Clinical Characteristics and Induced Pluripotent Stem Cells (iPSCs) Disease Model of Fabry Disease Caused by a Novel GLA Mutation.

BACKGROUND: Fabry disease (FD) is a rare X-linked inherited disease caused by mutations in the GLA gene. We established a cohort of FD patients and performed whole-exome sequencing (WES) to identify some novel mutations. AIM: The aim of this study is to investigate the etiology of the novel mutation (c.72G > A, p.Trp24*）in the GLA gene in affected patients by using induced pluripotent stem cells (iPSCs) as a valuable tool. METHODS: We explored the clinical implications of this proband and examined the deleteriousness and conservation of the mutation site through bioinformatics analysis. Simultaneously, we collected the peripheral blood mononuclear cells (PBMCs) of the affected patient, then reprogrammed them into iPSCs and assessed their enzymatic activity to confirm the function of lysosomal enzyme α-galactosidase A (α-Gal A). RESULTS: Clinical examination of the patient demonstrated a classical FD, such as neuropathic pain, gastrointestinal disorders, deficiency of α-Gal A activity, and accumulation of Lyso-Gb-3. The novel mutation located on the N-terminal region, leading to a truncation of the protein and remaining only 24 amino acids. The α-Gal A activity of the patient-specific iPSC (iPS-FD) was significantly lower (60%) than that of normal iPSCs derived from healthy donors (iPS-B1). CONCLUSION: This work not only elucidated the etiology of novel mutations in affected patients but also highlighted the utility of iPSCs as a valuable tool for clarifying the molecular mechanisms and providing new insights into the therapy of FD.

Lipidomic profiles in serum and urine in children with steroid sensitive nephrotic syndrome.

BACKGROUND: Steroid-sensitive nephrotic syndrome (SSNS) accounts for approximately 80% of cases of nephrotic syndrome. The involvement of aberrant lipid metabolism in early SSNS is poorly understood, warranting further investigation. This study aimed to explore alterations in lipid metabolism associated with SSNS pathogenesis. METHODS: A screening cohort containing serum (50 SSNS, 37 controls) and urine samples (27 SSNS, 26 controls) was analyzed by untargeted lipidomic profiling using UHPLC-QTOF-MS. Then, a validation cohort (20 SSNS, 56 controls) underwent further analysis to check the potential clinical application by ROC curve analysis. RESULTS: Lipidomic profiling of serum and urine samples revealed significant lipid alterations in SSNS patients, with the alterations in the serum samples being more significant. An elevated concentration of PE and PG and downregulated concentration of FA were observed in SSNS serum. A total of 38 dysregulated lipids and 5 lipid metabolic pathways were identified in the serum samples in SSNS patients. Validation in the second cohort confirmed differential regulation of nine kinds of lipids, including 5 up-regulated substances [SM d33:2 (m/z = 686.5361), SHexCer d34:1 (m/z = 779.521), PI 20:4_22:4 (m/z = 934.5558), Cer_NS d18:1_23:0 (m/z = 635.6216), and GM3 d36:1 (m/z = 1180.7431)], as well as 4 down-regulated substances: [CE 18:1 (m/z = 650.601), PE 38:6 (m/z = 763.5205), PC 17:0_20:4 (m/z = 795.5868) and EtherPC 16:2e_20:4 (m/z = 763.5498)]. CONCLUSIONS: Untargeted lipidomic analysis successfully identified specific lipid class changes in patients with SSNS, providing a deeper understanding of lipid alterations and underlying mechanisms associated with SSNS.

Dent disease 1-linked novel CLCN5 mutations result in aberrant location and reduced ion currents.

