Positional cloning of the gene LIMBIN responsible for bovine chondrodysplastic dwarfism - PubMed (original) (raw)

. 2002 Aug 6;99(16):10549-54.

doi: 10.1073/pnas.152337899. Epub 2002 Jul 22.

Marika Takami, Tomoko Oguni, Takehito Tsuji, Kazuhiro Yoneda, Hiroaki Sato, Naoya Ihara, Tomohito Itoh, Srinivas R Kata, Yuji Mishina, James E Womack, Yasuo Moritomo, Yoshikazu Sugimoto, Tetsuo Kunieda

Affiliations

Positional cloning of the gene LIMBIN responsible for bovine chondrodysplastic dwarfism

Haruko Takeda et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Chondrodysplastic dwarfism in Japanese brown cattle is an autosomal recessive disorder characterized by short limbs. Previously, we mapped the locus responsible for the disease on the distal end of bovine chromosome 6. Here, we narrowed the critical region to approximately 2 cM by using linkage analysis, constructed a BAC and YAC contig covering this region, and identified a gene, LIMBIN (LBN), that possessed disease-specific mutations in the affected calves. One mutation was a single nucleotide substitution leading to an activation of a cryptic splicing donor site and the other was a one-base deletion resulting in a frameshift mutation. Strong expression of the Lbn gene was observed in limb buds of developing mouse embryos and in proliferating chondrocytes and bone-forming osteoblasts in long bones. These findings indicate that LBN is responsible for bovine chondrodysplastic dwarfism and has a critical role in a skeletal development.

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Figures

Fig 1.

Fig 1.

Identification of bovine LBN gene. (A) Physical map consisting of YAC and BAC clones in the critical region on BTA 6. Informative and uninformative markers in our pedigree, STS markers for genes and ends of the clones are shown in blue, green, red, and black, respectively. Dotted lines show regions corresponding to HSA 4. (B) Transcription units on the human orthologous region on HSA 4p16. Genes and UniGene clusters localized by using the GRL database are shown in red and purple, respectively. C4000112, C4000113, and C4000114 are putative exons on the UCSC human genome database. The black boxes indicate a part of the human LBN gene. (C) Genomic structure of bovine LBN. M, translation initiation codon; *, stop codon.

Fig 2.

Fig 2.

Pedigrees with BCD and haplotypes consisting of nine markers in the critical region on BTA 6. Green, haplotype associated with a C1356T mutation; yellow, haplotype associated with a 2054–2055delCAinsG mutation. The number of animals is shown in parentheses. (A) This pedigree includes grandsire A and sire B, which were identified as carriers of the disease, 90 affected offspring, 90 dams of the affected offspring, and some ancestors. The shaded diamonds represent affected offspring. White with diagonal line denotes animals not genotyped. (B) Haplotypes of nine microsatellites (Left) for grandsire A, sire B, and the 119 affected offspring are shown.

Fig 3.

Fig 3.

Mutation analysis in bovine LBN gene. Sequence chromatograms of affected calves having heterozygous haplotypes (Left), normal calves (Center), and subcloned mutant alleles (Right). Amino acid sequence changes caused by mutations. (A) cDNA sequence in exon 11. A 56-bp deletion is observed at position 1355–1410 in the mutant allele, which is schematically illustrated. The deletion causes a frameshift, producing a premature termination at codon 464 with an extension of 10 aberrant amino acids. (B) Genomic sequence in exon 11. C to T substitution is observed at position 1356 in the affected calf (C1356T), which creates a cryptic splice donor site in exon 11 and leads to the improper splicing identified in the affected cDNA sequence. (C) cDNA and genomic sequences in exon 14. CA to G substitution is observed at positions 2054 and 2055 in the mutant allele (2054–2055delCAinsG), which causes a frameshift producing a premature termination at codon 706 with an extension of 21 aberrant amino acids.

Fig 4.

Fig 4.

Lbn expression in mouse tissues. (A) Northern blot analysis. An ≈4.5-kb Lbn mRNA can be seen in heart and kidney in adult, long bone, and cranial bone at P14 and whole embryos at all stages tested. An additional ≈7.0-kb band is faintly visible in kidney. (Lower) Loading controls with a G3pdh probe. (B) Whole-mount in situ hybridization with Lbn antisense probe (Left) and sense probe as a control (Right). A definite signal is observed in fore- and hindlimb buds (arrow), branchial arches, and facial primordia. The magnification is ×12.5.

Fig 5.

Fig 5.

Lbn expression in mouse long bones. In situ hybridization with Lbn antisense probe (Left) and sense probe as a control (Right). (A_–_D) Mouse tibia at E17. Lbn is expressed strongly in proliferating chondrocytes and weakly in other resting and hypertrophic chondrocytes in the epiphysial growth plates. (E_–_J) Mouse tibia at P35. Lbn is expressed strongly in proliferating chondrocytes in the epiphysial growth plates, osteoblasts, and osteoclasts and weakly in osteocytes in the metaphysis. RC, resting chondrocytes; PC, proliferating chondrocytes; HC, hypertrophic chondrocytes; OB, osteoblasts; OCL, osteoclasts; OCY, osteocytes; BM, bone marrow. The magnification is shown on the right.

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