Treating cartilage injuries and degenerations represents an open surgical challenge. The recent advances in cell therapies have raised the need for a potent off-the-shelf cell source. Intra-articular injections of TGF-β transduced polydactyly chondrocytes have been proposed as a chronic osteoarthritis treatment but despite promising results, the use of gene therapy still raises safety concerns. In this study, we characterized infant, polydactyly chondrocytes during in vitro expansion and chondrogenic re-differentiation. Polydactyly chondrocytes have a steady proliferative rate and re-differentiate in 3D pellet culture after up to five passages. Additionally, we demonstrated that polydactyly chondrocytes produce cartilage-like matrix in a hyaluronan-based hydrogel, namely transglutaminase cross-linked hyaluronic acid (HA-TG). We utilized the versatility of TG cross-linking to augment the hydrogels with heparin moieties. The heparin chains allowed us to load the scaffolds with TGF-β1, which induced cartilage-like matrix deposition both in vitro and in vivo in a subcutaneous mouse model. This strategy introduces the possibility to use infant, polydactyly chondrocytes for the clinical treatment of joint diseases.

Polydactyly is one of the most common hereditary congenital limb malformations in chickens and other vertebrates. The zone of polarizing activity regulatory sequence (ZRS) is critical for the development of polydactyly. The causative mutation of polydactyly in the Silkie chicken has been mapped to the ZRS; however, the causative mutations of other chicken breeds are yet to be established. To understand whether the same mutation decides the polydactyly phenotype in other chicken breeds, we detected the single-nucleotide polymorphism in 26 different chicken breeds, specifically, 24 Chinese indigenous breeds and 2 European breeds. The mutation was found to have fully penetrated chickens with polydactyly in China, indicating that it is causative for polydactyly in Chinese indigenous chickens. In comparison, the mutation showed no association with polydactyly in Houdan chickens, which originate from France, Europe. Based on the different morphology of polydactyly in Chinese and European breeds, we assumed that the trait might be attributable to different genetic foundations. Therefore, we subsequently performed genome-wide association analysis (GWAS) to locate the region associated with polydactyly. As a result, a ~0.39 Mb genomic region on GGA2p was identified. The region contains six candidate genes, with the causative mutation found in Chinese indigenous breeds also being located in this region. Our results demonstrate that polydactyly in chickens from China and Europe is caused by two independent mutation events that are closely located in the chicken genome.

Defects of ciliogenesis have been implicated in a wide range of human phenotypes and play a crucial role in signal transduction and cell-cycle coordination. We used homozygosity mapping in two families with autosomal-recessive short-rib polydactyly syndrome Majewski type to identify mutations in NEK1 as an underlying cause of this lethal osteochondrodysplasia. NEK1 encodes a serine/threonine kinase with proposed function in DNA double-strand repair, neuronal development, and coordination of cell-cycle-associated ciliogenesis. We found that absence of functional full-length NEK1 severely reduces cilia number and alters ciliar morphology in vivo. We further substantiate a proposed digenic diallelic inheritance of ciliopathies by the identification of heterozygous mutations in NEK1 and DYNC2H1 in an additional family. Notably, these findings not only increase the broad spectrum of ciliar disorders, but suggest a correlation between the degree of defective microtubule or centriole elongation and organization and the severity of the resulting phenotype.

Functional characterization of a gene often requires the discovery of the full spectrum of its associated phenotypes. Mutations in the human GLI3 gene have been identified in Greig cepalopolysyndactyly, Pallister-Hall syndrome (PHS), and postaxial polydactyly type-A (PAP-A). We studied the involvement of GLI3 in additional phenotypes of digital abnormalities in one family (UR003) with preaxial polydactyly type-IV (PPD-IV), three families (UR014, UR015, and UR016) with dominant PAP-A/B (with PPD-A and -B in the same family), and one family with PHS. Linkage analysis showed no recombination with GLI3-linked polymorphisms. Family UR003 had a 1-nt frameshift insertion, resulting in a truncated protein of 1,245 amino acids. A frameshift mutation due to a 1-nt deletion was found in family UR014, resulting in a truncated protein of 1,280 amino acids. Family UR015 had a nonsense mutation, R643X, and family UR016 had a missense mutation, G727R, in a highly conserved amino acid of domain 3. The patient with PHS had a nonsense mutation, E1147X. These results add two phenotypes to the phenotypic spectrum caused by GLI3 mutations: the combined PAP-A/B and PPD-IV. These mutations do not support the suggested association between the mutations in GLI3 and the resulting phenotypes. We propose that all phenotypes associated with GLI3 mutations be called "GLI3 morphopathies," since the phenotypic borders of the resulting syndromes are not well defined and there is no apparent genotype-phenotype correlation.

