Publications by authors named "Yukio Nakamura"

349 Publications

Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo.

Stem Cell Res 2021 Sep 3;56:102531. Epub 2021 Sep 3.

Cell Engineering Division, BioResource Research Center, RIKEN, Tsukuba, Japan.

Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.
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http://dx.doi.org/10.1016/j.scr.2021.102531DOI Listing
September 2021

Efficacy of Romosozumab for Osteoporosis in a Patient With Osteogenesis Imperfecta: A Case Report.

Mod Rheumatol Case Rep 2021 Aug 28. Epub 2021 Aug 28.

Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, Japan.

The efficacy of romosozumab for severe osteoporosis is uncertain in patients with osteogenesis imperfecta (OI). This report introduced a severe osteoporotic case of OI to examine the effect of romosozumab on bone fragility. A 64-year-old man with OI was referred to our department for finding out the cause of his repeated fractures. He was medicated with alendronate for only one year, eight years ago, but it did not prevent repeated fractures, and thus he had not received any treatments for osteoporosis since then. However, recently, the frequency of fractures had become increased. At presentation, his lumbar and bilateral total hip bone mineral density (BMD) values were severely decreased at 0.546 and 0.209 g/cm2, respectively. Because of his severe osteoporosis, we started romosozumab treatment with eldecalcitol. Romosozumab (210 mg) was injected subcutaneously every month. At 12 months after drug initiation, his lumbar and total hip BMD increased by 22.0% and 136.4% versus pre-treatment levels, respectively. Bone formation markers increased, and bone resorption markers decreased at 12 months of the therapy. Neither hypocalcemia nor any other severe adverse effects were observed in this severe osteoporotic case. This study revealed good responses of BMD and bone turnover markers to romosozumab treatment, which can be considered as an effective treatment option for osteoporotic OI patients.
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http://dx.doi.org/10.1093/mrcr/rxab018DOI Listing
August 2021

Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics.

Mol Ther Methods Clin Dev 2021 Sep 12;22:26-39. Epub 2021 Jun 12.

School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.

Developing robust methodology for the sustainable production of red blood cells is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34 cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34 cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.
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http://dx.doi.org/10.1016/j.omtm.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390520PMC
September 2021

Short- to Mid-Term Clinical Outcomes of Posterior-Stabilized Cementless Total Knee Arthroplasty with Trabecular Metal Components.

Ther Clin Risk Manag 2021 10;17:809-816. Epub 2021 Aug 10.

Department of Orthopaedic Surgery, Marunouchi Hospital, Matsumoto, Nagano, 390-8601, Japan.

Objective: This study aimed to evaluate the short- to mid-term clinical results of posterior-stabilized trabecular metal total knee arthroplasty (TKA) with cementless fixation of all components and investigate the radiographic changes of tibial and patellar components and cut bone surfaces over time.

Methods: We retrospectively collected the data of 128 knees from 88 consecutive patients who had undergone initial TKA with NexGen LPS-Flex TM implants. A total of 66 knees from 45 patients (mean ± standard deviation age: 70.3 ± 7.5 years) met the selection criteria, which had been employed cementless fixation of all parts and at least 3 years of postoperative follow-up duration. Clinical evaluations included range of motion, conventional knee score, function score, postoperative complications, and revision. For radiological evaluations, the bone contact surface of each implant was divided into 7 zones for tibial component and 2 zones for patellar component. Each region was examined immediately after surgery, at 6 and 12 months, and then every year afterwards.

Results: The mean observation period of 45 subjects was 4.2 years. Adequate fixation of tibial components was maintained during follow-up, although the patellar components of 2 knees required revision after repeated falls. No loosening was observed in any implants. The initial gap in tibial components disappeared in all knees, and a reactive line remained in 4 knees. There were no revisions, except for 2 cases, which were ascribed to patellar component fracture caused by repeated falls.

Conclusion: Cementless posterior-stabilized trabecular metal TKA appears to be a good surgical option. Longer-term examination for revision cases is required to validate our results.
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http://dx.doi.org/10.2147/TCRM.S320941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8364354PMC
August 2021

Transcriptional silencing of fetal hemoglobin expression by NonO.

Nucleic Acids Res 2021 Sep;49(17):9711-9723

The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and Urology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing, China.

Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult β-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.
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http://dx.doi.org/10.1093/nar/gkab671DOI Listing
September 2021

Associations of homocysteine metabolism with the risk of spinal osteoarthritis progression in postmenopausal women.

