Bioinformatics Analysis and Experimental Verification Identify Downregulation of COL27A1 in Poor Segmental Congenital Scoliosis.

BACKGROUND: Congenital scoliosis (CS) represents the congenital defect disease, and poor segmental congenital scoliosis (PSCS) represents one of its types. Delayed intervention can result in disability and paralysis. In this study, we would identify the core biomarkers for PSCS progression through bioinformatics analysis combined with experimental verification. METHODS: This work obtained the GSE11854 expression dataset associated with somite formation in the GEO database, which covers data of 13 samples. Thereafter, we utilized the edgeR of the R package to obtain DEGs in this dataset. Then, GO annotation, KEGG analyses, and DO annotation of DEGs were performed by "clusterProfiler" of the R package. This study performed LASSO regression for screening the optimal predicting factors for somite formation. Through RNA sequencing based on peripheral blood samples from healthy donors and PSCS cases, we obtained the RNA expression patterns and screen out DEGs using the R package DESeq2. The present work analyzed COL27A1 expression in PSCS patients by the RT-PCR assay. RESULTS: A total of 443 genes from the GSE11854 dataset were identified as DEGs, which were involved in BP associated with DNA replication, CC associated with chromosomal region, and MF associated with ATPase activity. These DEGs were primarily enriched in the TGF-ß signaling pathway and spinal deformity. Further, LASSO regression suggested that 9 DEGs acted as the signature markers for somite formation. We discovered altogether 162 DEGs in PSCS patients, which were involved in BP associated with cardiac myofibril assembly and MF associated with structural constituent of muscle. However, these 162 DEGs were not significantly correlated with any pathways. Finally, COL27A1 was identified as the only intersected gene between the best predictors for somite formation and PSCS-related DEGs, which was significantly downregulated in PSCS patients. CONCLUSION: This work sheds novel lights on DEGs related to the PSCS pathogenic mechanism, and COL27A1 is the possible therapeutic target for PSCS. Findings in this work may contribute to developing therapeutic strategies for PSCS.

Identification of a homozygous frameshift variant in RFLNA in a patient with a typical phenotype of spondylocarpotarsal synostosis syndrome.

Spondylocarpotarsal synostosis syndrome, a rare syndromic skeletal disorder characterized by disrupted vertebral segmentation with vertebral fusion, scoliosis, short stature, and carpal/tarsal synostosis, has been associated with biallelic truncating mutations in the filamin B gene or monoallelic mutations in the myosin heavy chain 3 gene. We herein report the case of a patient with a typical phenotype of spondylocarpotarsal synostosis syndrome who had a homozygous frameshift mutation in the refilin A gene (RFLNA) [c.241delC, p.(Leu81Cysfs*111)], which encodes one of the filamin-binding proteins. Refilins, filamins, and myosins play critical roles in forming perinuclear actin caps, which change the nuclear morphology during cell migration and differentiation. The present study implies that RFLNA is an additional causative gene for spondylocarpotarsal synostosis syndrome in humans and a defect in forming actin bundles and perinuclear actin caps may be a critical mechanism for the development of spondylocarpotarsal synostosis syndrome.

A postnatal role for embryonic myosin revealed by MYH3 mutations that alter TGFß signaling and cause autosomal dominant spondylocarpotarsal synostosis.

Spondylocarpotarsal synostosis (SCT) is a skeletal disorder characterized by progressive vertebral, carpal and tarsal fusions, and mild short stature. The majority of affected individuals have an autosomal recessive form of SCT and are homozygous or compound heterozygous for nonsense mutations in the gene that encodes the cytoskeletal protein filamin B (FLNB), but a subset do not have FLNB mutations. Exome sequence analysis of three SCT patients negative for FLNB mutations identified an autosomal dominant form of the disease due to heterozygosity for missense or nonsense mutations in MYH3, which encodes embryonic myosin. Cells transfected with the MYH3 missense mutations had reduced TGFß signaling, revealing a regulatory role for embryonic myosin in the TGFß signaling pathway. In wild-type mice, there was persistent postnatal expression of embryonic myosin in the small muscles joining the neural arches of the spine suggesting that loss of myosin function in these muscles contribute to the disease. Our findings demonstrate that dominant mutations in MYH3 underlie autosomal dominant SCT, identify a postnatal role for embryonic myosin and suggest that altered regulation of signal transduction in the muscles within the spine may lead to the development of vertebral fusions.

Ihh and PTH1R signaling in limb mesenchyme is required for proper segmentation and subsequent formation and growth of digit bones.

