Krüppel-like factors in bone biology.

The krüppel-like factor (KLF) family is a group of zinc finger transcription factors and contributes to different cellular processes such as differentiation, proliferation, migration, and apoptosis. While different studies show the roles of this family in skeletal development-specifically in chondrocyte and osteocyte development and bone homeostasis-there are few reviews summarizing their importance. To fill this gap, this review discusses current knowledge on different functions of the KLF family during skeletal development, including their roles in stem cell maintenance and differentiation, cell apoptosis, and cell cycle. To understand the importance of the KLF family, we also review genotype-phenotype correlations in different animal models. We also discuss how KLF proteins function through different signaling pathways and display their paramount importance in skeletal development. To highlight their roles in cartilage- or bone-related cells, we also use single-cell RNA sequencing publicly available data on mouse hindlimb. We also challenge our knowledge of how the KLF family is epigenetically regulated-e.g., using DNA methylation, histone modifications, and noncoding RNAs-during chondrocyte and osteocyte development.

Functional Analysis of RELN S2486G Mutation and its Contribution to Pathogenesis of Ankylosing Spondylitis.

BACKGROUND: Ankylosing spondylitis (AS; OMIM:106300) is a common complex inflammatory disease; in a previous study, we introduced a novel mutation in the RELN gene (OMIM: 600514) which was associated with AS. This study is designed to investigate the potential effect of RELN S2486G mutation on reelin secretion; additionally, we objected to evaluate the phospholipase A2 (PLA2G7) gene (OMIM: 601690) expression and platelet-activating factor-acetylhydrolase (PAF-AH) concentration as the downstream gene and the encoded protein. METHODS: The impact of the S2486G on reelin protein secretion was investigated in CHO-K1 and HEK-293T cells by constructing wild-type and mutant plasmids. Besides, the possible effect of the mutation on expression and concentration of PLA2G7 and PAF-AH in THP1 cells was assessed by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The study was performed at Tarbiat Modares University, Tehran, Iran, from 2016 to 2018. RESULTS: Our results showed that S2486G not only causes a significant reduction in reelin secretion in both HEK-293T and CHO-K1 cells, but also it leads to a significant reduction in PLA2G7 gene expression (P value < 0.001) and protein level of PAF-AH in THP-1 cells (P value < 0.003). CONCLUSION: The S2486G mutation in RELN can alter inflammatory and, to some extent, osteogenesis pathways mediated by reduced secretion of reelin and also reduced expression of the PLA2G7 gene.

Whole-exome sequencing identified a novel variant in an Iranian patient affected by pycnodysostosis.

BACKGROUND: Whole-exome sequencing (WES) has emerged as a successful diagnostic tool in molecular genetics laboratories worldwide. In this study, we aimed to find the potential genetic cause of skeletal disease, a heterogeneous disease, revealing the obvious short stature phenotype. In an Iranian family, we used solo-WES in a suspected patient to decipher the potential genetic cause(s). METHODS: A comprehensive clinical and genotyping examination was applied to suspect the disease of the patient. The solo clinical WES was exploited, and the derived data were filtered according to the standard pipelines. In order to validate the WES finding, the region harboring the candidate variant in the CTSK gene was amplified from genomic DNA and sequenced directly by Sanger sequencing. RESULTS: Sequence analysis revealed a rare novel nonsense variant, p.(Trp320*); c.905G>A, in the CTSK gene (NM_000396.3). In silico analysis shed light on the contribution of the variant to the pathogenicity of pycnodysostosis. This variant was confirmed by Sanger sequencing and further clinical examinations of the patient confirmed the disease. CONCLUSION: The present study shows a rare variant of the CTSK gene, which inherited as autosomal recessive, in an Iranian male patient with pycnodysostosis. Taken together, the novel nonsense CTSK variant meets the criteria of being likely pathogenic according to the American College of Medical Genetics and Genomics-the Association for Molecular Pathology (ACMG-AMP) variant interpretation guidelines.

S3440P Substitution in C-Terminal Region of Human Reelin Dramatically Impairs Secretion of Reelin from HEK 293T cells.

Reelin is a large extracellular glycoprotein secreted by Cajal-Retzius cells and has a main role during brain development, especially in neuronal migration. Reelin is comprised of N-terminal F-Spondin like domain, eight tandem repeats, and a highly conserved basic C-Terminal Region (CTR). The CTR main role in the secretion of Reelin has been investigated by advertently inducing deletion in whole or a part of this region; however, the role of CTR point mutations on the secretion of Reelin is shrouded in mystery. In this study, we performed experimental analyses on a sub-region of Human Reelin containing 5th and 6th repeats (R5-R6), a part of 8th repeat and the CTR which were amplified from cDNA of K562 and HEPG2(HepatocellularG2) cells and cloned into a mammalian expressional plasmid (pVP22/myc-His). Bioinformatics investigation was performed on the CTR at both level of nucleotide and amino acid as well as mutant type. Random mutagenesis by error-prone PCR method was utilized to induce mutation in the CTR. The secretion efficiency of recombinant wild-type and mutant Reelin constructs compared in cell lysate and supernatant isolated from the transiently transfected HEK 293T cells using 6XHistag ELISA method. In-vitro study demonstrated that the CTR alteration (S3440P) leads to impairment of Reelin secretion even after overexpression. Our results indicate that S3440P substitution is the highly conserved structure of the CTR has an important effect on Reelin secretion.