J Clin Endocrinol Metab 2021 Aug 10. Epub 2021 Aug 10.

Research Institute and Practice for Involutional Diseases, Azumino, Nagano, Japan.

Purpose: Although homocysteine accumulation is a reported risk factor for several age-related disorders, little is known on its relationship with osteoarthritis (OA). We therefore investigated for associations of homocysteine and C677T polymorphism in methylenetetrahydrofolate reductase (MTHFR), which is involved in homocysteine clearance, with the development and progression of spinal OA, through a combined cross-sectional and longitudinal cohort study.

Methods: A total of 1306 Japanese postmenopausal outpatients participating in the Nagano Cohort Study were followed for a 9.7-year mean period. Cross-sectional multiple logistic regression for spinal OA prevalence at registration by serum homocysteine level was performed with adjustment for confounders. In addition to Kaplan-Meier analysis, multivariate Cox regression was employed to examine the independent risk of MTHFR C677T variant for spinal OA progression.

Results: Multivariate regression analysis revealed a significant association between homocysteine and spinal OA prevalence (odds ratio 1.38; 95% confidence interval [CI] 1.14-1.68). Kaplan-Meier curves showed a gene dosage effect of the T allele in MTHFR C677T polymorphism on the accelerated progression of spinal OA severity (P = 0.003). A statistically significant independent risk of the T allele for spinal OA advancement was validated by Cox regression analysis. Respective adjusted hazard ratios for the CT/TT and TT genotypes were 1.68 (95% CI 1.16-2.42) and 1.67 (95% CI 1.23-2.28).

Conclusions: Circulating homocysteine and C677T variant in MTHFR are associated with the prevalence rate and ensuing progression, respectively, of spinal OA. These factors may represent potential interventional targets to prevent OA development and improve clinical outcomes.
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http://dx.doi.org/10.1210/clinem/dgab591DOI Listing
August 2021

Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis.

Epigenetics Chromatin 2021 Jul 30;14(1):37. Epub 2021 Jul 30.

Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester, Rochester, NY, USA.

Background: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis.

Results: We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X.

Conclusions: We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed.
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http://dx.doi.org/10.1186/s13072-021-00408-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325214PMC
July 2021

Deletion of Jdp2 enhances Slc7a11 expression in Atoh-1 positive cerebellum granule cell progenitors in vivo.

Stem Cell Res Ther 2021 06 29;12(1):369. Epub 2021 Jun 29.

Graduate Institute of Medicine, Regenerative Medicine and Cell Therapy Research Center, School of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.

Background: The cerebellum is the sensitive region of the brain to developmental abnormalities related to the effects of oxidative stresses. Abnormal cerebellar lobe formation, found in Jun dimerization protein 2 (Jdp2)-knockout (KO) mice, is related to increased antioxidant formation and a reduction in apoptotic cell death in granule cell progenitors (GCPs). Here, we aim that Jdp2 plays a critical role of cerebellar development which is affected by the ROS regulation and redox control.

Objective: Jdp2-promoter-Cre transgenic mouse displayed a positive signal in the cerebellum, especially within granule cells. Jdp2-KO mice exhibited impaired development of the cerebellum compared with wild-type (WT) mice. The antioxidation controlled gene, such as cystine-glutamate transporter Slc7a11, might be critical to regulate the redox homeostasis and the development of the cerebellum.

Methods: We generated the Jdp2-promoter-Cre mice and Jdp2-KO mice to examine the levels of Slc7a11, ROS levels and the expressions of antioxidation related genes were examined in the mouse cerebellum using the immunohistochemistry.

Results: The cerebellum of Jdp2-KO mice displayed expression of the cystine-glutamate transporter Slc7a11, within the internal granule layer at postnatal day 6; in contrast, the WT cerebellum mainly displayed Sla7a11 expression in the external granule layer. Moreover, development of the cerebellar lobes in Jdp2-KO mice was altered compared with WT mice. Expression of Slc7a11, Nrf2, and p21 was higher in the cerebellum of Jdp2-KO mice than in WT mice.

Conclusion: Jdp2 is a critical regulator of Slc7a11 transporter during the antioxidation response, which might control the growth, apoptosis, and differentiation of GCPs in the cerebellar lobes. These observations are consistent with our previous study in vitro.
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http://dx.doi.org/10.1186/s13287-021-02424-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8243712PMC
June 2021

Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing.

