A gene(s) for all-trans-retinoic acid-induced forelimb defects mapped and confirmed to murine chromosome 11

Abstract

All-trans-retinoic acid (RA) induces various anatomical limb dysmorphologies in mice dependent on the time of exposure. During early limb development, RA induces forelimb ectrodactyly (digital absence) with varying susceptibilities for different inbred mouse strains; C57BL/6N are highly susceptible while SWV are resistant. To isolate the genetic basis of this defect, a full-genome scan was performed in 406 backcross fetuses of F(1) males to C57BL/6N females. Fetuses were exposed via a maternal injection of 75 mg of RA per kilogram of body weight on gestational day 9.25. The genome-wide analysis revealed significant linkage to a chromosome 11 locus near D11Mit39 with a maximum LOD score of 9.0 and to a chromosome 4 locus near D4Mit170. An epistatic interaction was detected between loci on chromosome 11 (D11Mit39) and chromosome 18 (D18Mit64). Linkage to the chromosome 11 locus (D11Mit39) was confirmed in RA-treated backcross fetuses of F(1) females to C57BL/6N males. Loci associated with bone density/mass in both human and mouse were previously detected in the same region, suggesting a mechanistic linkage with bone homeostasis. The human syntenic region of this locus has been previously linked to Meckel syndrome; the phenotype includes postaxial polydactyly, an ectopic digital defect hypothesized to be induced by a common molecular pathway with ectrodactyly.

Figure 1.—

Forelimb defects induced by RA on GD 9.25. (A) Normal paw of right limb, score of 1; (B) right limb ectrodactyly digit 1, score of 3; (C) right limb ectrodactyly digits 4 and 5 and reduction of ulna, score of 6; and (D) right limb ectrodactyly digits 3, 4, and 5 and absence of ulna, score of 9.

Figure 2.—

Whole-genome scan of RA-induced forelimb defects in BC1 fetuses generated by QTL Cartographer. The solid curve and solid horizontal line show the LOD score and the 95% LOD score significance threshold, respectively, for the All-1 trait, whereas the dashed curve and the dashed horizontal line show the LOD score and the 95% LOD score significance threshold for the QT-1 trait. The thresholds established by Map Manager QTX and QTL Cartographer were similar.

Figure 3.—

Chromosome 11 linkage maps of RA-induced forelimb defects generated by QTL Cartographer (A) in BC1 fetuses and (B) in BC2 fetuses. The marker positions correspond to linkage distances found in the Mouse Genome Informatics Database (MGI; http://www.informatics.jax.org). The solid horizontal line shows the 95% LOD score significance threshold, whereas the dashed horizontal line shows the 99.9% LOD score significance threshold. According to the linkage map distance in MGI, D11Mit293 is located at 75 cM, but this marker is located more distally than D11Mit48 according to the physical map distance.

Figure 3.—

Chromosome 11 linkage maps of RA-induced forelimb defects generated by QTL Cartographer (A) in BC1 fetuses and (B) in BC2 fetuses. The marker positions correspond to linkage distances found in the Mouse Genome Informatics Database (MGI; http://www.informatics.jax.org). The solid horizontal line shows the 95% LOD score significance threshold, whereas the dashed horizontal line shows the 99.9% LOD score significance threshold. According to the linkage map distance in MGI, D11Mit293 is located at 75 cM, but this marker is located more distally than D11Mit48 according to the physical map distance.

Figure 4.—

The two-LOD confidence interval of the Rafar locus, its syntenic region in humans, and candidate genes in this region. Linkage map distances in mice were derived from MGI (http://www.informatics.jax.org), and physical map distances were derived from Ensembl Mouse and Human Genome Database (http://www.ensembl.org). Identified QTL for osteoporosis measuring bone mineral density (BMD), peak bone mass (PBM), periosteal circumference (PC), or bone size variation (BSV) and for Meckel syndrome are included.