Dent disease is a rare renal tubular disease with X-linked recessive inheritance characterized by low molecular weight proteinuria (LMWP), hypercalciuria, and nephrocalcinosis. Mutations disrupting the 2Cl-/1H+ exchange activity of chloride voltage-gated channel 5 (CLCN5) have been causally linked to the most common form, Dent disease 1 (DD1), although the pathophysiological mechanisms remain unclear. Here, we conducted the whole exome capture sequencing and bioinformatics analysis within our DD1 cohort to identify two novel causal mutations in CLCN5 (c.749 G > A, p. G250D, c.829 A > C, p. T277P). Molecular dynamics simulations of ClC-5 homology model suggested that these mutations potentially may induce structural changes, destabilizing ClC-5. Overexpression of variants in vitro revealed aberrant subcellular localization in the endoplasmic reticulum (ER), significant accumulation of insoluble aggregates, and disrupted ion transport function in voltage clamp recordings. Moreover, human kidney-2 (HK-2) cells overexpressing either G250D or T277P displayed higher cell-substrate adhesion, migration capability but reduced endocytic function, as well as substantially altered transcriptomic profiles with G250D resulting in stronger deleterious effects. These cumulative findings supported pathogenic role of these ClC-5 mutations in DD1 and suggested a cellular mechanism for disrupted renal function in Dent disease patients, as well as a potential target for diagnostic biomarker or therapeutic strategy development.

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Low skeletal muscle mass as an early sign in children with fabry disease.

BACKGROUND & AIMS: Fabry disease (FD) is a rare X-linked metabolic storage disorder due to the deficiency of lysosomal α-galactosidase A which causes the accumulation of glycosphingolipids throughout the body. Underweight and low BMI have been occasionally reported in FD patients previously. Whether underweight is common in the early stage of FD and body composition analysis to determine the cause have not been reported. METHODS: Children who were diagnosed with FD in the Children's Hospital of Zhejiang University School of Medicine from July 2014 to December 2022 were enrolled. Clinical data were obtained from medical records. Whole body dual energy X-ray absorptiometry scans (DXA) were used to assess body composition (fat mass, FM; fat free mass, FFM and bone mass) according to the International Society of Clinical Densitometry's standard operating method. Whole body muscle mass was calculated as fat-free mass minus bone mass. Appendicular skeletal muscle mass (ASM) was calculated as the sum of the arm and the leg muscle mass. The FM, FFM, ULSM and LLSM indices were calculated by dividing the total FM, FFM, and upper and lower limb skeletal muscle mass (ULSM and LLSM) by the height squared. RESULTS: A total of eighteen children (14 boys and 4 girls) were enrolled. Thirteen boys had the classical phenotype, and five children (1 boy with the N215S mutation and 4 girls) had the late-onset phenotype. Seven children with the classical phenotype (53.8%) and two of the five children (40%) with the late-onset phenotype had abnormal BMIs. Sixteen of the eighteen children (88.9%) had a height in the normal range, suggesting that low BMI was mainly due to underweight. By DXA body composition analysis, the FMI was abnormal in 3 children (2 boys and 1 girl), and the FFMI was abnormal in 12 children (9 boys and 3 girls). For the classical phenotype, 2 of the 13 children (15.4%) had abnormal FMI values, while 10 (76.9%) had abnormal FFMI values. Eight patients (61.5%) with the classical phenotype had a significant reduction in muscle mass index, ASM index and LLSM index values compared with age- and sex- matched Chinese controls. Late-onset patients also had mild low skeletal muscle mass compared to controls. The results suggested that low skeletal muscle mass is common in early FD. CONCLUSIONS: This is the first study to examine body composition and muscle mass in early Fabry disease patients. Low skeletal muscle mass is a common early symptom in children with Fabry disease, suggesting that skeletal muscle is significantly affected in the early stages of FD.

A Review of Inactivated COVID-19 Vaccine Development in China: Focusing on Safety and Efficacy in Special Populations.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been widespread globally, and vaccination is critical for preventing further spread or resurgence of the outbreak. Inactivated vaccines made from whole inactivated SARS-CoV-2 virus particles generated in Vero cells are currently the most widely used COVID-19 vaccines, with China being the largest producer of inactivated vaccines. As a result, the focus of this review is on inactivated vaccines, with a multidimensional analysis of the development process, platforms, safety, and efficacy in special populations. Overall, inactivated vaccines are a safe option, and we hope that the review will serve as a foundation for further development of COVID-19 vaccines, thus strengthening the defense against the pandemic caused by SARS-CoV-2.

Early onset of nephrogenic diabetes insipidus due to fabry disease in a child with GLA N215S mutation: Case report and literature review.