Stem Cell Res 2021 05 22;53:102363. Epub 2021 Apr 22.

iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Electronic address:

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.
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http://dx.doi.org/10.1016/j.scr.2021.102363DOI Listing
May 2021

Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines.

Commun Biol 2021 06 3;4(1):677. Epub 2021 Jun 3.

Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.
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http://dx.doi.org/10.1038/s42003-021-02202-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175573PMC
June 2021

Denosumab versus romosozumab for postmenopausal osteoporosis treatment.

Sci Rep 2021 Jun 3;11(1):11801. Epub 2021 Jun 3.

Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.

Denosumab and romosozumab, a recently approved new drug, are effective and widely known molecular-targeted drugs for postmenopausal osteoporosis treatment. However, no studies have directly compared their therapeutic effects or safety in postmenopausal osteoporosis. This retrospective observational registry study compared the efficacy of 12-month denosumab or romosozumab treatment in postmenopausal osteoporosis patients. The primary outcome was the change in bone mineral density (BMD) at the lumbar spine. Secondary outcomes included BMD changes at the total hip and femoral neck, changes in bone turnover markers, and adverse events. Propensity score matching was employed to assemble patient groups with similar baseline characteristics. Sixty-nine patients each received either denosumab or romosozumab for 12 months. The mean 12-month percentage change from baseline in lumbar spine BMD was 7.2% in the denosumab group and 12.5% in the romosozumab group, indicating a significant difference between the groups. The percentage changes in BMD at both the total hip and femoral neck were also significantly higher at 12 months in the romosozumab group than in the denosumab group. In denosumab patients, bone formation and bone resorption markers were significantly decreased at 6 and 12 months from baseline. In the romosozumab group, the bone formation marker was significantly increased at 6 months and then returned to baseline, while the bone resorption marker was significantly decreased at both time points. Adverse events were few and predominantly minor in both groups, with no remarkable difference in the incidence of new vertebral fractures. Romosozumab showed a higher potential for improving BMD than denosumab in this clinical study of postmenopausal osteoporosis patient treatment.
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http://dx.doi.org/10.1038/s41598-021-91248-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175428PMC
June 2021

Zinc Pharmacotherapy for Elderly Osteoporotic Patients with Zinc Deficiency in a Clinical Setting.

Nutrients 2021 May 27;13(6). Epub 2021 May 27.

Department of Orthopaedic Surgery, School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.

Although there have been reported associations between zinc and bone mineral density (BMD), no reports exist on the effect of zinc treatment in osteoporotic patients. Therefore, we investigated the efficacy and safety of zinc pharmacotherapy in Japanese elderly patients. The present investigation included 122 osteoporotic patients with zinc deficiency, aged ≥65 years, who completed 12 months of follow-up. In addition to standard therapy for osteoporosis in a clinical setting, the subjects received oral administration of 25 mg zinc (NOBELZIN, an only approved drug for zinc deficiency in Japan) twice a day. BMD and laboratory data including bone turnover markers were collected at 0 (baseline), 6, and 12 months of zinc treatment. Neither serious adverse effects nor incident fractures were seen during the observation period. Serum zinc levels were successfully elevated by zinc administration. BMD increased significantly from baseline at 6 and 12 months of zinc treatment. Percentage changes of serum zinc showed significantly positive associations with those of BMD. Bone formation markers rose markedly from the baseline values, whereas bone resorption markers displayed moderate or no characteristic changes. Additive zinc supplementation may contribute to BMD augmentation ensuing the prevention of fracture occurrence in elderly osteoporotic patients with zinc deficiency.
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http://dx.doi.org/10.3390/nu13061814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229639PMC
May 2021

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with β-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Br J Haematol 2021 06 17;193(6):1220-1227. Epub 2021 May 17.

Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat β-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with β-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34 cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with β-thalassaemia.
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http://dx.doi.org/10.1111/bjh.17511DOI Listing
June 2021

Real-world effects and adverse events of romosozumab in Japanese osteoporotic patients: A prospective cohort study.

Bone Rep 2021 Jun 16;14:101068. Epub 2021 Apr 16.

Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.

