Dwarfism and Altered Craniofacial Development in Rabbits Is Caused by a 12.1 kb Deletion at the HMGA2 Locus

Abstract

The dwarf phenotype characterizes the smallest of rabbit breeds and is governed largely by the effects of a single dwarfing allele with an incompletely dominant effect on growth. Dwarf rabbits typically weigh under 1 kg and have altered craniofacial morphology. The dwarf allele is recessive lethal and dwarf homozygotes die within a few days of birth. The dwarf phenotype is expressed in heterozygous individuals and rabbits from dwarf breeds homozygous for the wild-type allele are normal, although smaller when compared to other breeds. Here, we show that the dwarf allele constitutes a ∼12.1 kb deletion overlapping the promoter region and first three exons of the HMGA2 gene leading to inactivation of this gene. HMGA2 has been frequently associated with variation in body size across species. Homozygotes for null alleles are viable in mice but not in rabbits and probably not in humans. RNA-sequencing analysis of rabbit embryos showed that very few genes (4-29 genes) were differentially expressed among the three HMGA2/dwarf genotypes, suggesting that dwarfism and inviability in rabbits are caused by modest changes in gene expression. Our results show that HMGA2 is critical for normal expression of IGF2BP2, which encodes an RNA-binding protein. Finally, we report a catalog of regions of elevated genetic differentiation between dwarf and normal-size rabbits, including LCORL-NCAPG, STC2, HOXD cluster, and IGF2BP2 Levels and patterns of genetic diversity at the LCORL-NCAPG locus further suggest that small size in dwarf breeds was enhanced by crosses with wild rabbits. Overall, our results imply that small size in dwarf rabbits results from a large effect, loss-of-function (LOF) mutation in HMGA2 combined with polygenic selection.

Keywords: IGF2BP2; RNA-seq; body size; mtDNA; whole-genome sequencing.

Figure 1

The dwarf and wild-type phenotypes in rabbits. (A) A dwarf individual (Dw/dw) side-by-side with a normal-sized New Zealand white rabbit. (B) Dwarf individual (Dw/dw). These animals typically weigh < 1 kg, have compact and rounded bodies, short noses, a disproportionately large head when compared to rest of the body, and small ears. (C and D) Newborn dw/dw individual (peanut) side by side with a Dw/dw (dwarf) littermate. dw/dw animals are born smaller than their littermates and have a disproportionate body conformation with cone-shaped heads, small ears and limbs, and prominent eyes, which are evident at the time of birth. (E) Detail of the head of a dw/dw individual. Photo credits: (A) Sara Gutiérrez Albarran; (B–E) Javier Lopez.

Figure 2

Mapping of the dwarf locus to a region on chromosome 4. (A) Homozygosity mapping and genetic differentiation. The red and black lines represent two statistics that summarize levels and patterns of homozygosity across chromosome 4. The red line represents the proportion of SNPs previously identified in domestic rabbits that were monomorphic in dw/dw individuals (summarized in windows of 2000 SNPs iterated every 400 SNPs); the black line represents the proportion of SNPs polymorphic in Dw/dw individuals that were monomorphic in dw/dw individuals (summarized in windows of 500 SNPs iterated every 100 SNPs). The blue line represents F_ST (a measure of genetic differentiation) between dw/dw and Dw/dw individuals. (B) A deletion overlapping the promoter region and the first three exons of HMGA2 associated with the dwarf allele. The lines represent the normalized number of reads mapping to nonoverlapping windows of 1000 bp across the HMGA2 region. The data for the dw/dw and Dw/dw pools are given in red at the top, while the remaining panels summarize normalized read depth for six breeds that do not carry the dwarf allele (Belgian hare, Champagne d’argent, Dutch, Flemish giant, French lop, and New Zealand white). The gene model for HMGA2 is drawn in red across the top of the figure and is based on the Ensembl annotation.

Figure 3

Differential gene expression among the three HMGA2 genotypes and mtDNA copy number. (A) Statistically significant changes in gene expression between HMGA2 genotypes evaluated using DESeq2 (Love et al. 2014). Log fold change vs. mean expression for the 21,588 genes included in our analysis for the different contrasts (upper panel: dw/dw vs. Dw/dw; middle panel: dw/dw vs. Dw/Dw; lower panel: Dw/dw vs. Dw/Dw). Red indicates significant overexpression, blue indicates significant underexpression, and gray indicates no significant change. Mitochondrial genes are circled. The data points for HMGA2 and IGF2BP2 are indicated. Mean FPKM values for all transcripts are given in File S3. (B) Doughnut charts summarizing levels of expression for HMGA2, IGF2BP2, and protein-coding genes encoded in mtDNA. Both technical replicates have been combined. Each chart represents one gene and the different individuals are represented by different colors. Black bars separate the genotypes (green: Dw/Dw; brown: dw/dw; and cyan: Dw/dw). Numbers outside of each chart indicate the mean FPKM value for each genotype. (C) mtDNA abundance in embryos of the different HMGA2 genotypes. Barplot indicating the number of mtDNA copies relative to the autosomes for the three HMGA2 genotypes. The bars reflect the average values and SD among three dw/dw (peanut), four Dw/dw (dwarf), and three Dw/Dw (wild-type) individuals. qPCR1 and qPCR2 represent two independent reactions based on different sets of primers targeting the rabbit mitochondrial genome. FPKM, fragments per kilobase million; Ind, individual; mtDNA, mitochondrial DNA; qPCR, quantitative PCR.

Figure 4

Selective sweep screen for additional genomic regions associated with small size in Netherland dwarf rabbits. (A) Genome-wide distribution of d_i. Values were summarized in 500 kb windows iterated every 250 kb. The red and blue lines indicate the top 1 and 5% of the empirical distribution, respectively. Unmapped contigs were included in the analysis but are not represented in the figure. (B) F_ST and nucleotide diversity (π) in the LCORL-NCAPG region. Each π and F_ST data point is based on a sliding window analysis using 500 kb windows with 250 kb steps. Annotated protein-coding genes are drawn across the bottom of each region, and putative candidate genes are highlighted. Nucleotide diversities of wild rabbits were averaged over three populations of wild rabbits from France (Caumont, La Roque, and Villemolaque) and two populations of wild rabbits from the Iberian Peninsula (Guadalajara and Zaragoza) belonging to the Oryctolagus cuniculus cuniculus subspecies; data from Carneiro et al. (2014). BH, Belgian hare; CH, Champagne d’argent; Chr, chromosome; DU, Dutch; FG, Flemish giant; FL, French lop; ND, Netherland dwarf; NZ, New Zealand white.