Background: Fabry disease (FD) is a rare X-linked lysosomal storage disorder. Renal involvement in FD is characterized by proteinuria and progressive renal decline. Reports on FD with nephrogenic diabetes insipidus as the initial manifestation are rare. In this paper, we report a pediatric case with an N215S variant. Results: A boy with an onset of polydipsia and polyuria at approximately 4 years of age was diagnosed with nephrogenic diabetes insipidus. Whole-exome sequencing showed a GLA N215S variation with no secondary cause of diabetes insipidus. No family history of polydipsia or polyuria was reported; however, the patient's maternal grandmother and her two younger brothers had hypertrophic cardiomyopathy. Both brothers required surgery due to severe cardiac involvement, and the youngest brother died of heart disease at the age of 50 years. The patient's polydipsia and polyuria worsened over the next 7 years. Serum sodium was normal, but the patient required high-dose potassium chloride to maintain normal serum potassium levels. His physical and intellectual development was normal, with no common complications of nephrogenic diabetes insipidus, such as anemia, malnutrition, vomiting, high fever, or convulsions. Dried blood spot testing showed α-galactosidase A (α-gal A) activity of 0.6 µmol/L/h and a Lyso-GL-3 level of 7.01 ng/ml. The patient developed mild proteinuria and mild myocardial hypertrophy. Renal biopsy showed myeloid bodies and zebra bodies. After more than 1 year of ERT, his urine specific gravity increased to 1.005-1.008, which was a new sign reflecting the efficacy of ERT, although urine output was maintained at 3-5 ml/kg/hour. We will continue to monitor the patient's renal tubular function and urine output. Conclusions: Nephrogenic diabetes insipidus may be the initial manifestation in children with FD and/or N215S variation. In FD, the same mutation in a family may present a completely different phenotype.

Metabolomic profiles in serum and urine uncover novel biomarkers in children with nephrotic syndrome.

BACKGROUND: Nephrotic syndrome is common in children and adults worldwide, and steroid-sensitive nephrotic syndrome (SSNS) accounts for 80%. Aberrant metabolism involvement in early SSNS is sparsely studied, and its pathogenesis remains unclear. Therefore, the goal of this study was to investigate the changes in initiated SSNS patients-related metabolites through serum and urine metabolomics and discover the novel potential metabolites and metabolic pathways. METHODS: Serum samples (27 SSNS and 56 controls) and urine samples (17 SSNS and 24 controls) were collected. Meanwhile, the non-targeted analyses were performed by ultra-high-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-QTOF-MS) to determine the changes in SSNS. We applied the causal inference model, the DoWhy model, to assess the causal effects of several selected metabolites. An ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to validate hits (D-mannitol, dulcitol, D-sorbitol, XMP, NADPH, NAD, bilirubin, and α-KG-like) in 41 SSNS and 43 controls. In addition, the metabolic pathways were explored. RESULTS: Compared to urine, the metabolism analysis of serum samples was more clearly discriminated at SSNS. 194 differential serum metabolites and five metabolic pathways were obtained in the SSNS group. Eight differential metabolites were identified by establishing the diagnostic model for SSNS, and four variables had a positive causal effect. After validation by targeted MS, except XMP, others have similar trends like the untargeted metabolic analysis. CONCLUSION: With untargeted metabolomics analysis and further targeted quantitative analysis, we found seven metabolites may be new biomarkers for risk prediction and early diagnosis for SSNS.

A protocol for the generation of patient-specific iPSC lines from peripheral blood mononuclear cells.

The current procedure thoroughly explains how to reprogram induced pluripotent stem cells (iPSCs) from the patient's peripheral blood mononuclear cells (PBMCs), a less invasive source; e.g., somatic cells. We describe how to isolate PBMCs and reprogram them into iPSCs by electroporation. Furthermore, we provide an alternative approach to generating iPSC using Geltrex or Matrigel matrix to replace MEF-feeder. The challenge of this process is the relatively lower cell survival rates of PBMCs due to the damage of electroporation. For complete details on the use and execution of this protocol, please refer to Hu et al. (2021).