- Background: Polydactyly is one of the most common congenital limb dysplasia in many animal species. - Results: Our results showed that the PPD in the sampled pigs were not related to previously reported variants. - Polydactyly is one of the most commonly observed con- genital limb malformations and ciliopathies. - Based on the anatomic position of the additional digits, polydactyly can be classified into preaxial polydactyly (PPD), postaxial polydactyly and central polydactyly [3].. - Previous pathological researches have shown that most of the PPD abnormal cases follow an autosomal dominant in- heritance pattern and a few express an autosomal recessive pattern of inheritance [4]. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - In the process of limb development, the Zone of Polarizing Activity (ZPA) signaling center determines the formation of anterior- posterior axis of limb buds [7]. - The ectopic expression of SHH gene in the anterior part of limb bud is the main causative agent of PPD. - have found that the disruption of Zone of Polarizing Activ- ity Regulatory Sequence (ZRS), a long-range cis-regulator for SHH located in the fifth intron of LMBR1 gene, results in the ectopic expression of SHH responsible for PPD.. - In the early stages of embryonic development, Gli3 po- larizes the limb into anterior-posterior axis through the antagonism with HAND2 [16, 17]. - Among all of these identified candidate genes, majority of them are involved in the SHH signaling pathway or ciliogenesis process.. - All of the four PPD pigs were affected at one side of the forelimb, the F1-a1 male (Fig. - 1c) were af- fected on the left side of the forelimb, the F1-a2 female (Fig. - 1e) were affected on the right side of the forelimb.. - To show the detail of the additional digit and for fur- ther classification, two affected pigs (F1-a1 and F1-a3) were euthanized to get the hooves for radiograph ana- lysis. - Except for an extra toe on anterior side of the forelimb, there were no additional abnormal phenotypes observed in appearance and behavior.. - We scanned the homologue genes of all these candidate regions, but there were no mutations in the protein coding regions, untranslated regions (UTR) se- quences, transcription factor binding sites and other highly conserved sequences surrounding the PPD affected individual’s candidate genes. - Variants in intergenic region (IGR), intron and other regions were not significantly dif- ferentiated between PPD affected and normal pigs, so we ruled out the possibility of the previously reported candi- date genes as the causing loci for this PPD phenotype in our study.. - between PPD affected and unaffected groups based on whole-genome SNPs. - Moreover, the fixation index (F ST ) [35] analyses results showed that the region of ABCC4 gene on SSC11 was highly differentiated between the affected and normal groups based on both SNPs (Fig. - 1 Pedigree and phenotypes information of the four PPD affected pigs. - a Pedigree of the three PPD affected pigs and one random case.. - b Phenotypes of the affected male (F1-a1) which expressing a preaxial polydactyly on left forelimb. - f Radiograph and phenotype of the affected male (F1-a1). - g Radiograph and phenotype of the affected male (F1-a3). - G) in the third exon of ABCC4 (Additional file 6: Figure S1). - b F ST plot between the affected and normal individuals based on whole-genome SNPs. - c F ST plot between the affected and normal individuals based on whole-genome INDELs. - From these evidences, we con- cluded that ABCC4 gene on SSC11 is probably associated with an important role in the early limb formation stage and may affect the formation of PPD.. - In order to further compare the genetic differentiation be- tween PPD affected and normal groups in the two major candidate genes (SHH and LMBR1 (ZRS region was cov- ered)) and ABCC4, pairwise F ST analysis was carried out based on SNPs and INDELs (Fig. - For further identification of the causing mutation of PPD, the top ten highly associated SNPs identified through whole genome association study (Table 1) were selected to calculate the derived allele frequency (DAF) (Fig. - Furthermore, to investigate the genotypes of the highly differentiated INDELs between affected and normal pigs, 14 INDELs from the highly differentiated window re- gions (Additional file 7: Table S6) with which F ST >. - Table 1 Genome association analysis and genotyping results of the top 10 SNPs. - G) on the third exon of ABCC4, which resulting in the change of isoleucine to threonine. - Moreover, our result showed that the allele frequency of this mutation is 0.3529 in the two families, but we didn’t detect this homozygous mutation (G/G) in the large unpublished samples of normal individuals (559 in- dividuals covering 66 domestic pig breeds and wild boars) from our lab (Additional file 13: Table S9). - a-c F ST plot based on SNPs of three candidate genes. - d-f F ST plot based on INDELs of these genes. - g DAF analysis of the top ten highly associated SNPs. - The statistical results of the derived allele frequency showed that there was a great difference between population in this study and unpublished data.. - The potential association between this nonsynonymous mutation and PPD phenotype by genotyping illustrated that all of the four affected pigs were mutant homozy- gous (G/G). - In addition, the most significant SNP (rs791053563, P identified by association study was located in the intron region, and interestingly, all the affected in- dividuals were homozygous wild-type at this locus, while the normal individuals were mutant genotypes at this. - Besides, we have verified this SNP in the large normal population (n = 559. - 5a and all of the ten highly associated and significant SNPs are shown in Fig. - 4 Conservation