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BackgroundThe aim is to evaluate the clinical utility of a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in prenatal diagnosis of thalassemia.MethodsA total of 278 fetuses from at-risk pregnancies identified in thalassemia carrier screening by PCR-based methods were recruited from 9 hospitals, and PCR-based methods were employed for prenatal diagnosis. CATSA was performed retrospectively and blindly for all 278 fetuses.ResultsAmong the 278 fetuses, 263 (94.6%) had concordant results and 15 (5.4%) had discordant results between the 2 methods. Of the 15 fetuses, 4 had discordant thalassemia variants within the PCR detection range and 11 had additional variants identified by CATSA. Independent PCR and Sanger sequencing confirmed the CATSA results. In total, CATSA and PCR-based methods correctly detected 206 and 191 fetuses with variants, respectively. Thus, CATSA yielded a 7.9% (15 of 191) increment as compared with PCR-based methods. CATSA also corrected the predicted phenotype in 8 fetuses. Specifically, a PCR-based method showed one fetus had homozygous HBB c.52A > T variants, while CATSA determined the variant was heterozygous, which corrected the predicted phenotype from β-thalassemia major to trait, potentially impacting the pregnancy outcome. CATSA additionally identified α-globin triplicates in 2 fetuses with the heterozygous HBB c.316-197C > T variant, which corrected the predicted phenotype from β-thalassemia trait to intermedia and changed the disease prognosis.ConclusionsCATSA represents a more comprehensive and accurate approach that potentially enables more informed genetic counseling and improved clinical outcomes compared to PCR-based methods.

Erythropoiesis is a highly complex and sophisticated multistage process regulated by many transcription factors, as well as noncoding RNAs. Anthrax toxin receptor 1 (ANTXR1) is a type I transmembrane protein that binds the anthrax toxin ligands and mediates the entry of its toxic part into cells. It also functions as a receptor for the Protective antigen (PA) of anthrax toxin, and mediates the entry of Edema factor (EF) and Lethal factor (LF) into the cytoplasm of target cells and exerts their toxicity. Previous research has shown that ANTXR1 inhibits the expression of γ-globin during the differentiation of erythroid cells. However, the effect on erythropoiesis from a cellular perspective has not been fully determined. This study examined the role of ANTXR1 on erythropoiesis using K562 and HUDEP-2 cell lines as well as cord blood CD34+ cells. Our study has shown that overexpression of ANTXR1 can positively regulate erythrocyte proliferation, as well as inhibit GATA1 and ALAS2 expression, differentiation, and apoptosis in K562 cells and hematopoietic stem cells. ANTXR1 knockdown inhibited proliferation, promoted GATA1 and ALAS2 expression, accelerated erythrocyte differentiation and apoptosis, and promoted erythrocyte maturation. Our study also showed that ANTXR1 may regulate the proliferation and differentiation of hematopoietic cells, though the Wnt/β-catenin pathway, which may help to establish a possible therapeutic target for the treatment of blood disorders.

Background: Arterial calcification in the aortic arch, carotid bifurcation, or siphon on computed tomography was associated with cardiovascular disease. The association between arterial calcification prevalence and progression of branch atheromatous disease (BAD) in intracranial artery atherosclerosis was little investigated. Methods: This study included 310 patients with ischemic stroke from one stroke center. Patients were divided into BAD (110) and non-BAD groups (200). Baseline characteristics, lipids, and arterial calcification were measured. The primary outcome was the prevalence of arterial calcification in BAD progression, and the secondary outcome was the prevalence of calcification in arterial stenosis. The association or correlation among calcification prevalence, lipid markers, and BAD progression was analyzed using logistic regression, receiver operating characteristic curve, and linear regression. Results: Our study found that carotid siphon calcification on computed angiography was more prevalent ( P =0.01) in patients with BAD and also more prevalent ( P P =0.01). Furthermore, a strong linear correlation between oxidized lipid and calcification density was found (beta=−0.73, P =0.0048) in patients with BAD, a subtype (B-type) of intracranial arterial atherosclerotic disease. Conclusions: We found that carotid siphon calcification was associated with BAD and its computed tomography values could predict the symptomatic progression in patients with intracranial arterial atherosclerotic disease and BAD, indicating the important role of carotid calcification in B-type intracranial arterial atherosclerotic disease. Registration: URL: http://www.chictr.org.cn ; Unique identifier: ChiCTR1800018315