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

Polydactyly is one of the most common hereditary limb malformation characterized by additional digits in hands and/or feet. With extra fingers/toes, which could be very problematic, polydactyly patients are usually treated in early childhood by removing of extra digits with surgery. Genetically, polydactyly is caused by mutations of genes that involve in digit formation.

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis.

Polydactyly, a common heritable limb malformation in vertebrates, is characterized by supernumerary digits. In chickens, basic characteristics and rough dominant genes have been explored in past decades; however, the elaborate pattern of inheritance and the determinant gene remain obscure. In this study, different types of polydactylism were classified by the numbers and the shapes of toes, including the newly defined subtypes of B' and G, for the Beijing fatty chicken, a native breed of chicken from China. Through experiments on hybridization, we demonstrated a complete dominant inheritance of polydactyly instead of an incomplete penetrance or genetic modification of the previous conjecture. In particular, by using the F2 population of the five-digit purebred line of Beijing fatty chicken backcrossed to Shiqiza chicken and by using restriction-site associated DNA based markers, we performed a genome-wide association study on the trait of polydactyly. Furthermore, whole genome resequencing strategy was applied to sweep SNPs across the whole genome. An outlier-based Fst approach was employed to search for signatures of selection, and results indicated that the determinant mutation was found in the region ranging from 8.3 Mb to 8.7 Mb, where the polydactyly candidate gene LMBR1 was located. The G/T mutation of rs80659072 was identified to be highly associated with polydactyly in our resequencing and was validated in random samples from an expanded population. Thus, we confirmed that LMBR1 was the causative gene of polydactyly in the Beijing fatty chicken by using GWAS with restriction-site associated DNA based markers and resequencing.

Polydactyly occurs in some chicken breeds, but the molecular mechanism remains incompletely understood. Combined genome-wide linkage analysis and association study (GWAS) for chicken polydactyly helps identify loci or candidate genes for the trait and potentially provides further mechanistic understanding of this phenotype in chickens and perhaps other species. The linkage analysis and GWAS for polydactyly was conducted using an F2 population derived from Beijing-You chickens and commercial broilers. The results identified two QTLs through linkage analysis and seven single-nucleotide polymorphisms (SNPs) through GWAS, associated with the polydactyly trait. One QTL located at 35 cM on the GGA2 was significant at the 1% genome-wise level and another QTL at the 1% chromosome-wide significance level was detected at 39 cM on GGA19. A total of seven SNPs, four of 5% genome-wide significance (P < 2.98 × 10(-6)) and three of suggestive significance (5.96 × 10(-5)) were identified, including two SNPs (GGaluGA132178 and Gga_rs14135036) in the QTL on GGA2. Of the identified SNPs, the eight nearest genes were sonic hedgehog (SHH), limb region 1 homolog (mouse) (LMBR1), dipeptidyl-peptidase 6, transcript variant 3 (DPP6), thyroid-stimulating hormone, beta (TSHB), sal-like 4 (Drosophila) (SALL4), par-6 partitioning defective 6 homolog beta (Caenorhabditis elegans) (PARD6B), coenzyme Q5 (COQ5), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, etapolypeptide (YWHAH). The GWAS supports earlier reports of the importance of SHH and LMBR1 as regulating genes for polydactyly in chickens and other species, and identified others, most of which have not previously been associated with limb development. The genes and associated SNPs revealed here provide detailed information for further exploring the molecular and developmental mechanisms underlying polydactyly.

The short rib polydactyly syndromes (SRPSs) are a heterogeneous group of autosomal recessive, perinatal lethal skeletal disorders characterized primarily by short, horizontal ribs, short limbs and polydactyly. Mutations in several genes affecting intraflagellar transport (IFT) cause SRPS but they do not account for all cases. Here we identify an additional SRPS gene and further unravel the functional basis for IFT. We perform whole-exome sequencing and identify mutations in a new disease-producing gene, cytoplasmic dynein-2 light intermediate chain 1, DYNC2LI1, segregating with disease in three families. Using primary fibroblasts, we show that DYNC2LI1 is essential for dynein-2 complex stability and that mutations in DYNC2LI1 result in variable length, including hyperelongated, cilia, Hedgehog pathway impairment and ciliary IFT accumulations. The findings in this study expand our understanding of SRPS locus heterogeneity and demonstrate the importance of DYNC2LI1 in dynein-2 complex stability, cilium function, Hedgehog regulation and skeletogenesis.

