The Assessment of CDX1, IHH, SHH, GATA4, FOXA2, FOXF1 in Congenital Intra-Abdominal Adhesions.

Congenital abdominal adhesions are a rare condition that can result in a small bowel obstruction at any age, more frequently in pediatric populations. The cause remains unknown, and the importance of aberrant congenital bands is related to the difficulty of diagnosis, and cases of death with late detection have been documented. This research examines the expression of Caudal Type Homeobox 1 (CDX1), Indian Hedgehog (IHH), Sonic Hedgehog (SHH), GATA Binding Protein 4 (GATA4), Forkhead Box A2 (FOXA2) and Forkhead Box F1 (FOXF1) gene expression in human abdominal congenital adhesion fibroblast and endothelium cells by chromogenic in situ hybridization, with the aim of elucidating their potential association with the etiology of congenital intra-abdominal adhesion band development. The potential genes' signals were examined using a semi-quantitative approach. Significant correlations were observed between the expression of CDX1 (p <.001) and SHH (p=0.032) genes in fibroblasts from congenital intra-abdominal adhesions compared to fibroblasts from control peritoneal tissue. Statistically significant very strong correlations were found between the CDX1 and IHH comparing endothelium and fibroblast cells in congenital abdominal adhesion bands. There was no statistically significant difference found in the distribution of IHH, FOXA2, GATA4, and FOXF1 between the fibroblasts and endothelium of the patients compared to the control group. The presence of notable distinctions and diverse associations suggests the potential involvement of numerous morpho-pathogenetic processes in the development of intraabdominal adhesions.

Variants in both the N- or C-terminal domains of IHH lead to defective secretion causing short stature and skeletal defects.

BACKGROUND: Heterozygous Indian Hedgehog gene (IHH) variants are associated with brachydactyly type A1 (BDA1). However, in recent years, numerous variants have been identified in patients with short stature and more variable forms of brachydactyly. Many are located in the C-terminal domain of IHH (IHH-C), which lacks signaling activity but is critical for auto-cleavage and activation of the N-terminal (IHH-N) peptide. The absence of functional studies of IHH variants, particularly for those located in IHH-C, has led to these variants being classified as variants of uncertain significance (VUS). OBJECTIVE: To establish a simple functional assay to determine the pathogenicity of IHH VUS and confirm that variants in the C-terminal domain affect protein function. DESIGN/METHODS: In vitro studies were performed for 9 IHH heterozygous variants, to test their effect on secretion and IHH intracellular processing by western blot of cells expressing each variant. RESULTS: IHH secretion was significantly reduced in all mutants, regardless of the location. Similarly, intracellular levels of N-terminal and C-terminal IHH peptides were severely reduced in comparison with the control. Two variants present at a relatively high frequency in the general population also reduced secretion but to a lesser degree in the heterozygous state. CONCLUSIONS: These studies provide the first evidence that variants in the C-terminal domain affect the secretion capacity of IHH and thus, reduce availability of IHH ligand, resulting in short stature and mild skeletal defects. The secretion assay permits a relatively easy test to determine the pathogenicity of IHH variants. All studied variants affected secretion and interestingly, more frequent population variants appear to have a deleterious effect and thus contribute to height variation.

Short stature with brachydactyly caused by a novel mutation in the IHH gene and response to 4-year growth hormone therapy: a case report.