The aim of the study was to assess the clinical utility of a third-generation sequencing (TGS) approach termed comprehensive analysis of thalassemia alleles (CATSA) for identifying both α and β thalassemia genetic carrier status. Prospective blood samples (n = 1759) with abnormal hemoglobin parameters were screened for pathogenic thalassemia variants by CATSA on the PacBio TGS platform. In 1159 individuals, a total of 1317 pathogenic thalassemia variants were identified and confirmed by independent PCR-based tests. Of the total thalassemia variants detected, the α-variant --SEA (35.4%) and β-variant c.126_129delCTTT (15%) were the most common. CATSA was also able to detect three types of rare HBA structural variants as well as five rare HBA2, three HBA1, and 10 HBB single-nucleotide variations/insertions and deletions. Compared with standard thalassemia variant PCR panel testing, CATSA identified all panel variants present, with no false-negative results. Carrier assignment was improved through identification of rare variants missed by the panel test. On the basis of allelic coverage, reliability, and accuracy, TGS with long-range PCR presents a comprehensive approach with the potential to provide a universal solution for thalassemia genetic carrier screening. It is proposed that CATSA has immediate clinical utility as an effective carrier screening approach for at-risk couples.

Background Oxidized low-density lipoprotein (ox-LDL) is closely related to the recrudescence and prognosis of acute ischemic stroke (AIS). However, there are limited studies about the effect of a long-term elevation of ox-LDL in AIS. In this research, our porpose was to explore the relevence between cumulative ox-LDL exposure and the short-term prognosis of AIS. Methods This study recruited patients with AIS. AIS severity was evaluated by infarct volumes and National Institute of Health Stroke Scale (NIHSS) scores at admission. AIS prognosis was assessed by Modified Rankin Scale (mRS) scores at 90 days and the difference between NIHSS scores at admission and discharge. Cumulative ox-LDL exposure was defined as ox-LDL level (pg/mL) multiplied by age (y). A multivariate logistic regression model was employed to investigate the correlation between exposure factors and the prognosis of AIS. The predictive ability of cumulative ox-LDL exposure for the prognosis of AIS was compared with cumulative LDL exposure by the receiver operating characteristic curve (ROC). Results A total of 175 AIS patients were included with an average age of 66.00 (61.00, 73.00). Patients with higher cumulative ox-LDL exposure tended to manifest worse prognosis, including neurological worsening at discharge (NIHSS increasing more than 2 points) (OR = 3.02, 95% CI, 1.30–6.98, P = 0.01) and poor functional prognosis at 90 days (mRS ≥ 3) (OR = 21.21, 95% CI, 4.72–95.36, P

Purpose This study aims to identify genetic causes of female infertility associated with recurrent failure of assisted reproductive technology (ART) characterized by embryonic developmental arrest. Methods We recruited infertile patients from two consanguineous families from the Reproductive Medicine Center of Guizhou Provincial People’s Hospital. Peripheral blood was collected for genomic DNA extraction. Two affected individuals and their family members were performed with whole-exome sequencing and Sanger validation in order to identify possible causative genes. For further analyzing the effect of splicing mutation on mRNA integrity in vivo, TLE6 cDNA from the peripheral blood lymphocyte of the affected individual was sequenced. In addition, the possible impact of the pathogenic mutation on the structure and function of the protein were also assessed. Results Two novel homozygous mutations in the peptidylarginine deiminase type VI (PADI6) and the transducin-like enhancer of split 6 (TLE6) genes were identified in the two families. One patient carried the frameshift deletion mutation c.831_832del:p.S278Pfs*59 of the PADI6 gene and the other patient carried the splicing mutation c.1245-2 A>G of the TLE6 gene. The analysis of the mRNA from the proband’s peripheral blood leukocytes confirmed aberrant splicing. Conclusions Our findings expand the mutational spectrum of PADI6 and TLE6 associated with embryonic developmental arrest and deepen our understanding of the genetic causes of infertility with recurrent ART failure.