Jeune asphyxiating thoracic dystrophy (ATD) is an autosomal-recessive chondrodysplasia characterized by short ribs and a narrow thorax, short long bones, inconstant polydactyly, and trident acetabular roof. ATD is closely related to the short rib polydactyly syndrome (SRP) type III, which is a more severe condition characterized by early prenatal expression and lethality and variable malformations. We first excluded IFT80 in a series of 26 fetuses and children belonging to 14 families diagnosed with either ATD or SRP type III. Studying a consanguineous family from Morocco, we mapped an ATD gene to chromosome 11q14.3-q23.1 in a 20.4 Mb region and identified homozygous mutations in the cytoplasmic dynein 2 heavy chain 1 (DYNC2H1) gene in the affected children. Compound heterozygosity for DYNC2H1 mutations was also identified in four additional families. Among the five families, 3/5 were diagnosed with ATD and 2/5 included pregnancies terminated for SRP type III. DYNC2H1 is a component of a cytoplasmic dynein complex and is directly involved in the generation and maintenance of cilia. From this study, we conclude that ATD and SRP type III are variants of a single disorder belonging to the ciliopathy group.

Polydactyly is a highly heterogeneous group of skeletal deformities in clinical and genetic background. The variation spectrum in Chinese sporadic polydactyly has not been comprehensively analyzed. To elucidate genetic variation spectrum and genotype-phenotype correlations in Chinese patients with polydactyly, we conducted comprehensive genetic analysis of patients nationwide using targeted sequencing.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a pathogenic gene of selective tooth agenesis-7 (OMIM#616724). Although the malformation of the digits and fore- and hindlimbs has been reported in Lrp6-deficient mice, it has been rarely discovered in humans with LRP6 mutations. Here, we demonstrate an unreported autosomal dominant LRP6 heterozygous mutation (c.2840 T > C;p.Met947Thr) in a tooth agenesis family with hand polydactyly, and another unreported autosomal dominant LRP6 heterozygous mutation (c.1154 G > C;p.Arg385Pro) in a non-syndromic tooth agenesis family. Bioinformatic prediction demonstrated the deleterious effects of the mutations, and LRP6 structure changes suggested the corresponding functional impairments. Analysis on the pattern of LRP6-related tooth agenesis demonstrated the maxillary lateral incisor was the most affected. Our study report that LRP6 mutation might be associated with hand preaxial polydactyly in humans, which broaden the phenotypic spectrum of LRP6-related disorders, and provide valuable information on the characteristics of LRP6-related tooth agenesis.

Short-rib polydactyly syndromes (SRPS) arise from mutations in genes involved in retrograde intraflagellar transport (IFT) and basal body homeostasis, which are critical for cilia assembly and function. Recently, mutations in WDR34 or WDR60 (candidate dynein intermediate chains) were identified in SRPS. We have identified and characterized Tctex1d2, which associates with Wdr34, Wdr60 and other dynein complex 1 and 2 subunits. Tctex1d2 and Wdr60 localize to the base of the cilium and their depletion causes defects in ciliogenesis. We propose that Tctex1d2 is a novel dynein light chain important for trafficking to the cilium and potentially retrograde IFT and is a new molecular link to understanding SRPS pathology.

The Beijing You chicken is a Chinese native breed with superior meat quality and a unique appearance. The G/T mutation of SNP rs80659072 in the Shh long-range regulator of GGA2 is highly associated with the polydactyly phenotype in some chicken breeds. In the present study, this SNP was genotyped using the TaqMan detection method, and its association with the number of toes was analyzed in a flock of 158 birds of the Beijing You population maintained at the Beijing Academy of Agriculture and Forestry Sciences. Furthermore, the skeletal structure of the digits was dissected and assembled in 113 birds. The findings revealed that the toes of Beijing You chickens were rich and more complex than expected. The plausible mutation rs80659072 in the zone of polarizing activity regulatory sequence (ZRS) in chickens was an essential but not sufficient condition for polydactyly and polyphalangy in Beijing You chickens. Several individuals shared the T allele but showed normal four-digit conformations. However, breeding trials demonstrated that the T allele could serve as a strong genetic marker for five-toe selection in Beijing You chickens.

