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β-thalassemia is an inherited blood disease caused by reduced or inadequate β-globin synthesis due to β-globin gene mutation. Our previous study developed a gene-edited mice model (β654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the βIVS2-654 (C > T) mutation site and the 3ʹ splicing acceptor site at 579 and corrected abnormal β-globin mRNA splicing in the β654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from β654-ER mice on β-thalassemia by consecutive HSC transplantation. The results indicated that HSC transplantation derived from gene-edited mice can significantly improve the survival rate of mice after lethal radiation doses and effectively achieve hematopoietic reconstruction and long-term hematopoiesis. Clinical symptoms, including hematologic parameters and tissue pathology of transplanted recipients, were significantly improved compared to the non-transplanted β654 mice. The therapeutic effect of gene-edited HSC transplantation demonstrated no significant difference in hematological parameters and tissue pathology compared with wild-type mouse-derived HSCs. Our data revealed that HSC transplantation from gene-edited mice completely recovered the β-thalassemia phenotype. Our study systematically investigated the therapeutic effect of HSCs derived from β654-ER mice on β-thalassemia and further confirmed the efficacy of our gene-editing approach. Altogether, it provided a reference and primary experimental data for the clinical usage of such gene-edited HSCs in the future. [ABSTRACT FROM AUTHOR]

Limb‐girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium‐dependent neutral cysteine protease calpain‐3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild‐type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild‐type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA‐sequencing results demonstrated that the expression levels of the mitochondrial‐related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function. [ABSTRACT FROM AUTHOR]

Pathogenic variants of zinc finger C4H2-type containing (ZC4H2) on the X chromosome cause a group of genetic diseases termed ZC4H2-associated rare disorders (ZARD), including Wieacker-Wolff Syndrome (WRWF) and Female-restricted Wieacker-Wolff Syndrome (WRWFFR). In the current study, a de novo c.352C>T (p.Gln118*) mutation in ZC4H2 (NM_018684.4) was identified in a female neonate born with severe arthrogryposis multiplex congenita (AMC) and Pierre-Robin sequence (cleft palate and micrognathia). Plasmids containing the wild-type (WT), mutant-type (MT) ZC4H2, or GFP report gene (N) were transfected in 293T cell lines, respectively. RT-qPCR and western blot analysis showed that ZC4H2 protein could not be detected in the 293T cells transfected with MT ZC4H2. The RNA seq results revealed that the expression profile of the MT group was similar to that of the N group but differed significantly from the WT group, indicating that the c.352C>T mutation resulted in the loss of function of ZC4H2. Differentially expressed genes (DEGs) enrichment analysis showed that c.352C>T mutation inhibited the expression levels of a series of genes involved in the oxidative phosphorylation pathway. Subsequently, expression levels of ZC4H2 were knocked down in neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) by lentiviral-expressed small hairpin RNAs (shRNAs) against ZC4H2. The results also demonstrated that decreasing the expression of ZC4H2 significantly reduced the growth of NSCs by affecting the expression of genes related to the oxidative phosphorylation signaling pathway. Taken together, our results strongly suggest that ZC4H2 c.352C>T (p.Gln118*) mutation resulted in the loss of protein function and caused WRWFFR. [ABSTRACT FROM AUTHOR]

Pantothenate kinase associated neurodegeneration (PKAN) is a severe autosomal recessive rare disease and characterized by iron accumulation in the basal ganglia. To investigate the pathogenesis of this disease in two sibling patients with PANK in a Chinese family, whole-exome variant detection and functional analysis were performed. Clinical and radiographic investigations were performed in the two brother patients. Whole exome sequencing (WES) was used in mutation detection, and the mutations were confirmed by Sanger sequencing. A longevity cohort genetic database was applied as Chinese urban controls. Bioinformatic analysis was performed to predict the pathogenicity. Compound heterozygous mutations of PANK2 were detected in two sibling brothers with PKAN in a Chinese family: c.510_522del (p.A170fs) and c.1319G > C (p.R440P) in the transcript NM_153638. PANK2: c.510_522del (p.A170fs) was absent in public data and the Chinese urban controls. Bioinformatics analysis showed that the above two variants were pathogenicity. We identified a rare compound heterozygous combination of PANK2 mutations found in a Chinese family in which two sibling brothers suffered from PKAN. PANK2 c.510_522del (p.A170fs) was the first reported to be a PKAN pathogenic variant. [ABSTRACT FROM AUTHOR]

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte''s response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach. [Copyright &y& Elsevier]

Objectives: This study explored whether TALENs‐mediated non‐homologous end joining (NHEJ) targeting the mutation site can correct the aberrant β‐globin RNA splicing, and ameliorate the β‐thalassaemia phenotype in β654 mice. Material and methods: TALENs vectors targeted to the human β‐globin gene (HBB) IVS2‐654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs+/β654 mice. The gene editing and off‐target effects were analysed by sequencing analysis. β‐globin expression was identified by RT‐PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs+/β654 mice. Results: Sequencing analysis revealed that the HBBIVS2‐654C >T point mutation was deleted in over 50% of the TALENs+/β654 mice tested, and off‐target effects were not detected. RT‐PCR and Western blot analysis confirmed the expression of normal β‐globin in TALENs+/β654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs+/β654 mice. Conclusion: These results suggest effective treatment of the anaemia phenotype in TALENs+/β654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of β654‐thalassaemia in the future. [ABSTRACT FROM AUTHOR]

Thalassemia is a recessive monogenic hematological disease associated with reduced amounts of functional hemoglobin caused by mutations/deletions in at least one of the globin genes. This disease has attracted significant attention throughout the years in terms of genetic diagnosis and developments in gene and cell therapy. Here, recent progress is reviewed in the genetic diagnosis and development of therapeutics for thalassemia, particularly β-thalassemia, in China and around the world. [ABSTRACT FROM AUTHOR]