents the site of the translocation where there is identical sequence on both chromosomes 4 and 17 and may designate the origin of the translocation.
Figure  3
Figure 3
Autopsy study of patient 2. A, Micrognathia as part of Robin sequence. B, Cone-shaped fingers and hypoplastic thumb, with poorly formed distal interphalangeal creases and hypoplastic nails. C, Normal female external genitalia. D, Absence of oocytes.
Figure  4
Figure 4
Interphase FISH colocalization experiment. A, Physical map of the region surrounding SOX9. BAC clones are depicted as horizontal bars, with the colors corresponding to the probes in D and E. The upstream and downstream breakpoints are shown as vertical arrows. B, Interphase FISH with two probes, RAI1-specific BAC clone RP11-525O11 (red) and RP11-28B23 (green), located ∼1 Mb upstream from RAI1, in a male control individual without CD. The distance between the red and green signals was measured in 50 cells. The average distance between these two probes was 1.35 μm. There was no significant correlation (r=0.55) between the signal distance and the cell diameter. These measurements served as the negative control for the colocalization experiment. C, FISH with BAC clones that encompass the gene POU3F4 (RP11-246G22 [red]) and its suspected enhancer (RP11-54M14 [green]). The average distance between these markers was 0.67 μm. This constituted the positive control for the experiment. D, FISH with SOX9-specific BAC clone RP11-1116I6 (red) and the ∼1 Mb upstream breakpoint–spanning clone RP11-879D6 (green). The average distance between these probes was 0.62 μm. E, FISH with SOX9-specific BAC clone RP11-1116I6 (red) and the BAC clone RP11-661C3 (green), which is adjacent to the downstream breakpoint on the proximal side. The average distance between these probes was 0.61 μm. F, Graphic representation of the results of the experiment. The average distances between markers were analyzed using a one-way ANOVA test. The graph is centered around the grand mean (0.81 μm). The boxes contain 75% of the data for each group. The bars contain 95% of the data, and the line in the box is the mean. The distances from SOX9 to SOX9cre1 and from SOX9 to the downstream breakpoint are significantly shorter than the negative control (P=1.56×10-8) and are highly similar to the positive control. All distances were measured with the aid of a stage micrometer for accuracy (0.01 mm).
Figure  5
Figure 5
Computer analysis of the regulatory elements around SOX9. A, Graphical representation of the region ∼1.35 Mb upstream of SOX9. The evolutionary conserved sequences (ECS) regions described by Qin et al. (2004) and the nearby region of high RP values are shown as blue and pink boxes, respectively. B, A more detailed view of the region of high RP values, with the UCSC Genome Browser map of three-way (human, mouse, and rat) RP analysis of the candidate cis-regulatory element SOX9cre1, ∼1.1 Mb upstream of SOX9. The presence of such an element has been hypothesized elsewhere (Pfeifer et al. ; Bagheri-Fam et al. ; Pop et al. ; Qin et al. 2004). C, UCSC map of the region surrounding the downstream breakpoint within the SDK2 gene in 17q25.1. There are several regions of high RP scores throughout the breakpoint area, signifying areas of potential regulatory elements of SOX9. Most RP peaks fall outside coding regions. BLAST results of SOX9cre1 suggest that this may be a pseudogene of the PAI-1 mRNA binding protein from chromosome 1. PhyloHMM Cons = phylogenetic hidden Markov model. URLs for SWISS-PROT, TrEMBL, and RefSeq can be found in the Electronic-Database Information section. For information on Multiz, see Blanchette et al. (2004).

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