No abstract available

Cases of polydactyly in natural populations of amphibians have attracted great interest from biologists. At the end of the 1940s, the French biologist Jean Rostand discovered a polymorphic syndrome in some water frog (Anura: Pelophylax) populations that included polydactyly and some severe morphological anomalies (he called it 'anomaly P'). The cause of this anomaly remains unknown for 70 years. In a previous study, we obtained anomaly P in the laboratory in tadpoles of water frogs that developed together with molluscs Planorbarius corneus (Mollusca: Gastropoda) collected in the field. We thus proposed the 'trematode hypothesis', according to which the infectious agent responsible for anomaly P is a trematode species.

In the past decade, mesenchymal stem cells (MSCs) have been widely used for the treatment of osteoarthritis (OA), and exosomes may play a major role. Here, we acquired a special kind of MSCs from the bone marrow of surgically resected tissue from the hand of a patient with polydactyly. Experiments were focused on the role of polydactyly bone marrow-derived MSCs (pBMSCs) in osteoarthritis. The results showed that the pBMSCs had a greater ability than the BMSCs to differentiate into chondrocytes. Mechanistically, the expression of BMP4 was significantly higher in the pBMSCs than it was in the BMSCs. Furthermore, we showed that the migration and proliferation of chondrocytes were stimulated by exosomes secreted by pBMSC (pBMSC-EXOs). Notably, the downregulation of BMP4 in pBMSCs by siRNA inhibited both the chondrogenic differentiation potential of the MSCs and the function of the chondrocytes. In addition, the injection of pBMSC-EXOs and BMSC-EXOs attenuated OA in an OA mouse model, but the pBMSC-EXOs had a superior therapeutic effect compared with that of the BMSC-EXOs. Taken together, the data indicate that pBMSCs have greater ability to differentiate into chondrocytes and regulate chondrocyte formation through BMP4 signaling. Therefore, pBMSC-EXOs may represent a novel treatment for OA.

MYCN, a member of the MYC proto-oncogene family, regulates cell growth and proliferation. Somatic mutations of MYCN are identified in various tumors, and germline loss-of-function variants are responsible for Feingold syndrome, characterized by microcephaly. In contrast, one megalencephalic patient with a gain-of-function variant in MYCN, p.Thr58Met, has been reported, and additional patients and pathophysiological analysis are required to establish the disease entity. Herein, we report two unrelated megalencephalic patients with polydactyly harboring MYCN variants of p.Pro60Leu and Thr58Met, along with the analysis of gain-of-function and loss-of-function Mycn mouse models. Functional analyses for MYCN-Pro60Leu and MYCN-Thr58Met revealed decreased phosphorylation at Thr58 which reduced protein degradation mediated by FBXW7 ubiquitin ligase. The gain-of-function mouse model recapitulated the human phenotypes of megalencephaly and polydactyly, while brain analyses revealed excess proliferation of intermediate neural precursors during neurogenesis, which we determined to be the pathomechanism underlying megalencephaly. Interestingly, the kidney and female reproductive tract exhibited overt morphological anomalies, possibly as a result of excess proliferation during organogenesis. In conclusion, we confirm a MYCN gain-of-function-induced megalencephaly-polydactyly syndrome, which shows a mirror phenotype of Feingold syndrome, and reveal that MYCN plays a crucial proliferative role, not only in the context of tumorigenesis, but also organogenesis.

Apparently balanced chromosomal inversions may lead to disruption of developmentally important genes at the breakpoints of the inversion, causing congenital malformations. Characterization of such inversions may therefore lead to new insights in human development. Here, we report on a de novo inversion of chromosome 7 (p15.2q36.3) in a patient with postaxial polysyndactyly. The breakpoints do not disrupt likely candidate genes for the limb phenotype observed in the patient. However, on the p-arm the breakpoint separates the HOXA cluster from a gene desert containing several conserved noncoding elements, suggesting that a disruption of a cis-regulatory circuit of the HOXA cluster could be the underlying cause of the phenotype in this patient.