Background: The etiology of short stature is heterogeneous. The disturbance of endochondral ossification and cartilage matrix synthesis caused by genetic mutations often causes short height combined with skeletal deformities in children. Some patients with minor skeletal abnormalities, such as short fingers and mild limb shortening, may be overlooked by clinicians and misdiagnosed as idiopathic short stature (ISS) or growth hormone deficiency (GHD). Case Description: We conducted a detailed investigation of laboratory and imaging examinations on a family with short stature and non-classical brachydactyly type A1 (BDA1) and summarized the clinical features. They received whole exome sequencing (WES) to reveal the possible genetic variation. A heterozygous mutation in the Indian hedgehog gene (IHH) (c.387_388insC, p.Thr130Hisfs*18) was found in the two siblings and their mother. The siblings both started recombinant human growth hormone (rhGH) therapy (rhGH: 33 µg/kg/day) and followed up for 4 years. After treatment, the siblings' height improved significantly, and they acquired a significant increase in the height standard deviation score (SDS) (the boy: +2.54, the girl: +1.86) during the 4-year therapy. No noticeable adverse effect was observed during rhGH treatment. Conclusions: We found a novel heterozygous pathogenic mutation in the IHH gene in a family and detailed the phenotype with short stature and non-classical BDA1. The therapy of rhGH showed promising effects. To avoid misdiagnosis, clinicians should not overlook minor skeletal anomalies in patients with short stature, especially those with a family history.

Hedgehog Signaling Controls Chondrogenesis and Ectopic Bone Formation via the Yap-Ihh Axis.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by abnormal bone formation due to ACVR1 gene mutations. The identification of the molecular mechanisms underlying the ectopic bone formation and expansion in FOP is critical for the effective treatment or prevention of HO. Here we find that Hh signaling activation is required for the aberrant ectopic bone formation in FOP. We show that the expression of Indian hedgehog (Ihh), a Hh ligand, as well as downstream Hh signaling, was increased in ectopic bone lesions in Acvr1R206H; ScxCre mice. Pharmacological treatment with an Ihh-neutralizing monoclonal antibody dramatically reduced chondrogenesis and ectopic bone formation. Moreover, we find that the activation of Yap in the FOP mouse model and the genetic deletion of Yap halted ectopic bone formation and decreased Ihh expression. Our mechanistic studies showed that Yap and Smad1 directly bind to the Ihh promoter and coordinate to induce chondrogenesis by promoting Ihh expression. Therefore, the Yap activation in FOP lesions promoted ectopic bone formation and expansion in both cell-autonomous and non-cell-autonomous manners. These results uncovered the crucial role of the Yap-Ihh axis in FOP pathogenesis, suggesting the inhibition of Ihh or Yap as a potential therapeutic strategy to prevent and reduce HO.

Pth1r in Neural Crest Cells Regulates Nasal Cartilage Differentiation.

Neural crest cells (NCC) arise from the dorsal margin of the neural plate border and comprise a unique cell population that migrates to and creates the craniofacial region. Although factors including Shh, Fgf8, and bone morphogenetic proteins have been shown to regulate these biological events, the role of parathyroid hormone 1 receptor (Pth1r) has been less studied. We generated an NCC-specific mouse model for Pth1r and researched gene expression, function, and interaction focusing on nasal cartilage framework and midfacial development. Wnt1-Cre;Pth1rfl/fl;Tomatofl/+ mice had perinatal lethality, but we observed short snout and jaws, tongue protrusion, reduced NCC-derived cranial length, increased mineralization in nasal septum and hyoid bones, and less bone mineralization at interfrontal suture in mutants at E18.5. Importantly, the mutant nasal septum and turbinate cartilage histologically revealed gradual, premature accelerated hypertrophic differentiation. We then studied the underlying molecular mechanisms by performing RNA seq analysis and unexpectedly found that expression of Ihh and related signaling molecules was enhanced in mutant nasomaxillary tissues. To see if Pth1r and Ihh signaling are associated, we generated a Wnt1-Cre; Ihhfl/fl;Pth1rfl/fl;Tomatofl/+ (DKO) mouse and compared the phenotypes to those of each single knockout mouse: Wnt1-Cre; Ihhfl/fl;Pth1rfl/+;Tomatofl/+ (Ihh-CKO) and Wnt1-Cre;Ihhfl/+;Pth1rfl/fl;Tomatofl/+ (Pth1r-CKO). Ihh-CKO mice displayed a milder effect. Of note, the excessive hypertrophic conversion of the nasal cartilage framework observed in Pth1r-CKO was somewhat rescued DKO embryos. Further, a half cAMP responsive element and the 4 similar sequences containing 2 mismatches were identified from the promoter to the first intron in Ihh gene. Gli1-CreERT2;Pth1rfl/fl;Tomatofl/+, a Pth1r-deficient model targeted in hedgehog responsive cells, demonstrated the enlarged hypertrophic layer and significantly more Tomato-positive chondrocytes accumulated in the nasal septum and ethmoidal endochondral ossification. Collectively, the data suggest a relevant Pth1r/Ihh interaction. Our findings obtained from novel mouse models for Pth1r signaling illuminate previously unknown aspects in craniofacial biology and development.