Intellectual disability (ID) represents a neurodevelopmental disorder, which is characterized by marked defects in the intellectual function and adaptive behavior, with an onset during the developmental period. ID is mainly caused by genetic factors, and it is extremely genetically heterogeneous. This study aims to identify the genetic cause of ID using trio-WES analysis.We recruited four pediatric patients with unexplained ID from non-consanguineous families, who presented at the Department of Pediatrics, Guizhou Provincial People's Hospital. Whole-exome sequencing (WES) and Sanger sequencing validation were performed in the patients and their unaffected parents. Furthermore, conservative analysis and protein structural and functional prediction were performed on the identified pathogenic variants.We identified five novel de novo mutations from four known ID-causing genes in the four included patients, namely COL4A1 (c.2786TA, p.V929D and c.2797GA, p.G933S), TBR1 (c.1639_1640insCCCGCAGTCC, p.Y553Sfs*124), CHD7 (c.7013AT, p.Q2338L), and TUBA1A (c.1350del, p.E450Dfs*34). These mutations were all predicted to be deleterious and were located at highly conserved domains that might affect the structure and function of these proteins.Our findings contribute to expanding the mutational spectrum of ID-related genes and help to deepen the understanding of the genetic causes and heterogeneity of ID.

Background. The use of chromosomal microarray analysis (CMA) in prenatal diagnosis of chromosomal and genetic diseases has resulted in a significant improvement in the diagnosis of genetically caused congenital malformations, neurodevelopmental disorders, and congenital anomalies, with a high diagnostic yield in selected prenatal cases. Objective. The objective of this study was to evaluate the application of CMA in the prenatal diagnosis of high-risk pregnant women. Method. A total of 576 pregnancies were selected from May 2018 to October 2020 in our hospital, including amniotic fluid chromosome, karyotype analysis, and CMA detection. The study group was divided into two groups based on the indications for testing: group A has 88 patients at the age of 35 years or older, and group B patients were in high-risk pregnancies, which consisted of 33 cases of bad pregnancy history, 252 high-risk serological screenings, 70 high-risk non-invasive prenatal testing (NIPT), 65 cases of B-ultrasound indicated fetal development abnormalities or ultrasonic soft marker abnormalities, and 68 other cases of pregnant women or both who have genetic or chromosomal abnormalities. At last, we have an analysis of the detection rate from different testing methods. Results. Based on the follow-up test, 576 high-risk pregnant women showed an amniotic fluid chromosome karyotype rate of 18.1% (104/576), and the remaining 472 of these cases suffered a CNV ratio of 14.2% (67/472). 472 women of low clinical relevance are at 4.87% (23/472), 16 people showed a clear cause ratio = 3.39% (16/472), and 28 of the 472 (5.93%) cases showed polymorphism. Conclusions. In our study, CMA significantly improved the fetal detection rate and diagnosis rate in high-risk pregnant women, which proved to be a very useful method in the diagnosis of genetically caused neurodevelopmental disorders and congenital anomalies. The use of CMA in high-risk pregnant women is justified, and these women can detect an additional (3.40%, 16/472) of pathogenic microdeletions and microduplications in the cases.

Beta-hemoglobinopathies are caused by mutations in the β-globin gene. One strategy to cure this disease relies on re-activating the γ-globin expression. BCL11A is an important transcription factor that suppresses the γ-globin expression, which makes it one of the most promising therapeutic targets in β-hemoglobinopathies. Here, we performed single-gene editing and multiplex gene editing via CRISPR/Cas9 technology to edit BCL11A erythroid-specific enhancer and BCL11A binding site on γ-globin gene promoter in HUDEP-2 cells and adult human CD34

Erythropoiesis is a complex and sophisticated multi-stage process regulated by a variety of factors, including the transcription factor GATA1 and non-coding RNA. GATA1 is regarded as an essential transcriptional regulator promoting transcription of erythroid-specific genes—such as long non-coding RNAs (lncRNA). Here, we comprehensively screened lncRNAs that were potentially regulated by GATA1 in erythroid cells. We identified a novel lncRNA—PCED1B-AS1—and verified its role in promoting erythroid differentiation of K562 erythroid cells. We also predicted a model in which PCED1B-AS1 participates in erythroid differentiation via dynamic chromatin remodeling involving GATA1. The relationship between lncRNA and chromatin in the process of erythroid differentiation remains to be revealed, and in our study we have carried out preliminary explorations.