analysis and structure prediction of the missense mutation site. - a Cross-species alignment of amino acids sequences of the SNP in ABCC4 . - b Changes in the primary structure of this amino acid residues. - c The full 3D structure of the protein encoded by ABCC4 . - In addition, we predicted whether this amino acid substitution has an impact on the biological function of the protein encoded by ABCC4 through PRO- VEAN [42]. - The results showed that the mu- tated residue is located in the alpha helices and is smaller than the wild-type residue. - SHH signal of anterior-posterior axial plays an import- ant role in the early limb development and patterning.. - 5 Haplotype pattern around ABCC4 and the genotype of the top ten highly associated and significant SNPs. - b The genotype of the top ten highly associated and significant SNPs. - ABCC4, a member of the ATP-binding cassette trans- porter family is also known as multidrug resistance- associated protein 4 (MRP4) [50]. - As an important transmembrane protein, the mutation or mis-regulation of ABCC4 results in the disorder of PGE2 transmembrane transport and further results in the misfunction of the stimulation of anterograde IFT through EP4 and protein kinase A (PKA). - reported that the T804M mutation in ABCC4 showed cilium loss and cilium-associated phenotypes in zebra- fish, including ventrally curved body axis, hydrocephalus, abnormal otolith number and laterality defects of the brain and other organs.. - In our study, based on two uncorrelated families’ asso- ciation analysis showed that some SNPs and INDELs in ABCC4 were highly associated with PPD phenotype. - Mutation of the residue might have disturbed this. - function, resulting in the inactivation of ABCC4 and further into the disorder of ciliogenesis.. - Quality control for removing the low-quality reads was done based on the following criteria: Up to 10% of the read bases include “N” content in each sequenced read, up to 50% of the read bases include low quality (Q <. - RealignerTargetCreator, IndelRealigner, and BaseRecali- brator tools in the Genome Analysis Toolkit (GATK) (ver.. - between the affected and normal groups based on whole- genome SNPs and INDELs with 10 kb window and 2 kb overlapping slides using VCFtools . - In addition, F ST of the three candidate genes (ABCC4, LMBR1 and SHH) were calculated based on every SNP and INDEL loci.. - Meanwhile, based on the identified highly associated variations, genotyping and derived allele fre- quency were performed. - cmbi.ru.nl/hope/) to predict this amino acid substitution’s impact on the biological function and structural effect of the protein. - PROVERAN [42] will give a score of the vari- ant and the default threshold is − 2.5, that is: variants with a score equal to or below − 2.5 are considered “deleteri- ous” and variants with a score above − 2.5 are considered. - All the prediction was based on the protein sequence of UniPortKB (ID: A0A287A6F6_PIG) or Ensembl (Transcript ID: ENSSSCT . - ENCODE indicate that the SNPs which were located in the homologous sequences of human counterparts have ENCODE annotations. - in the homologous sequences of transcription factor DNA-binding motifs of human counterparts. - The highly differentiated windows based on whole-genome SNPs. - The distribution of the highly differentiated SNPs.. - The highly differentiated regions based on INDELs between PPD affected and normal pigs. - Plots of whole-genome screening of puta- tively loci which associated with PPD in the large family. - (B) F ST plot of the whole selected SNPs between 3 affected and normal individuals. - (C) F ST plot between the 3 affected and normal individuals based on whole selected INDELs. - (D) Manhattan plot of whole-genome association analysis based on whole SNPs of PPD phenotype.. - Schematic diagram of the relative position of ABBC4 and DZIP1 . - The F ST comparison between three candidate genes based on all SNPs and INDELs. - (A-C) F ST plot based on all SNPs. - (D-F) F ST plot based on all INDELs.. - Phased genotyping results of the top ten highly associated SNPs. - represent the missense mutation SNP which located in the third exon of ABCC4 . - The genotyping of the highly differentiated INDELs between PPD affected and normal pigs. - The allele frequency of the top ten highly associated SNPs between population in this study and unpublished data.. - The genotype of the top ten highly associated SNPs between population in this study and the 559 unpublished data set. - The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - We obtained written informed consent to use the animals in this study from two owners of the pigs respectively. - The care and treatment of the pigs comply with the guidelines of animal use protocols approved by the Animal Care and Ethics Committee of Kunming Institute of Zoology, Chinese Academy of Science, China (Approval ID: SYDW-2015012).. - Polydactyly of the hand. - The results of surgery for polydactyly of the thumb. - Advances in the Molecular Genetics of Non-syndromic Syndactyly. - Sonic hedgehog mediates the polarizing activity of the ZPA. - A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. - Preaxial polydactyly: interactions among ETV, TWIST1 and HAND2 control anterior-posterior patterning of the limb. - Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. - Hoxd and Gli3 interactions modulate digit number in the amniote limb. - Ciliary abnormalities due to defects in the retrograde transport protein DYNC2H1 in short-rib polydactyly syndrome. - Struhl G: hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen. - Ingham PW: iguana encodes a novel zinc-finger protein with coiled-coil domains essential for hedgehog signal transduction in the zebrafish embryo
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