LncRNA Malat1 regulates iPSC-derived ß-cell differentiation by targeting the miR-15b-5p/Ihh axis.

BACKGROUND: Differentiation of induced pluripotent stem cells (iPSCs)-derived ß-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in ß-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic ß-cells and may improve the quality of ß-like cells for stem cell therapy. METHODS: ß-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of LncRNA Malat1. Quantitative real-time PCR (qRT-PCR), Western blot (WB) analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic ß-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in ß-like cells. Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of ß-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p/Ihh were detected by dual luciferase reporter assays (LRAs). RESULTS: We found that the expression of lncRNA Malat1 declined during differentiation, and overexpression (OE) of lncRNA Malat1 notably impaired the differentiation and maturation of ß-like cells derived from iPSCs in vitro and in vivo. Most importantly, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments. CONCLUSION: This study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into ß-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

Mechanical stress can regulate temporomandibular joint cavitation via signalling pathways.

The temporomandibular joint (TMJ), composed of temporal fossa, mandibular condyle and a fibrocartilage disc with upper and lower cavities, is the biggest synovial joint and biomechanical hinge of the craniomaxillofacial musculoskeletal system. The initial events that give rise to TMJ cavities across diverse species are not fully understood. Most studies focus on the pivotal role of molecules such as Indian hedgehog (Ihh) and hyaluronic acid (HA) in TMJ cavitation. Although biologists have observed that mechanical stress plays an irreplaceable role in the development of biological tissues and organs, few studies have been concerned with how mechanical stress regulates TMJ cavitation. Based on the evidence from human or other animal embryos today, it is implicated that mechanical stress plays an essential role in TMJ cavitation. In this review, we discuss the relationship between mechanical stress and TMJ cavitation from evo-devo perspectives and review the clinical features and potential pathogenesis of TMJ dysplasia.

Intermittent Hypobaric Hypoxia Ameliorates Autistic-Like Phenotypes in Mice.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by persistent deficits in social communication and stereotyped behaviors. Although major advances in basic research on autism have been achieved in the past decade, and behavioral interventions can mitigate the difficulties that individuals with autism experience, little is known about the many fundamental issues of the interventions, and no specific medication has demonstrated efficiency for the core symptoms of ASD. Intermittent hypobaric hypoxia (IHH) is characterized by repeated exposure to lowered atmospheric pressure and oxygen levels, which triggers multiple physiological adaptations in the body. Here, using two mouse models of ASD, male Shank3B -/- and Fmr1 -/y mice, we found that IHH training at an altitude of 5,000 m for 4 h per day, for 14 consecutive days, ameliorated autistic-like behaviors. Moreover, IHH training enhanced hypoxia inducible factor (HIF) 1α in the dorsal raphe nucleus (DRN) and activated the DRN serotonergic neurons. Infusion of cobalt chloride into the DRN, to mimic IHH in increasing HIF1α expression or genetically knockdown PHD2 to upregulate HIF1α expression in the DRN serotonergic neurons, alleviated autistic-like behaviors in Shank3B -/- mice. In contrast, downregulation of HIF1α in DRN serotonergic neurons induced compulsive behaviors. Furthermore, upregulating HIF1α in DRN serotonergic neurons increased the firing rates of these neurons, whereas downregulation of HIF1α in DRN serotonergic neurons decreased their firing rates. These findings suggest that IHH activated DRN serotonergic neurons via upregulation of HIF1α, and thus ameliorated autistic-like phenotypes, providing a novel therapeutic option for ASD.