Background: β-thalassemia and sickle cell disease (SCD) are chronic hemolytic anemias caused by mutation or deletion of the β-globin gene. Reactivation of fetal γ-globin gene (HBG) expression can ameliorate the clinical course of β-hemoglobinopathies. Our current understanding of the globin switching mechanism is not comprehensive, and research into the roles of long non-coding RNAs (lncRNAs) in this process remains limited. Results: We collected umbilical cord blood samples from preterm and full-term infants, from which we enriched and isolated nucleated red blood cells (NRBCs) for transcriptome sequencing. We identified mRNAs, lncRNAs, and microRNAs that were differentially expressed in the preterm and full-term samples, and constructed hub gene network of differentially expressed transcription factors. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis revealed the biological significance of differentially expressed mRNAs and the targets of differentially expressed lncRNAs and microRNAs. Our analyses revealed that the Fanconi anemia pathway may be involved in the regulation of globin switching at birth. Using quantitative polymerase chain reaction, we confirm that BCL11A is more highly expressed in full-term samples than in preterm samples and is statistically differentially expressed based on biological sex. Results of RNA sequencing and quantitative polymerase chain reaction demonstrated that the lncRNA H19 was expressed at a statistically lower level in full-term samples than in preterm samples. Furthermore, we established the H19‑related lncRNA/miRNA/mRNA network to reveal the potential regulatory mechanisms of H19.Conclusions: Our results suggest that the Fanconi anemia pathway and the H19/let-7/LIN28B axis may be involved in globin switching in umbilical cord blood. The differentially expressed transcripts and related pathways may constitute novel therapeutic targets for β-thalassemia.

Background: Increasing studies suggest that tumor immune infiltration is a relative factor of prognosis in ovarian cancer (OvCa). The present study explored the composition of tumor-infiltrating immune cells (TIICs) in OvCa using CIBERSORT algorithm and further assessed their values for prognosis and therapeutic strategies by molecular subtypes. Methods: Publicly available databases including The Cancer Genome Atlas (TCGA) and GTEx were searched. Ovarian tumor samples were available from TCGA, and normal ovarian samples were obtained from the GTEx dataset. The relative proportions of immune cell profiling in OvCa and normal samples were evaluated by CIBERSORT algorithm. Association between each immune cell subtype and survival was inferred by the fractions of 22 immune cell types. “CancerSubtypes” R-package was employed to identify the three types of molecular classification and analyze the functional enrichment in each subclass. Response to immunotherapy and anticancer drug targets was predicted via TIDE algorithm and GDSC dataset. Results: Substantial variation reflecting individual difference was identified between cancer and normal tissues in the immune infiltration profiles. T cells CD4 memory activated, macrophages M1 were associated with improved overall survival (OS) as evaluated by univariate Cox regression and multivariate Cox. Three subtypes were identified by ´CancerSubtypes’ R-package and every sub-cluster possessed specific immune cell characterization. Meanwhile, Cluster II exhibited poor prognosis and sensitive response to immunotherapy. Conclusions: The cellular component of immune infiltration shows remarkable variation in OvCa. Profiling of immune infiltration is useful in prediction of prognosis of OvCa. The results from profiling might be considered in therapeutic modulation.

Alterations in messenger RNAs (mRNAs) of protein-coding genes can influence the malignant behaviors of acute lymphoblastic leukemia (ALL) cells. According to the prediction from The Cancer Genome Atlas (TCGA) database, we discovered that glutathione peroxidase 1 (GPX1) was up-regulated in acute myeloid leukemia (LAML) tissues, which pushed us to explore the feasible role and its related modulatory mechanism of GPX1 in ALL. In this research, we first proved the high expression of GPX1 in ALL cells compared with normal cells. Functional assays further revealed that the proliferation was obstructed and the apoptosis was facilitated in ALL cells with silenced GPX1. Then, both miR-491-5p and miR-214-3p that were down-regulated in ALL cells were affirmed to target GPX1. Subsequently, VPS9D1 antisense RNA 1 (VPS9D1-AS1) was recognized as the upstream regulator of miR-491-5p-miR-214-3p/GPX1 axis in a competing endogenous RNA (ceRNA) model. Importantly, we proved that VPS9D1-AS1 served as a tumor promoter in ALL through elevating GPX1. In conclusion, VPS9D1-AS1 contributed to ALL cell proliferation through miR-491-5p-miR-214-3p/GPX1 axis, hinting an optional choice for the treatment of ALL.