Real-world data on the new anti-sclerostin antibody drug, romosozumab, remain scarce. There is a strong need to accumulate and analyze data on romosozumab treatment for such conditions as osteoporosis. The purpose of this study was to investigate the therapeutic and adverse effects of romosozumab for osteoporosis treatment in clinical practice. Of the 230 osteoporosis patients prescribed romosozumab from September 2019 in this prospective multicenter cohort study, 204 patients completed 12 months of treatment. The primary outcome of interest was the rate of change in bone mineral density (BMD) of the lumbar spine, total hip, and femoral neck as measured by dual-energy X-ray absorptiometry. Secondary outcomes included changes in bone turnover markers and serum-corrected calcium level as well as the incidence of adverse events. At 6 and 12 months of romosozumab treatment, the respective percentage change in BMD from baseline was 7.4% and 12.2% for the lumbar spine, 1.8% and 5.8% for the total hip, and 2.9% and 6.0% for the femoral neck, all of which were significantly higher ( < 0.001) than baseline values. Patients who switched from another osteoporosis regimen exhibited significantly lower lumbar spine BMD gains versus treatment-naïve patients, especially for cases switching from denosumab. P1NP was significantly increased at 6 months (58.9%;  < 0.01), while TRACP-5b was significantly decreased at 6 months (-14.7%;  < 0.001) and 12 months (-18.8%;  < 0.001) versus baseline values. The largest rate of decrease in serum-corrected calcium was 3.7% at 12 months. Sixty-four (27.8%) of 230 patients experienced an adverse event, and 7 (3.0%) new fractures were recorded. In sum, romosozumab treatment for 12 months significantly improved lumbar spine, total hip, and femoral neck BMD according to real-world data.
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http://dx.doi.org/10.1016/j.bonr.2021.101068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085670PMC
June 2021

Molecular analysis of the erythroid phenotype of a patient with BCL11A haploinsufficiency.

Blood Adv 2021 05;5(9):2339-2349

Academic Center for Hemoglobinopathies and Rare Anemias.

The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach.
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http://dx.doi.org/10.1182/bloodadvances.2020003753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114548PMC
May 2021

Generation of two human induced pluripotent stem cell lines derived from two X-linked adrenoleukodystrophy patients with ABCD1 mutations.

Stem Cell Res 2021 05 15;53:102337. Epub 2021 Apr 15.

iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Electronic address:

Adrenoleukodystrophy (ALD) is an X-linked genetic disorder, characterized by demyelination in the central nervous system and adrenal insufficiency. Human induced pluripotent stem cell (hiPSC) lines derived from two Japanese male patients with ALD were generated from skin fibroblasts using retroviral vectors. The generated hiPSC lines showed self-renewal and pluripotency, and carried either a missense or a nonsense mutation in ABCD1 gene. Since the molecular pathogenesis caused by ABCD1 dysfunction remains unclear, these cell resources provide useful tools to establish disease models and to develop new therapies for X-ALD.
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http://dx.doi.org/10.1016/j.scr.2021.102337DOI Listing
May 2021

Reference values for bone metabolism in a Japanese cohort survey randomly sampled from a basic elderly resident registry.

Sci Rep 2021 Apr 9;11(1):7822. Epub 2021 Apr 9.

Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.

The aim of this study was to provide definitive reference values for bone mineral density (BMD) and bone turnover markers in the general elderly population. Registered citizens of 50 to 89 years old were targeted for this survey. After random sampling from the resident registry of Obuse town, we established eight groups based on age (50 s, 60 s, 70 s, and 80 s) and gender. A total of 411 people were enrolled. We used a dual-energy x-ray absorptiometry device to measure and evaluate BMD. The bone formation marker bone alkaline phosphatase (BAP) was measured as a bone turnover marker. Bone quality marker pentosidine, and bone resorption markers including urinary total deoxypyridinoline (DPD), tartrate-resistant acid phosphatase 5b (TRACP-5b), 25-hydroxyvitamin D (25[OH]D), and whole parathyroid hormone (PTH) were also measured as bone turnover markers. Sixty-three people (15.3%) were diagnosed as osteoporosis. BMD decreased with age in the femoral neck and total hip. On the other hand, there was no characteristic change with age in the lumber spine. As for bone markers, pentosidine and DPD increased with aging, although 25(OH)D, whole PTH, and BAP showed no characteristic associations with gender and aging. In terms of the relationship between low BMD and bone markers, there was a significant independent association between low BMD and TRACP-5b in females. In conclusions, hip BMD decreased with aging in men and women. However, there was no characteristic decline with aging in the lumbar spine. All bone markers showed no significant independent characteristics associated with age or gender in a multivariate analysis model, except for a significant association between low BMD and TRACP-5b in females. TRACP-5b was a potentially useful marker for the detection of low BMD.
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http://dx.doi.org/10.1038/s41598-021-87393-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035137PMC
April 2021

Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells.