Hepatocyte-targeted siTAZ therapy lowers liver fibrosis in NASH diet-fed chimeric mice with hepatocyte-humanized livers.

Nonalcoholic steatohepatitis (NASH) is emerging as the most common cause of liver disease. Although many studies in mouse NASH models have suggested therapies, translation to humans is poor, with no approved drugs for NASH. One explanation may lie in differences between mouse and human hepatocytes. We used NASH diet-fed chimeric mice reconstituted with human hepatocytes (hu-liver mice) to test a mechanism-based hepatocyte-targeted small interfering RNA (siRNA), GalNAc-siTaz, shown previously to block the progression to fibrotic NASH in mice. Following ablation of endogenous hepatocytes, male mice were reconstituted with human hepatocytes from a single donor with the rs738409-C/G PNPLA3 risk variant, resulting in ∼95% human hepatocyte reconstitution. The mice were then fed a high-fat choline-deficient l-amino acid-defined diet for 6 weeks to induce NASH, followed by six weekly injections of GalNAc-siTAZ to silence hepatocyte-TAZ or control GalNAc-siRNA (GalNAc-control) while still on the NASH diet. GalNAc-siTAZ lowered human hepatic TAZ and IHH, a TAZ target that promotes NASH fibrosis. Most important, GalNAc-siTAZ decreased liver inflammation, hepatocellular injury, hepatic fibrosis, and profibrogenic mediator expression versus GalNAc-control, indicating that GalNAc-siTAZ decreased the progression of NASH in mice reconstituted with human hepatocytes. In conclusion, silencing TAZ in human hepatocytes suppresses liver fibrosis in a hu-liver model of NASH.

Application of Human Menopausal Gonadotropins in the Treatment of Idiopathic Hypogonadotropic Hypogonadism (IHH)-Based Infertility in Females: A Case Report.

Rationale: Idiopathic hypogonadotropic hypogonadism (IHH) is a prevalent congenital genetic disorder with multiple inheritance patterns. IHH can manifest as normal hypogonadotrophic sexual hypofunction (nIHH) or with an abnormal sense of smell, known as Kallmann. It primarily affects the production and effectiveness of gonadotropin-releasing-hormone (GnRh), leading to reduced follicle-stimulating hormone and luteinizing hormone levels. This results in infertility and underdeveloped secondary sexual characteristics. Patient Concerns: A 29-year-old female presented with infertility. Diagnosis: IHH diagnosis was confirmed through magnetic resonance (MR) scan, endocrine tests, physical examination, and B ultrasonic inspection. Additionally, genetic studies, including chromosome analysis, were conducted for the patient. The results confirmed no genetic abnormalities or concerns. Interventions: The patient underwent multiple ovulation induction programs. Outcome: After several ovulation induction cycles, the patient conceived and delivered a live baby. Lessons: For IHH patients, a tailored human menopausal gonadotropin (HMG) dose is recommended. High-dose HMG can benefit those with poor follicular response. The addition of letrozole (5-7.5mg) may enhance follicular response during stimulation. Our approach, which emphasizes the combined use of high-dose HMG, letrozole, and the adjustment of FSH and LH ratios, offers a unique perspective compared to traditional treatments. If HMG treatment is ineffective, alternative ovulation induction methods, such as r-fsh combined with r-lh or HMG combined with rLH, can be considered. Adjusting the FSH and LH ratio and varying rFSH and rLH additions might help achieve dominant follicles and live birth in resistant cases. This case report underscores the potential benefits of our regimen, suggesting its consideration for future research and clinical applications.

Clinical and molecular features of 40 Chinese patients with idiopathic hypogonadotropic hypogonadism.