Background: β-thalassemia and sickle cell disease (SCD) are chronic hemolytic anemias caused by mutation or deletion of the β-globin gene. Reactivation of fetal γ-globin gene (HBG) expression can ameliorate the clinical course of β-hemoglobinopathies. Our current understanding of the globin switching mechanism is not comprehensive, and research into the roles of long non-coding RNAs (lncRNAs) in this process remains limited. Results: We collected umbilical cord blood samples from preterm and full-term infants, from which we enriched and isolated nucleated red blood cells (NRBCs) for transcriptome sequencing. We identified mRNAs, lncRNAs, and microRNAs that were differentially expressed in the preterm and full-term samples. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis revealed the biological significance of differentially expressed mRNAs and the targets of differentially expressed lncRNAs and microRNAs. Our analyses revealed that the Fanconi anemia pathway may be involved in the regulation of globin switching at birth. Using quantitative polymerase chain reaction, we confirm that BCL11A is more highly expressed in full-term samples than in preterm samples and is statistically differentially expressed based on biological sex. Results of RNA sequencing and quantitative polymerase chain reaction demonstrated that the lncRNA H19 was expressed at a statistically lower level in full-term samples than in preterm samples.Conclusions: Our results suggest that the Fanconi anemia pathway and the H19/let-7/LIN28B axis may be involved in globin switching in umbilical cord blood. The differentially expressed transcripts and their related pathways may constitute novel therapeutic targets for β-thalassemia.

To detect mutation of LBR gene in a pedigree affected with Pelger-Huёt anomaly (PHA) and to explore its clinical characteristics.Genomic DNA was extracted from the pedigree and healthy controls. The 14 exons of the LBR gene were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified in other family members and 100 healthy controls. Polyphen-2 and SIFT software were used to predict the effect of the mutation, and Swiss-model software was used to simulate the protein structure.Three patients were found to carry a c.893GA mutation in exon 8 of the LBR gene, which resulted in substitution of the 298th amino acid residue glycine by glutamic acid (p.Gly298Glu). The same mutation was not found in healthy family members and 100 healthy controls. The mutation was predicted to be damaging. Bioinformatic simulation showed the mutation has altered the 3D structure of the LBR protein.The c.893GA (p.Gly298Glu) mutation in the LBR gene probably underlies the PHA in this pedigree and has enriched the spectrum of LBR gene mutations.

Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) β-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.

MicroRNAs (miRNAs) are present in plasma and have emerged as critical regulators of gene expression at posttranscriptional level, and thus are involved in various human diseases, including diabetes. The objective of this study was to screen and validate differentially expressed plasma miRNAs in prediabetes and newly diagnosed type 2 diabetes (T2D). In this study, we screened differentially expressed plasma miRNAs in prediabetes and newly diagnosed T2D by miRNA microarray analysis, and validated the expression of candidate miRNAs using quantitative reverse transcription polymerase chain reaction assays. Furthermore, we performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses to disclose functional enrichment of genes predicted to be regulated by the differentially expressed miRNAs. Notably, our results revealed that hsa-miR-1249, hsa-miR-320b, and hsa-miR-572 (P

Two 21-year old dizygotic twin men of Iraqi descent were homozygous for HBB codon 8, deletion of two nucleotides (-AA) frame-shift β(0) -thalassaemia mutation (FSC8; HBB:c25_26delAA). Both were clinically well, had splenomegaly, and were never transfused. They had mild microcytic anaemia (Hb 120-130 g/l) and 98% of their haemoglobin was fetal haemoglobin (HbF). Both were carriers of Hph α-thalassaemia mutation. On the three major HbF quantitative trait loci (QTL), the twins were homozygous for G>A HBG2 Xmn1 site at single nucleotide polymorphism (SNP) rs7482144, homozygous for 3-bp deletion HBS1L-MYB intergenic polymorphism (HMIP) at rs66650371, and heterozygous for the A>C BCL11A intron 2 polymorphism at rs766432. These findings were compared with those found in 22 other FSC8 homozygote patients: four presented with thalassaemia intermedia phenotype, and 18 were transfusion dependent. The inheritance of homozygosity for HMIP 3-bp deletion at rs66650371 and heterozygosity for Hph α-thalassaemia mutation was found in the twins and not found in any of the other 22 patients. Further studies are needed to uncover likely additional genetic variants that could contribute to the exceptionally high HbF levels and mild phenotype in these twins.