Digit formation is a process, which requires the proper segmentation, formation and growth of phalangeal bones and is precisely regulated by several important factors. One such factor is Ihh, a gene linked to BDA1 and distal symphalangism in humans. In existing mouse models, mutations in Ihh have been shown to cause multiple synostosis in the digits but lead to perinatal lethality. To better study the exact biological and pathological events which occur in these fused digits, we used a more viable Prx1-Cre;Ihh(fl/fl) model in which Cre recombinase is expressed during mesenchymal condensation in the earliest limb buds at E9.5 dpc and found that mutant digits continuously fuse postnatally until phalanges are finally replaced by an unsegmented "one-stick bone". Mutant mice displayed osteocalcin-positive mature osteoblasts, but had reduced proliferation and abnormal osteogenesis. Because of the close interaction between Ihh and PTHrP during endochondral ossification, we also examined the digits of Prx1-Cre;PTH1R(fl/fl) mice, where the receptor for PTHrP was conditionally deleted. Surprisingly, we found PTH1R deletion caused symphalangism, demonstrating another novel function of PTH1R signaling in digit formation. We characterized the symphalangism process whereby initial cartilaginous fusion prevented epiphyseal growth plate formation, resulting in resorption and replacement of the remaining cartilage by bony tissue. Chondrocyte differentiation displayed abnormal directionality in both mutants. Lastly, Prx1-Cre;Ihh(fl/fl);Jansen Tg mice, in which a constitutively active PTH1R allele was introduced into Ihh mutants, were established to address the possible involvement of PTH1R signaling in Ihh mutant digits. These rescue mice failed to show significantly improved phenotype, suggesting that PTH1R signaling in chondrocytes is not sufficient to restore digit formation. Our results demonstrate that Ihh and PTH1R signaling in limb mesenchyme are both essential to regulate proper development of digit structures, although they appear to use different mechanisms.

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal.

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six-generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild-type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG-mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain-of-function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development.

Mutations in MECOM, Encoding Oncoprotein EVI1, Cause Radioulnar Synostosis with Amegakaryocytic Thrombocytopenia.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome, characterized by thrombocytopenia and congenital fusion of the radius and ulna. A heterozygous HOXA11 mutation has been identified in two unrelated families as a cause of RUSAT. However, HOXA11 mutations are absent in a number of individuals with RUSAT, which suggests that other genetic loci contribute to RUSAT. In the current study, we performed whole exome sequencing in an individual with RUSAT and her healthy parents and identified a de novo missense mutation in MECOM, encoding EVI1, in the individual with RUSAT. Subsequent analysis of MECOM in two other individuals with RUSAT revealed two additional missense mutations. These three mutations were clustered within the 8(th) zinc finger motif of the C-terminal zinc finger domain of EVI1. Chromatin immunoprecipitation and qPCR assays of the regions harboring the ETS-like motif that is known as an EVI1 binding site showed a reduction in immunoprecipitated DNA for two EVI1 mutants compared with wild-type EVI1. Furthermore, reporter assays showed that MECOM mutations led to alterations in both AP-1- and TGF-ß-mediated transcriptional responses. These functional assays suggest that transcriptional dysregulation by mutant EVI1 could be associated with the development of RUSAT. We report missense mutations in MECOM resulting in a Mendelian disorder that provide compelling evidence for the critical role of EVI1 in normal hematopoiesis and in the development of forelimbs and fingers in humans.

A comprehensive review of reported heritable noggin-associated syndromes and proposed clinical utility of one broadly inclusive diagnostic term: NOG-related-symphalangism spectrum disorder (NOG-SSD).

The NOG gene encodes noggin, a secreted polypeptide that is important for regulating multiple signaling pathways during human development, particularly in cartilage and bone. The hallmark of NOG-related syndromes is proximal symphalangism, defined by abnormal fusion of the proximal interphalangeal joints of the hands and feet. Many additional features secondary to NOG mutations are commonly but inconsistently observed, including a characteristic facies with a hemicylindrical nose, congenital conductive hearing loss due to stapes fixation, and hyperopia. The variable clinical presentations led to the designation of five different autosomal dominant syndromes, all subsequently found to have resulted from NOG mutations. These include (1) proximal symphalangism; (2) multiple synostoses syndrome 1; (3) stapes ankylosis with broad thumbs and toes; (4) tarsal-carpal coalition syndrome; and (5) brachydactyly type B2. Herein, we review the phenotypic features associated with mutations in the NOG gene, demonstrating the overlapping characteristics of these syndromes. Due to the variable phenotypic spectrum within families and among families with the same mutation, we propose a unifying term, NOG-related symphalangism spectrum disorder (NOG-SSD), to aid in the clinical recognition and evaluation of all affected individuals with these phenotypes. These NOG gene variants are available in a new locus-specific database (https://NOG.lovd.nl).

Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation.

Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway.