Jeune Asphyxiating Thoracic Dystrophy Research Articles

DYNC2H1 hypomorphic or retina-predominant variants cause nonsyndromic retinal degeneration

PurposeDetermining the role of DYNC2H1 variants in nonsyndromic inherited retinal disease (IRD). MethodsGenome and exome sequencing were performed for five unrelated cases of IRD with no identified variant. In vitro assays were developed to validate the variants identified (fibroblast assay, induced pluripotent stem cell [iPSC] derived retinal organoids, and a dynein motility assay). ResultsFour novel DYNC2H1 variants (V1, g.103327020_103327021dup; V2, g.103055779A>T; V3, g.103112272C>G; V4, g.103070104A>C) and one previously reported variant (V5, g.103339363T>G) were identified. In proband 1 (V1/V2), V1 was predicted to introduce a premature termination codon (PTC), whereas V2 disrupted the exon 41 splice donor site causing incomplete skipping of exon 41. V1 and V2 impaired dynein-2 motility in vitro and perturbed IFT88 distribution within cilia. V3, homozygous in probands 2–4, is predicted to cause a PTC in a retina-predominant transcript. Analysis of retinal organoids showed that this new transcript expression increased with organoid differentiation. V4, a novel missense variant, was in trans with V5, previously associated with Jeune asphyxiating thoracic dystrophy (JATD). ConclusionThe DYNC2H1 variants discussed herein were either hypomorphic or affecting a retina-predominant transcript and caused nonsyndromic IRD. Dynein variants, specifically DYNC2H1 variants are reported as a cause of non syndromic IRD.

IFT80 Improves Invasion Ability in Gastric Cancer Cell Line via ift80/p75NGFR/MMP9 Signaling.

The assembly and maintenance of cilia depend on intraflagellar transport (IFT) proteins, which play an important role in development and homeostasis. IFT80 is a newly defined IFT protein and partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III, both characterized by abnormal skeletal development. However, the role and mechanism of IFT80 in the invasion of gastric cancer is unknown. We established SGC-7901 and MKN-45 gastric cancer cell lines that stably overexpressed IFT80, as verified by quantitative reverse transcription-PCR, Western blot, and immunofluorescence. Matrix metalloproteinase-9 (MMP9) plays an important role in tumor invasion, and its expression was assessed by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence. The invasion ability of IFT80 on SGC-7901 and MKN-45 cells was examined by the Matrigel invasion assay. The relationship between p75NGFR, and the p75NGFR antagonists, PD90780 and IFT80, were detected by quantitative reverse transcription-PCR and Western blotting. We first detected an IFT80 expression pattern, and found that IFT80 was highly expressed in gastric cancer clinical samples. Overexpression of IFT80 in the gastric cancer cell lines, SGC-7901 and MKN-45, led to lengthening cilia. Additionally, overexpression of IFT80 significantly improved proliferation and invasion, but inhibited apoptosis, in gastric cancer cells. We further found that overexpression of IFT80 increased p75NGFR and MMP9 mRNA and protein expression. Treatment with the p75NGFR antagonist PD90780 inhibited the increased invasion ability resulting from overexpression of IFT80 in SGC-7901 and MKN-45 gastric cancer cells. Thus, these results suggest that IFT80 plays an important role in invasion of gastric cancer through regulating the ift80/p75NGFR/MMP9 signal pathways.

Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis

SummaryCiliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles.

Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes

BackgroundCiliopathies are an extensive group of autosomal recessive or X-linked disorders with considerable genetic and clinical overlap, which collectively share multiple organ involvement and may result in lethal or viable...

Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation.

Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates cartilage formation. To define the role and mechanism of IFT80 in chondrocyte function and cartilage formation, we generated a Col2α1; IFT80f/f mouse model by crossing IFT80f/f mice with inducible Col2α1-CreER mice, and deleted IFT80 in chondrocyte lineage by injection of tamoxifen into the mice in embryonic or postnatal stage. Loss of IFT80 in the embryonic stage resulted in short limbs at birth. Histological studies showed that IFT80-deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in the postnatal stage led to mouse stunted growth with shortened growth plate but thickened articular cartilage. Defects of ciliogenesis were found in the cartilage of IFT80-deficient mice and primary IFT80-deficient chondrocytes. Further study showed that chondrogenic differentiation was significantly inhibited in IFT80-deficient mice due to reduced hedgehog (Hh) signaling and increased Wnt signaling activities. These findings demonstrate that loss of IFT80 blocks chondrocyte differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction, which in turn alters epiphyseal and articular cartilage formation.