Int J Mol Sci 2021 Mar 31;22(7). Epub 2021 Mar 31.

Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia 2371, Cyprus.

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.
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http://dx.doi.org/10.3390/ijms22073626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037168PMC
March 2021

Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells.

Cells 2021 03 1;10(3). Epub 2021 Mar 1.

Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore 632002, Tamil Nadu, India.

Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34 hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from patients with red cell diseases is challenging as obtaining a sufficient number of CD34 cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.
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http://dx.doi.org/10.3390/cells10030523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999632PMC
March 2021

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1.

Biochem Biophys Res Commun 2021 05 19;552:157-163. Epub 2021 Mar 19.

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200438, China. Electronic address:

β-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, αβ), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat β-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, αγ) in β-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in β-thalassemia.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.051DOI Listing
May 2021

Integrated Collection of Stem Cell Bank Data, a Data Portal for Standardized Stem Cell Information.

Stem Cell Reports 2021 Apr 18;16(4):997-1005. Epub 2021 Mar 18.

Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Sho-goin, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address:

The past decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed the Integrated Collection of Stem Cell Bank data (ICSCB) (http://icscb.stemcellinformatics.org/), the largest database search portal for stem cell line information, based on the standardized data items and terms of the MIACARM framework. Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases, including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.
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http://dx.doi.org/10.1016/j.stemcr.2021.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072026PMC
April 2021

Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia.

Am J Hum Genet 2021 04 17;108(4):709-721. Epub 2021 Mar 17.

Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515 Guangdong, China; Guangdong Engineering and Technology Research Center for Molecular Diagnostics of Human Genetic Diseases, Guangzhou, 510515 Guangdong, China; Guangdong Engineering and Technology Research Center for Genetic Testing, Guangzhou, 510515 Guangdong, China. Electronic address:

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34 erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.
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http://dx.doi.org/10.1016/j.ajhg.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059375PMC
April 2021

Dimethyl sulfoxide stimulates the AhR-Jdp2 axis to control ROS accumulation in mouse embryonic fibroblasts.

Cell Biol Toxicol 2021 Mar 15. Epub 2021 Mar 15.

Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

The aryl hydrocarbon receptor (AhR) is a ligand-binding protein that responds to environmental aromatic hydrocarbons and stimulates the transcription of downstream phase I enzyme-related genes by binding the cis element of dioxin-responsive elements (DREs)/xenobiotic-responsive elements. Dimethyl sulfoxide (DMSO) is a well-known organic solvent that is often used to dissolve phase I reagents in toxicology and oxidative stress research experiments. In the current study, we discovered that 0.1% DMSO significantly induced the activation of the AhR promoter via DREs and produced reactive oxygen species, which induced apoptosis in mouse embryonic fibroblasts (MEFs). Moreover, Jun dimerization protein 2 (Jdp2) was found to be required for activation of the AhR promoter in response to DMSO. Coimmunoprecipitation and chromatin immunoprecipitation studies demonstrated that the phase I-dependent transcription factors, AhR and the AhR nuclear translocator, and phase II-dependent transcription factors such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) integrated into DRE sites together with Jdp2 to form an activation complex to increase AhR promoter activity in response to DMSO in MEFs. Our findings provide evidence for the functional role of Jdp2 in controlling the AhR gene via Nrf2 and provide insights into how Jdp2 contributes to the regulation of ROS production and the cell spreading and apoptosis produced by the ligand DMSO in MEFs.
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http://dx.doi.org/10.1007/s10565-021-09592-2DOI Listing
March 2021

Identification of characteristic proteins at late-stage erythroid differentiation in vitro.

Hum Cell 2021 May 22;34(3):745-749. Epub 2021 Feb 22.

Department of Research and Development, Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Tatsumi 2-1-67, Koto-ku, Tokyo , 135-8521, Japan.

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.
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http://dx.doi.org/10.1007/s13577-021-00503-5DOI Listing
May 2021

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Mol Ther 2021 04 29;29(4):1625-1638. Epub 2021 Jan 29.