To report on the result of thalassemia screening and genetic diagnosis for pregnant women from Guiyang region.Prenatal screening for thalassemia was carried out based on erythrocyte parameters and hemoglobin electrophoresis. Single-tube multiplex GAP-PCR and PCR-reverse dot blot hybridization were performed on suspected cases to identify common alpha- and beta- thalassemia mutations, and direct sequencing was used for identifying rare mutations.Among 13 738 pregnant women, 1745 (12.70%) were suspected as thalassemia. In terms of native place, the provinces with highest screening-positive rates were Guangxi, Guangdong, Jiangxi and Guizhou. And the ethnic groups with highest screening-positive rates were Zhuang, Li, and Buyi. Among 801 women subjected to genetic testing, 457 (57.05%) were diagnosed with thalassemia. In total 9 genotypes of alpha- thalassemia were detected, with the most common genotypes being --The screening-positive rate of thalassemia among pregnant women in Guiyang region is relatively high. The rates have shown substantial difference in terms of native place and ethnic group. Thalassemia-related mutations in Guizhou region have a diverse spectrum, which showed certain difference from those of other regions.

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

To identify potential mutation of TRAPPC2 gene in a Chinese family affected with X-linked spondyloepiphyseal dysplasia tarda (X-SEDL), and explore its underlying molecular mechanism.Peripheral blood samples were collected from 32 members of the family and 50 healthy adults to extract genomic DNA. DNA sequences of exons 3 to 6 and their exon/intron boundaries were amplified with PCR amplification. Direct bi-directional sequencing analysis was performed on the PCR products. The sequences were aligned to the reference sequences from the GenBank to determine mutation site and type.A nucleotide substitution of the splice-donor in TRAPPC2 intron 3, c.93+5GA, was detected in the proband, but no sequence change was detected in TRAPPC2 exons 3 to 6. All of the 6 male patients and 8 female carriers from the family were detected to have carried this mutation. The same mutation was not found in the remaining 18 family members with a normal phenotype and 50 healthy controls.We have detected a c.93+5GA mutation in the TRAPPC2 gene in a Chinese family affected with X-SEDL. Our results have expanded the spectrum of TRAPPC2 mutations and is helpful for presymptomatic and prenatal diagnoses of this disease.

To investigate the spectrum of β -thalassemia mutations in Guizhou Province.For 542 individuals suspected to have β -thalassemia by decreased mean corpuscular volume (MCV) and corpuscle hemoglobin (MCH) by routine blood test and hemoglobin electrophoresis, reverse dot blot hybridization (RDB) was performed to detect 17 known β -thalassemia mutations, including 8 common and 9 rare mutations. For cases where no mutation was identified, the entire human β -globin gene was screened to find other rare mutations. The distribution and frequencies of detected β -thalassemia mutations were then analyzed.A total of 460 individuals were diagnosed as β -thalassemia by DNA analysis, which included 352 heterozygotes, 67 compound heterozygotes and 41 mutant homozygotes. A total of 12 β -thalassemia mutations were detected in these individuals. The mutations have ranked from high to low frequency as: CD17 (40.74%), CD41-42 (33.69%), IVS-II-654 (13.76%), -28 (3.70%), β E (3.35%), CD71-72(1.94%), CD43 (1.06%), IVS-I-1 (0.71%), CD27-28 (0.35%), -29(0.35%), CAP (0.18%), and CD121 (0.18%). The former six mutations have accounted for 97.18% of all. CD121 (GAATAA) detected from a heterozygote, as a dominant mutation, has been firstly found in the Chinese population.The spectrum of β -thalassemia in Guizhou Province showed certain distinct characteristics, with CD17 being the most common mutation. The newly discovered mutation of CD121 has expanded the spectrum of β -thalassemia in Chinese population. Our result may provide valuable information for the prevention and control of β -thalassemia in Guizhou.