Background: Male idiopathic hypogonadotropic hypogonadism (IHH) is a heterogeneous clinical rare genetic disorder that can be divided into two forms: Kallmann syndrome (KS) and olfactory normal IHH (nIHH). Nearly half of unknown pathogenic genes and related pathogenic mechanisms have yet to be explored. Methods: Clinical data of 40 IHH patients (22 KS and 18 nIHH) were retrospectively recorded. All patients were diagnosed at the Department of Endocrinology of Jinling Hospital, Jiangsu Provincial People's Hospital, and the First Affiliated Hospital of the University of Science and Technology of China from 2014 to 2021. The proband genomic DNA (gDNA) was confirmed by whole exome sequencing (WES) and Sanger sequencing. Results: Ten new genetic mutations related to IHH in four families and eight sporadic unrelated IHH patients were identified. The total positive detection rate of 40 patients was 30% (nIHH 8/18 + KS 4/22), and the FGFR1 mutation rate accounted for 7.5% (3/40). Mutation rates of ANOS1, CHD7, and KISS1R were 5% (2/40), respectively. The mutation rates of SEMA3E, PROKR2, and SOX10 were 2.5% (1/40), respectively. After analysis by SIFT and PolyPhen-2 software, all missense mutation sites, such as SEMA3E (p.P323S), CHD7 (p.W1785C), PROKR2 (p.Y223D and p.R298C), were harmful; all nonsense mutation sites, such as FGFR1 (p.R661X) and KISS1R (p.R331X, p.Y103X), analyzed were pathogenic by Mutation Taster software. The comparison of MEGA5 software showed that all the variants had extremely high homology among different species and were extremely conservative in evolution. Conclusions: The study aims to expand the genotype mutation spectrum of IHH and provide evidence for the follow-up clinical treatment and genetic counseling of the disease.

Circulating indian hedgehog is a marker of the hepatocyte-TAZ pathway in experimental NASH and is elevated in humans with NASH.

Background & Aims: Non-alcoholic steatohepatitis (NASH)-induced liver fibrosis is emerging as the most common cause of liver disease. For evaluation of therapies, there is a pressing need to identify non-invasive, mechanism-based biomarkers. A pro-fibrotic process relevant to human NASH involves a pathway in which a transcriptional regulator called TAZ (WWTR1) in hepatocytes induces the secretion of pro-fibrotic Indian hedgehog (IHH). We therefore reasoned that circulating IHH may be a useful mechanism-based marker to assess changes in NASH fibrosis. Methods: Circulating IHH was assessed in wild-type and hepatocyte-TAZ-silenced NASH mice and in three separate cohorts of patients with mild-moderate NASH. Results: Circulating IHH was elevated in mice with diet-induced NASH compared with chow-fed mice or with NASH mice in which hepatocyte TAZ was silenced, which is an effective means to decrease NASH fibrosis. In patients with fatty liver disease with or without NASH, NASH fibrosis was associated with increased concentrations of circulating IHH. Conclusions: The results of these analyses support further investigation to determine whether circulating IHH may be useful as a mechanism-based indicator of target engagement in anticipated future clinical trials testing NASH fibrosis therapies that block the IHH pathway. Impact and implications: Non-alcoholic steatohepatitis (NASH)-induced liver fibrosis is a common cause of liver disease. Circulating biomarkers that reflect liver fibrosis in NASH would be very useful to evaluate therapies. One mechanism of NASH fibrosis with potential as a therapeutic target involves a liver-secreted protein called Indian hedgehog (IHH). We report that circulating levels of IHH in experimental and human NASH associates with NASH and NASH-associated liver fibrosis, providing the premise for further investigation into using circulating IHH to evaluate anticipated future NASH therapies that block the IHH pathway in liver.

Theoretical study on the vibrational spectra and potential energy curves for SiC (X3Π) and SiS (X1Σ+) molecules.

In this work, the vibrational constants (ωe,ωexe) calculated by the variational algebraic method (VAM) and some other molecular constants (De,re,Be,αe) were used to construct the improved Hulburt-Hirschfelder (IHH) analytical potential energy function (APEF). Not only that, but the calculated VAM potential points are used as the 'true' energies to determine the value of the variational parameter λ which is the pivotal fitting parameter in the IHH potential. With limited experimental data, high-precision IHH potential can be achieved by combining the VAM and the IHH APEF. This combination of the VAM and the IHH APEF is referred to be VAIHH APEF, which is employed to study the vibrational energies and potential energy curves (PECs) of SiC (X3Π) and SiS (X1Σ+) molecules, yielding full vibrational spectra and spectroscopic constants. The calculational results indicate that the VAIHH APEFs of SiC (X3Π) and SiS (X1Σ+) molecules are in good agreement with the experimental RKR potential points. Accurate PECs of SiC (X3Π) and SiS (X1Σ+) molecules imply that the VAIHH APEF is of high quality.