Division of Hematology, Children's Hospital of Philadelphia (CHOP), Philadelphia, PA, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Cell and Molecular Biology Affinity Group (CAMB), University of Pennsylvania, Philadelphia, PA, USA; Raymond G. Perelman Center for Cellular and Molecular Therapeutics, CHOP, Philadelphia, PA, USA; Penn Center for Musculoskeletal Disorders, CHOP, Philadelphia, PA, USA. Electronic address:

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34 cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058492PMC
April 2021

FGF-23 from erythroblasts promotes hematopoietic progenitor mobilization.

Blood 2021 03;137(11):1457-1467

Division of Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.

Fibroblast growth factor 23 (FGF-23) hormone is produced by bone-embedded osteocytes and regulates phosphate homeostasis in kidneys. We found that administration of granulocyte colony-stimulating factor (G-CSF) to mice induced a rapid, substantial increase in FGF-23 messenger RNA in bone marrow (BM) cells. This increase originated mainly from CD45-Ter119+CD71+ erythroblasts. FGF-23 protein in BM extracellular fluid was markedly increased during G-CSF-induced hematopoietic progenitor cell (HPC) mobilization, but remained stable in the blood, with no change in the phosphate level. Consistent with the BM hypoxia induced by G-CSF, low oxygen concentration induced FGF-23 release from human erythroblast HUDEP-2 cells in vitro. The efficient mobilization induced by G-CSF decreased drastically in both FGF-23-/- and chimeric mice with FGF-23 deficiency, only in hematopoietic cells, but increased in osteocyte-specific FGF-23-/- mice. This finding suggests that erythroblast-derived, but not bone-derived, FGF-23 is needed to release HPCs from BM into the circulation. Mechanistically, FGF-23 did not influence CXCL-12 binding to CXCR-4 on progenitors but interfered with their transwell migration toward CXCL-12, which was canceled by FGF receptor inhibitors. These results suggest that BM erythroblasts facilitate G-CSF-induced HPC mobilization via FGF-23 production as an intrinsic suppressor of chemoattraction.
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http://dx.doi.org/10.1182/blood.2020007172DOI Listing
March 2021

Genome-wide analysis of pseudogenes reveals HBBP1's human-specific essentiality in erythropoiesis and implication in β-thalassemia.

Dev Cell 2021 02 20;56(4):478-493.e11. Epub 2021 Jan 20.

State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China.

The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in β-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in β-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes.
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http://dx.doi.org/10.1016/j.devcel.2020.12.019DOI Listing
February 2021

Pentosidine and carboxymethyl-lysine associate differently with prevalent osteoporotic vertebral fracture and various bone markers.

Sci Rep 2020 12 16;10(1):22090. Epub 2020 Dec 16.

Research Institute and Practice for Involutional Diseases, 1610-1 Meisei, Misato, Azumino, Nagano, 399-8101, Japan.

Pentosidine (PEN) and carboxymethyl-lysine (CML) are well-recognized advanced glycation end products (AGEs). However, how these AGEs affect the pathophysiology of osteoporosis and osteoporotic fractures remains controversial. This cross-sectional study aimed to investigate the associations of PEN and CML with bone markers, bone mineral density (BMD), and osteoporotic fractures in postmenopausal women from the Nagano Cohort Study. A total of 444 Japanese postmenopausal outpatients (mean ± standard deviation age: 69.8 ± 10.2 years) were enrolled after the exclusion of patients with acute or severe illness or secondary osteoporosis. The relationships among urinary PEN and serum CML levels, various bone markers, lumbar and hip BMD, and prevalent vertebral and long-bone fractures were evaluated. PEN associated significantly with prevalent vertebral fracture after adjustment for other confounders (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.22-2.07; P < 0.001), but not with lumbar BMD. In contrast, a significant negative correlation was found between CML and lumbar BMD (r =  - 0.180; P < 0.001), and this relationship was significant after adjustment for confounders (OR 0.84, 95% CI 0.76-0.93; P < 0.01). Although patients with prevalent vertebral fracture had significantly higher CML levels, the association between CML and prevalent vertebral fracture did not reach significance in the multivariate regression model. Both PEN and CML may play important roles in bone health for postmenopausal women, possibly via different mechanisms.
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http://dx.doi.org/10.1038/s41598-020-78993-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744574PMC
December 2020
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