In the Arab-Indian (AI) β-globin gene (HBB) haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are higher and the disease phenotype milder than in African HBB haplotypes. To study the genetic basis of HbF regulation in the AI haplotype, whole genome sequencing (WGS) was done in 14 unrelated Saudi AI haplotype HbS homozygotes; 7 selected for low HbF (7.9±1.9%) and 7 for high HbF (18.5±3.2%). WGS was also done in 3 Indians with the AI haplotype (HbF 26.0±4.5%), 3 African Americans with the Benin haplotype selected because of the uncommonly high HbF for this haplotype (19.8±0.4%) and 1 African American homozygote with the Senegal haplotype (HbF 16.0%). Association of SNPs with HbF was tested using simple linear regression with an additive genetic model, and the Sequence Kernel Association Test using sliding windows of 5 SNPs. We found 465 SNPs that were significantly associated with HbF in a single SNP analysis (p≤10-5). Following annotation using SNPper and ENCODE RegulomeDB, PLINK was employed to remove all SNPs in high linkage disequilibrium (r2>0.8) ending with 128 SNPs for follow-up analysis. These prioritized variants were further investigated using functional evaluation tools such as SCAN, Hembase, ErythronDB and the results of WGS were followed by genome-wide SNP analysis, imputation of genotypes to 1000genomes, and targeted direct genotyping in the following cohorts with sickle cell anemia: 110 AI haplotype cases (HbF 18.0±7.0%); 71 Saudi Benin haplotype cases (HbF 11.4±4.1%); 44 Indian AI haplotype homozygotes (HbF 23.0±4.8%); 894 African Americans with diverse HBB haplotypes (HbF 5.2±5.6%). All cases were examined when the HbF levels had stabilized. None of the patients were using hydroxyurea when HbF was measured. Based on WGS, 2 SNPs, rs4527238 and rs35685045 (D'=1) in intron 9 of ANTXR1 (anthrax toxin receptor 1, 2p13.1) remained associated with HbF after correction for multiple comparisons. This result was replicated in the cohort of 110 additional AI haplotype Saudi patients but not in cohorts with other HBB haplotypes. The alternative alleles of these 2 SNPs had nearly equal frequencies in Saudis with the Saudi AI haplotype and lower frequencies in Indians, Saudi Benin haplotype and African Americans with sickle cell anemia. The T allele of rs4527238 and the C allele of rs35685045 are associated with high HbF. ANTXR1 SNPs explained 10% of the HbF variability compared with 8% for BCL11A; these genes had independent, additive effects on HbF and explained about 15% of HbF variability. RNA sequencing and gene expression microarray analysis at 3 time points during the erythroid differentiation of CD34+ and iPSCs isolated from the same subjects showed that only the long splice variant of ANTXR1 was expressed. Expression increased over time in CD34+ erythroid cells and decreased over time in iPSC-derived erythroid progenitors in a pattern similar to BCL11A expression, with increasing expression as CD34+ cells matured and HbF expression fell and declining expression as iPSCs differentiated and HbF expression increased. Immunofluorescence co-staining for ANTXR1 and HbF during the erythroid differentiation of sickle iPSCs showed that reduced expression of ANTXR1 was paralleled by increased expression of HbF. By GTEx analysis, eQTLs at rs4527238 and rs35685045 showed a trend for increased expression according to genotype with rs4527238 T/T having the lowest level of expression and T/C and C/C genotypes, higher expression levels (p= 0.08) and for rs35685045, C/C the lowest expression levels and C/T and T/T higher expression levels (p= 0.07); rs4527238 and rs35685045 had no effect on the expression of BCL11A, a repressor of HbF expression located at 2p16 (p=0.8). STRING 9.1 predicted protein interactions of ANTXR1 directly with LRP6, HDAC2, BCLX and BRAC1 with the highest level of confidence (0.95) with LRP6. The HbF phenotype in AI haplotype sickle cell anemia is unique and ANTXR1 is the first trans-acting element associated with HbF whose effects appear to be limited to the Saudi AI haplotype. ANTXR1 might have dual effects on HbF, indirectly affecting hematopoiesis via interactions with LRP6 and Wnt signaling, and directly modulating HBG expression through its effects on HDAC2. A dual effect on HbF expression has been suggested for MYB. Whether targeting ANTXR1 is a therapeutic option will require further studies. Disclosures No relevant conflicts of interest to declare.