CRC Therapy Identifies Indian Hedgehog Signaling in Mouse Endometrial Epithelial Cells and Inhibition of Ihh-KLF9 as a Novel Strategy for Treating IUA.

Intrauterine adhesion (IUA) causes menstrual disturbance and infertility. There is no effective treatment available for moderate to severe IUA cases. Stem cell-based therapy has been investigated for treating IUA but is limited in clinical applications due to issues including the precise induction of differentiation, tumorigenesis, and unclear molecular mechanisms. In our recent study, we isolated and expanded the long-term cultures of conditional reprogrammed (CR) mouse endometrial epithelial cells. Treating IUA mice with these CR cells (CRCs) restored the morphology and structure of the endometrium and significantly improved the pregnancy rate. In this study, our data with high-throughput sequencing, CRISPR knockout Ihh-/-CRCs, and transplantation identified for the first time that the Indian hedgehog (Ihh) gene plays a critical role in the regulation of endometrial epithelial cell proliferation. We also found that aberrant activated Ihh-krüppel-like factor 9 (KLF9) signaling contributes to the inhibition of normal progesterone receptor (PR) function in IUA mice. Thus, we hypothesized that inhibition of the Ihh-KLF9 pathway may be a novel strategy to treat IUA. Our data demonstrated that treatment with the hedgehog signaling inhibitor Vismodegib restored the morphology, structure, and microenvironment of the endometrium, and greatly improved the pregnancy rate in IUA mice. This study suggests a promising application of hedgehog inhibitors as a targeted drug in the IUA clinic.

Research on the variants of FGFR1 and CEP290 genes in idiopathic hypogonadotropin hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) is a rare endocrine disease characterized by gonadal dysplasia. According to whether the sense of smell is affected, this disorder is classified into Kallmann syndrome (KS) and normosmic isolated hypogonadotropic hypogonadism (nIHH). In this study, we reported a case of nIHH patient and explored the pathogenic mechanism of FGFR1 in nIHH. A FGFR1 variant (c.2008G>A, p.E670K) and a CEP290 variant (c.964G>A, p.D322N) were detected by the whole exome sequencing in this nIHH patient. Bioinformatic analysis revealed that this FGFR1 variant (c.2008G>A) causes structural perturbations in TK2 domain demonstrating that this variant result in FGFR1 loss-of-function and abnormal signaling. The identification of an additional CEP290 variant (c.964G>A) indicated that CEP290 might play a potential role in developmental abnormalities and inhibition of GnRH neuron release. A protein interaction network analysis showed that CEP290 was predicted to interact with FGFR1. In summary, our study identified the potential pathogenic mechanism(s) of the novel FGFR1 variant and indicated that CEP290 might play a role in the GnRH neuron migration route. Our findings expand the mutation spectrum of FGFR1 and CEP290 and provide a reference for clinical diagnosis and treatment of IHH.

Exogenous Indian hedgehog antagonist damages intervertebral discs homeostasis in adult mice.