A founder CEP120 mutation in Jeune asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies.

Jeune asphyxiating thoracic dystrophy (JATD) is a skeletal dysplasia characterized by a small thoracic cage and a range of skeletal and extra-skeletal anomalies. JATD is genetically heterogeneous with at least nine genes identified, all encoding ciliary proteins, hence the classification of JATD as a skeletal ciliopathy. Consistent with the observation that the heterogeneous molecular basis of JATD has not been fully determined yet, we have identified two consanguineous Saudi families segregating JATD who share a single identical ancestral homozygous haplotype among the affected members. Whole-exome sequencing revealed a single novel variant within the disease haplotype in CEP120, which encodes a core centriolar protein. Subsequent targeted sequencing of CEP120 in Saudi and European JATD cohorts identified two additional families with the same missense mutation. Combining the four families in linkage analysis confirmed a significant genome-wide linkage signal at the CEP120 locus. This missense change alters a highly conserved amino acid within CEP120 (p.Ala199Pro). In addition, we show marked reduction of cilia and abnormal number of centrioles in fibroblasts from one affected individual. Inhibition of the CEP120 ortholog in zebrafish produced pleiotropic phenotypes characteristic of cilia defects including abnormal body curvature, hydrocephalus, otolith defects and abnormal renal, head and craniofacial development. We also demonstrate that in CEP120 morphants, cilia are shortened in the neural tube and disorganized in the pronephros. These results are consistent with aberrant CEP120 being implicated in the pathogenesis of JATD and expand the role of centriolar proteins in skeletal ciliopathies.

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.

Combined NGS Approaches Identify Mutations in the Intraflagellar Transport Gene IFT140 in Skeletal Ciliopathies with Early Progressive Kidney Disease

Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.

IFT80 is essential for chondrocyte differentiation by regulating Hedgehog and Wnt signaling pathways

Partial mutation of intraflagellar transport 80 (IFT80) in humans causes Jeune asphyxiating thoracic dystrophy (JATD) and short-rib polydactyly (SRP) syndrome type III. These diseases are autosomal recessive chondrodysplasias that share clinical similarities, including shortened long bones and constricted thoracic cage. However, the role and mechanism of IFT80 in the regulation of chondrocyte differentiation and function remain largely unknown. We hypothesize that IFT80 is required for the formation and function of cilia and plays a critical role in chondrogenic differentiation by regulating Hedgehog (Hh) and Wingless (Wnt) signaling pathways. To test this hypothesis, we first analyzed the IFT80 expression pattern and found that IFT80 was predominantly expressed in growth plate chondrocytes and during chondrogenic differentiation. Silencing IFT80 impaired cilia formation and chondrogenic differentiation in mouse bone marrow derived stromal cells (BMSCs), and decreased the expression of chondrocyte marker genes—collagen II and aggrecan. Additionally, silencing IFT80 down-regulated Hh signaling activity whereas up-regulated Wnt signaling activity. The overexpression of Gli2 in IFT80-silenced cells promoted chondrogenesis and recovered the chondrogenic deficiency from IFT80 silencing. Overall, our results demonstrate that IFT80 is essential for chondrocyte differentiation by regulating the Hh and Wnt signaling pathways.

The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis

Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia, which play important roles in development and homeostasis. IFT80 is a newly defined IFT protein. Partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III with abnormal skeletal development. However, the role and mechanism of IFT80 in osteogenesis is unknown. Here, we first detected IFT80 expression pattern and found that IFT80 was highly expressed in mouse long bone, skull, and during osteoblast differentiation. By using lentivirus-mediated RNA interference (RNAi) technology to silence IFT80 in murine mesenchymal progenitor cell line-C3H10T1/2 and bone marrow derived stromal cells, we found that silencing IFT80 led to either shortening or loss of cilia and the decrease of Arl13b expression — a small GTPase that is localized in cilia. Additionally, silencing IFT80 blocked the expression of osteoblast markers and significantly inhibited ALP activity and cell mineralization. We further found that IFT80 silencing inhibited the expression of Gli2, a critical transcriptional factor in the hedgehog signaling pathway. Overexpression of Gli2 rescued the deficiency of osteoblast differentiation from IFT80-silenced cells, and dramatically promoted osteoblast differentiation. Moreover, introduction of Smo agonist (SAG) promotes osteoblast differentiation, which was partially inhibited by IFT80 silencing. Thus, these results suggested that IFT80 plays an important role in osteogenesis through regulating Hedgehog/Gli signal pathways.