The first case of NSHL by direct impression on EYA1 gene and identification of one novel mutation in MYO7A in the Iranian families.

OBJECTIVES: Targeted next-generation sequencing (NGS) provides a consequential opportunity to elucidate genetic factors in known diseases, particularly in profoundly heterogeneous disorders such as non-syndromic hearing loss (NSHL). Hearing impairments could be classified into syndromic and non-syndromic types. This study intended to assess the significance of mutations in these genes to the autosomal recessive/dominant non-syndromic genetic load among Iranian families. MATERIALS AND METHODS: Two families were involved in this research and two patients were examined by targeted next-generation sequencing. Here we report two novel mutations in the MYO7A and EYA1 genes in two patients detected by targeted NGS. They were confirmed by Sanger sequencing and quantitative real-time PCR techniques. RESULTS: In this investigation, we identified a novel mutation in MYO7A, c.3751G>C, p.A1251P, along with another previously identified mutation (c.1708C>T) in one of the cases. This mutation is located in the MYTH4 protein domain which is a pivotal domain for the myosin function. Another finding in this research was a novel de-novo deletion which deletes the entire EYA1 coding region (EX1-18 DEL). Mutations in EYA1 gene have been found in branchiootorenal (BOR) syndrome. Interestingly the patient with EYA1 deletion did not show any other additional clinical implications apart from HL. This finding might argue for the sole involvement of EYA1 function in the mechanism of hearing. CONCLUSION: This investigation exhibited that the novel mutations in MYO7A, c.3751G>C, p.A1251P, and EYA1, EX1-18 DEL, were associated with NSHL. Our research increased the mutation spectrum of hearing loss in the Iranian population.

Chitosan nanoparticles for siRNA delivery: optimization of processing/formulation parameters.

Chitosan nanoparticles were prepared using ultrasonication methodology at specific amplitudes and times of sonication. Subsequently, small interfering RNA (siRNA) was added to the solution at predetermined values of nitrogen to phosphorous ratio (N/P), and stirring time. Employing response surfaces generated from a statistical model, the effect of sonication time and amplitude, stirring time, and N/P ratio was studied on the particle size, polydispersity, and loading efficiency of prepared siRNA/chitosan nanoparticles. It was found that to obtain the smallest size, amplitude and time of sonication as well as stirring time should be kept at ∼45%, 165 seconds, and 50 minutes, respectively. Minimum polydispersity values were also obtained at similar values of sonication time/amplitude and stirring time in addition to N/P values of ∼28. Also, the maximum proportion of siRNA loading was observed at approximate values of 300 seconds, 80% and 280 for sonication time, amplitude, and N/P ratio, respectively. The optimum conditions (i.e., to prepare a sample with minimum values of particle size and polydispersity index and maximum values of loading efficiency) were determined as 60.6, 30.0 (seconds), 28.0, and 12.5 (minutes) for amplitude, time of sonication, N/P, and stirring time, respectively. In this scrutiny, the predicted values of optimum formulation were 456 nm size, 89.6% loading efficiency, and 0.4 polydispersity index.

cAMP response element-binding protein 1 is required for hydroxyurea-mediated induction of γ-globin expression in K562 cells.

1. Hydroxyurea (HU) is a drug used for the treatment of haemoglobinopathies. Hydroxyurea functions by upregulating γ-globin transcription and fetal haemoglobin (HbF) production in erythroid cells. The K562 erythroleukaemia cell line is widely used as a model system in which to study the mechanism of γ-globin induction by HU. However, the transcription factors required for the upregulation of γ-globin expression by HU in K562 cells have not been identified. Similarities between the HU and sodium butyrate (SB) pathways suggest cAMP response element-binding protein (CREB) 1 as a potential candidate. Thus, the aim of the present study was to investigate the possible role of CREB1 in the HU pathway. 2. Experiments were performed using transient and stable RNA interference (RNAi) to show that CREB1 is necessary for HU-mediated induction of γ-globin expression and haemoglobin production in K562 cells. 3. Furthermore, western blot analyses demonstrated that CREB1 becomes phosphorylated in a dose-dependent manner after HU (100-400 µmol/L) treatment of K562 cells for 72 h. 4. We also investigated role of a Gγ promoter CREB1 response element (G-CRE) in this pathway. Quantitative amplification refractory mutation system-polymerase chain reaction experiments were performed to demonstrate that HU induces the expression of both Gγ and Aγ in this cell line. In addition, electrophoretic mobility shift assays were used to show that levels of CREB1 complexes binding to the G-CRE site are increased following HU treatment and are decreased in CREB1-knockdown cells. 5. The results suggest that CREB1 is necessary for γ-globin induction by HU in K562 cells, a role that may be mediated, in part, through the G-CRE element.