Background: Vismodegib, as an exogenous Indian hedgehog (Ihh) antagonist, has been approved by the Food and Drug Administration (FDA) for the clinical treatment of patients with basal cell carcinoma, and previous observations implicate the potential therapeutic of vismodegib in osteoarthritis treatment. However, there is no direct evidence for the role of Ihh signaling in intervertebral discs (IVDs) homeostasis of adult mice. The aim of the present study is to assess the effect of systemic administration of Smoothened inhibitor (SMOi) - vismodegib on IVDs homeostasis during the adult stage. Methods: The expression of glioma-associated oncogene homolog 1 (Gli1), the downstream targeting gene of Ihh signaling, in IVDs of adult mice after receiving systemic administration of SMOi was examined by immunohistochemistry. The pathological changes of vertebral bodies after SMOi treatment were evaluated by X-ray and micro-CT. The effects of SMOi on homeostasis of IVDs including cartilaginous endplates (CEP), growth plates (GP) and annulus fibrous (AF) were evaluated by histological analysis. The expressions of Aggrecan, Matrix metalloproteinase 13 (MMP13) and Runt-related transcription factor 2 (Runx2), in IVDs were also investigated by immunohistochemistry. Changes in chondrocyte apoptosis and proliferation in IVDs were evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and analyzing the expression of the cell proliferation antigen Ki-67. Results: Systemic administration of SMOi significantly decreased the expression of Gli1 in IVDs that indicating effective inhibition of Ihh signaling. Bone mass of vertebral bodies was diminished after SMOi treatment. Moreover, IVDs degeneration (IDD) like defects including CEP sclerosis, degenerative nucleus pulposus (NP) and fissure within AF, as well as narrowed or fused GP and loss bone mass of vertebral bodies was observed in SMOi-treated mice. The severity of IDD was time-dependent with the administration of SMOi treatment after 2-8 weeks. The expressions of Aggrecan, MMP13 and Runx2 in IVDs of mice receiving SMOi treatment were significantly decreased. In addition, chondrocyte apoptosis was significantly enhanced, while chondrocyte proliferation was significantly inhibited. Conclusions: Our study propose that systemic administration of vismodegib damages IVDs homeostasis via inhibition of Ihh signaling in adult mice. The clinical application of Ihh signaling antagonists such as vismodegib should be careful considering these side adverse. The Translational Potential of this Article: Vismodegib as an exogenous antagonist of Ihh signaling has been approved by the FDA for the clinical treatment of patients with basal cell carcinoma. However, it is still unknown whether vismodegib will has adverse effects on the patient or animal model of IVDs cartilage homeostasis. Based on our study, systemic administration of vismodegib damages IVDs homeostasis via inhibition of Ihh signaling in adult mice and special attention should be paid to the clinical application of vismodegib.

The role of hypertrophic chondrocytes in regulation of the cartilage-to-bone transition in fracture healing.

Endochondral bone formation is an important pathway in fracture healing, involving the formation of a cartilaginous soft callus and the process of cartilage-to-bone transition. Failure or delay in the cartilage-to-bone transition causes an impaired bony union such as nonunion or delayed union. During the healing process, multiple types of cells including chondrocytes, osteoprogenitors, osteoblasts, and endothelial cells coexist in the callus, and inevitably crosstalk with each other. Hypertrophic chondrocytes located between soft cartilaginous callus and bony hard callus mediate the crosstalk regulating cell-matrix degradation, vascularization, osteoclast recruitment, and osteoblast differentiation in autocrine and paracrine manners. Furthermore, hypertrophic chondrocytes can become osteoprogenitors and osteoblasts, and directly contribute to woven bone formation. In this review, we focus on the roles of hypertrophic chondrocytes in fracture healing and dissect the intermingled crosstalk in fracture callus during the cartilage-to-bone transition.

Accuracy of various tests alone and in combination to differentiate IHH from CDGP.