An Ift80 mouse model of short rib polydactyly syndromes shows defects in hedgehog signalling without loss or malformation of cilia

IFT80, a protein component of intraflagellar transport (IFT) complex B, is required for the formation, maintenance and functionality of cilia. Mutations in IFT80 cause Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III. Both diseases are autosomal recessive chondrodysplasias and share clinical and radiological similarities, including shortening of the long bones and constriction of the thoracic cage. A murine Ift80 gene-trap line was used to investigate the role of Ift80 during development. The homozygote appears hypomorphic rather than a true null due to low level wild-type transcript production by alternative splicing around the gene-trap cassette. Hypomorphic levels of Ift80 result in embryonic lethality highlighting a key role for Ift80 in development. In rare cases, gene-trap homozygotes survive to postnatal stages and phenocopy both JATD and SRP type III by exhibiting growth retardation, shortening of the long bones, constriction of the ribcage and polydactyly. Mouse embryonic fibroblasts made from this line showed a significant reduction in hedgehog pathway activation in response to Hedgehog analog treatment. This defective signalling was not accompanied by the loss or malformation of cilia as seen in some knockout models of other IFT component genes. Phenotypes indicative of defects in cilia structure or function such as situs inversus, cystic renal disease and retinal degeneration were not observed in this line. These data suggest that there is an absolute requirement for Ift80 in hedgehog signalling, but low level expression permits ciliogenesis indicating separate but linked roles for this protein in formation and function.

The Intraflagellar Transport Protein Ift80 Is Essential for Photoreceptor Survival in a Zebrafish Model of Jeune Asphyxiating Thoracic Dystrophy

PURPOSE. Jeune's asphyxiating thoracic dystrophy (JATD) is an autosomal recessive disorder with symptoms of retinal degeneration, kidney cysts, and chondrodysplasia and results from mutations in the ift80 gene. This study was conducted to characterize zebrafish lacking ift80 function for photoreceptor degeneration and defects in ciliogenesis to establish zebrafish as a vertebrate model for visual dysfunction in JATD and to determine whether ift80 interacts genetically with Bardet-Biedl syndrome (BBS) genes. METHODS. Zebrafish were injected with morpholinos (MOs) targeted to the ift80 gene. Retinas were analyzed by histology, transmission electron microscopy, and immunohistochemistry. Ear and kidney cilia were analyzed by whole-mount immunostaining. Intraflagellar transport (IFT) particle composition was subjected to Western blot analysis. Genetic interactions were tested by coinjection of MOs against ift80 and bbs4 or bbs8 followed by in situ hybridization. RESULTS. Zebrafish lacking ift80 function exhibited defects in photoreceptor outer segment formation and photoreceptor death. Staining with opsin antibodies revealed opsin mislocalization in both rods and cones. Ultrastructural analysis showed abnormal disc stacking and shortened photoreceptor outer segments. The kinocilia of the ear and motile cilia in the kidney were shorter and reduced in number. Western blot analysis revealed a slight increase in the stability of other IFT proteins. Coinjection of MOs against ift80 and BBS genes led to convergent-extension defects. CONCLUSIONS. Zebrafish lacking ift80 exhibited defects characteristic of JATD. Because the developing outer segments degenerated, Ift80 could possibly act as a maintenance factor for the IFT particle.

Ciliopathy spectrum expanded? Jeune syndrome associated with foregut dysmotility and malrotation

We report the association and surgical management of gastrointestinal dysmotility and malrotation with Jeune asphyxiating thoracic dystrophy (JATD), an autosomal recessive condition that often results in respiratory failure due to a small rib cage. A 4-month-old male with JATD presented with vomiting and aspiration pneumonitis compounding already severe respiratory morbidity. A contrast study revealed esophageal and gastric dysmotility with associated malrotation. This was treated surgically with good results. Some cases of JATD are caused by missense mutations in the gene IFT80, which encodes a protein implicated in the process of intraflagellar transport of primary cilia. We speculate that these abdominal complications might also be part of the extending spectrum of ciliopathy.