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, 179 (4), 2163-72

Canine Polydactyl Mutations With Heterogeneous Origin in the Conserved Intronic Sequence of LMBR1

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Canine Polydactyl Mutations With Heterogeneous Origin in the Conserved Intronic Sequence of LMBR1

Kiyun Park et al. Genetics.

Abstract

Canine preaxial polydactyly (PPD) in the hind limb is a developmental trait that restores the first digit lost during canine evolution. Using a linkage analysis, we previously demonstrated that the affected gene in a Korean breed is located on canine chromosome 16. The candidate locus was further limited to a linkage disequilibrium (LD) block of <213 kb composing the single gene, LMBR1, by LD mapping with single nucleotide polymorphisms (SNPs) for affected individuals from both Korean and Western breeds. The ZPA regulatory sequence (ZRS) in intron 5 of LMBR1 was implicated in mammalian polydactyly. An analysis of the LD haplotypes around the ZRS for various dog breeds revealed that only a subset is assigned to Western breeds. Furthermore, two distinct affected haplotypes for Asian and Western breeds were found, each containing different single-base changes in the upstream sequence (pZRS) of the ZRS. Unlike the previously characterized cases of PPD identified in the mouse and human ZRS regions, the canine mutations in pZRS lacked the ectopic expression of sonic hedgehog in the anterior limb bud, distinguishing its role in limb development from that of the ZRS.

Figures

F<sc>igure</sc> 1.—
Figure 1.—
Genomic analysis of the PPD locus on CFA16. (A) The genomic structure around the MCS regions of the LMBR1 gene. The SNPs (solid triangles) and microsatellites (open triangles) are indicated below. The black bar and shaded regions within the LMBR1 gene represent exon and MCS regions, respectively. The yellow and pink bars represent human syntenic regions on HSA8p and HSA7q. The deduced candidate region (213 kb) around the LMBR1 gene for PPD is designated below. (B) The highly conserved region between human and dog, analyzed using VISTA (http://genome.lbl.gov/vista), is presented. The region with >50% identity analyzed with windows of 100 bp appears on the plot, while those >75% are shaded in pink. The identified PPD mutations, DC-1 and DC-2, are presented (solid triangles). The red and black arrows indicate the sites of two mouse mutations, Hx-G/A and M100081-A/G, and six human PPD mutations: Dutch-C/G (105 bp from 5′-ZRS), Belgian1-A/T (305), Belgian2-T/C (323), Cuban-G/A (404), family B-C/G (621), and families A and C-A/G (740) from northern European descent (Lettice et al. 2003; Gurnett et al. 2007). The phenotypic patterns of preaxial polydactyly are indicated below. (C) A sequence electropherogram of DC-1 (Sapsaree) and DC-2 (Great Pyrenees) mutations read in the reverse direction are shown with hind foot radiographs.
F<sc>igure</sc> 2.—
Figure 2.—
The genealogical relationships between different breeds and their haplotypes. (A) The backbone of the phylogenetic tree (including dotted lines) and bootstrap numbers are adopted from a previous report (Parker et al. 2004). The dashed line includes breeds analyzed previously (Kim et al. 2001) or in this study. The numbers with asterisks represent the affected haplotypes from Asian and Western breeds. (B) The SNP markers used to construct the haplotypes are Int16, Int15, Int6, ZRSi5, ShotR2, and Int3. The shaded circles represent haplotypes found in Western breeds, and the open circles (also for numbers in A) signify the unique haplotypes in Asian breeds. The relationships among different haplotypes were analyzed using the NETWORK program (http://www.fluxus-engineering.com) and are shown schematically. The double dashes specify two mismatches.
F<sc>igure</sc> 3.—
Figure 3.—
Transcriptional enhancer activities of the ZRS and pZRS regions. (A) The canine ZRS or pZRS sequence with or without the PPD mutation was inserted 269 bp upstream of the luciferase gene under the control of an SV40 promoter. The full-length insert includes a 1917-bp region containing pZRS and ZRS. The asterisks represent the mutational positions. The ZRS region (1218–1917 bp) is a highly conserved region among humans, dogs, and mice in which the core ZRS (1571–1750 bp) is located. The boxes with a solid line (left) represent the sizes of inserts with or without mutations (*). The broken line is used as a reference to designate the size of the full-length insert. After transfection of P19 embryonal carcinoma cells with the appropriate constructs, luciferase activities were determined, standardized with β-galactosidase, and shown as relative values calculated by setting the activity of an empty vector (pGL2, promoter only) as one. The solid bars (right) represent expressions from the constructs with mutations. Data were obtained in triplicate. (B) Transcriptional activity of constructs containing a human ZRS with or without mutations. The constructs with mutations contain nucleotide changes for Dutch-C/G, Belgian1-A/T, Belgian2-T/C, and Cuban-G/A. Data were obtained in triplicate.
F<sc>igure</sc> 4.—
Figure 4.—
Spatio-temporal patterns of gene expression in a developing embryo. (A, D, and E) Whole mounts, lateral views, and ventral at the left. (B, C, and F–K) Dorsal views and anterior at the top. (A) Shh expression in dog embryos at day 25, equivalent to mouse E11.0. (B and C) Shh expression in the ZPA region of the hind limb bud in wild-type (B, WT) and PPD (C, MUT) dogs. (D and E) β-Gal staining patterns of transgenic mouse embryos at E11.5 with the wild-type pZRS (D) and the wild-type pZRS plus ZRS (E). The extents of the inserts in the transgenic constructs are shown with boxes (solid lines) at the bottom. The asterisk in D indicates β-gal staining in the olfactory region. (F–I) β-Gal staining in the hind limb buds with wild-type pZRS-ZRS at E11.5 (F) and E13.5 (H) with mutant pZRS-ZRS at E11.5 (G) and E13.5 (I). The asterisk in I indicates the position of a developing second digit. (J and K) β-Gal staining of forelimb buds in wild-type (J) and mutant (K) embryos at E13.5.

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