Context: Constitutional delay in growth and puberty (CDGP) is a normal physiological variant of delayed puberty in both sexes and is the most common cause of delayed puberty. Idiopathic hypogonadotropic hypogonadism (IHH) is due to deficiency in or insensitivity to gonadotropin-releasing hormone (GnRH) with normal structure and function of the anterior pituitary after exclusion of secondary causes of hypogonadotropic hypogonadism. To differentiate CDGP from IHH is crucial because it not only helps in decision making in management but also lessen anxiety of the parents. Aim: In this study we aimed to find out the accuracy of hormonal tests used individually as well as in various combinations to distinguish cases of IHH from CDGP. Methods: A cohort of 34 boys with delayed puberty were recruited in this study. Detailed history, clinical examination, hormonal analysis including basal serum testosterone, inhibin-B, LH, FSH as well as GnRH analogue stimulated gonadotrophins and testosterone along with hCG stimulated testosterone was done. At 6 monthly follow-up, detailed clinical examination was repeated and the cohort was followed until 2 years. Results: Out of the 29 boys taken for final analysis, CDGP was diagnosed in 23 boys and IHH in 6 boys. Basal LH, basal inhibin-B, 3 hours post leuprolide LH and 72 hours post hCG testosterone were significantly higher in CDGP than IHH. However, no statistically significant difference was observed between basal FSH, basal testosterone and 3 hours post leuprolide FSH between these two groups. When basal LH (cut-off <0.565 IU/L) and basal inhibin-B (cut-off <105 pg/ml) were taken together the sensitivity and specificity were increased to 100% as was for the combination of basal LH (cutoff <0.565 IU/L) and 3 hours post leuprolide LH (cutoff <6.16 IU/L) for diagnosis of IHH. Both combinations have PPV of 100% and NPV of 100%. A combination of 3 hours post leuprolide LH with 72 hours post hCG testosterone also has good sensitivity (100%), specificity (96%), PPV (90%) and NPV (100%). Conclusion: Differentiating IHH from CDGP is a challenging task due to considerable overlap in their clinical as well as hormonal profiles. Therefore we suggest that a combination of basal LH and basal inhibin-B may be considered as a useful screening tool to differentiate IHH from CDGP rather than the cumbersome and invasive stimulation tests.

Case Report: Brachydactyly Type A1 Induced by a Novel Variant of in-Frame Insertion in the IHH Gene.

Brachydactyly type A1 (BDA1) is an autosomal dominant inherited disease characterized by the shortness/absence of the middle phalanges, which can be induced by mutations in the Indian hedgehog gene (IHH). Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by joint destruction, synovitis, and the presence of autoantibodies. In this study, the proband was diagnosed with both BDA1 and RA. We performed whole-exome sequencing in a four-generation Chinese family to investigate their inherited causal mutation to BDA1. A novel in-frame insertion variant in IHH: NM_002,181.4: c.383_415dup/p.(R128_H138dup) was identified in the BDA1 pedigree. This insertion of 11 amino acids was located in the highly conserved amino-terminal signaling domain of IHH and co-segregated with the disease status. This adds one to the total number of different IHH mutations found to cause BDA1. Moreover, we found a potential causal germline variant in CRY1 for a molecular biomarker of RA (i.e., a high level of anti-cyclic citrullinated peptide). Collectively, we identified novel variants in IHH for inherited BDA1, which highlights the important role of this gene in phalange development.

Genetic architectures and selection signatures of body height in Chinese indigenous donkeys revealed by next-generation sequencing.

Donkeys are widely distributed labour animals in the world. During the process of the domestication and artificial selection of domestic donkeys, body sizes show significant differences among different breeds of donkeys. Based on the genome resequencing data of 103 Chinese indigenous donkeys from 11 breeds (Biyang, Dezhou, Guangling, Hetian, Jiami, Kulun, Qingyang, Turfan, Tibetan, Xinjiang, and Yunnan), seven Spanish donkeys from two breeds (Zamorano~Leonés and Andalusian), and three wild donkeys, we investigated the population structures of Chinese domestic donkeys with different body sizes. We used FST and XP-EHH analyses to explore the selected regions related to body sizes. The results showed that Chinese indigenous donkeys have a closer relationship with African wild donkeys than with Asian wild donkeys. LCORL/NCAPG, FAM184B, TBX3, and IHH were identified as genes with strong signals in analysis of selection signature (FST and XP-EHH) in large and small donkeys. The seven identified variants can be served as candidate loci affecting the body size of Chinese donkeys. Five of seven loci were located in intron 9 of FAM184B and were in a haplotype block, and one of the identified variants (Chr03:112664848) located in the CDS region of the LCORL gene was found to cause stop-loss. These candidate genes and variants shed new light on the molecular basis of donkey body size and will facilitate the breeding activities of donkeys.