To investigate the association between the apolipoprotein E (apoE) gene polymorphism and the dose for warfarin individual maintenance.The genotypes of 249 patients with warfarin treatment in maintenance doses were determined by PCR/DHPLC assay. The doses for warfarin maintenance were compared among patients with different genotypes.In the total of 249 patients, the frequencies of 2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, ε4/ε4 genotype were 1.20%, 15.66%, 1.80%, 72.29%, 9.24%, 0.80%, respectively; the allele frequencies of ε2, ε3, ε4 were 9.44%, 84.74%, 5.82%, respectively. The warfarin dose of group ε2 (ε2/ε2, ε2/ε3) was (3.24 ± 1.36) mg/d, slightly higher than that of group ε3 (ε3/ε3, 2.91 ± 1.14 mg/d) or group ε4 [ε4/ε4, ε3/ε4, (2.98 ± 1.05) mg/d], but the difference of the warfarin doses among the 3 groups did not reach statistical significance (F=1.848,P0.05).ApoE polymorphism may be not a major genetic factor that influences the individual dose for warfarin maintenance.

Introduction It has been widely accepted that genetic factors were the major sources of the variation in warfarin dose. This study is intended to investigate whether the 3261G>A variation in GGCX gene influences stable warfarin dose in Chinese patient population. Materials and Methods A total of 217 patients with stable warfarin dose were enrolled. Genomic DNA was extracted from each subject and the genotype of GGCX 3261G>A was determined by using of denaturing high-performance liquid chromatography (DHPLC). Least significant difference tests (LSDs) were used to compare dose with genotypes. Analysis of variance (ANVOA) was used to calculate the proportion of warfarin dose that could be explained by variation in genotype. Results In the total of 217 subjects, 84 patients (38.7%) were GG homozygote, whereas 117 (53.9%) were GA heterozygote and 16 (7.4%) were AA homozygote. Patients with the GGCX 3261AA genotype had a significantly higher average daily maintenance dose (3.39 ± 1.40 mg) than those with the GG genotype (2.69 ± 1.07 mg; P = 0.027), and GGCX 3261G>A explains 2.3% of the univariate warfarin dose variance. Conclusion GGCX 3261G>A may affect warfarin dose requirements, and showed a small but significant effect on warfarin dose in a Chinese patient population.

To develop a warfarin-dosing algorithm that could be combined with pharmacogenomic and demographic factors, and to evaluate its effectiveness in a randomized prospective controlled clinical trial.A pharmacogenetics-based dosing model was derived using retrospective data from 266 Chinese patients and multiple linear regression analysis. To prospectively validate this model, 156 patients with an operation of heart valve replacement were enrolled and randomly assigned to the group of pharmacogenetics-guided or traditional dosing for warfarin therapy. All patients were followed up for 50 days after initiation of warfarin therapy. The log-rank test was compared with the time-to-event (Kaplan-Meier) curves. Cox proportional hazards-regression model was used to assess the hazard ratio of the time to reach stable dose.The linear regression model derived from the pharmacogenomic model correlated with 54.1% of warfarin dosing variance. The final multiple linear regression model included age, body surface area, VKORC1, and CYP2C9 genotype. The study showed that the hazard ratio for the time to reach stable dose was 1.932 for the traditional dosing group versus the model-based group and a close and highly significant relationship was observed to exist between the predicted and the actual warfarin dose (R=0.454).A pharmacogenetics-based dosing algorithm has been developed for improvement in the time to reach the stable dosing of warfarin. This model may be useful in helping the clinicians to prescribe warfarin with greater safety and efficiency.

In β-thalassemia major, an inherited disorder caused by homozygosity or compound heterozygosity for β-thalassemia mutations, most patients are severely anemic and require chronic RBC transfusions. Elevated levels of HbF can ameliorate the disease severity and 3 major HbF quantitative trait loci (QTL) on chr 11p, 6q (the HBS1L-MYB intergenic polymorphism) and 2p (BCL11A) are associated with HbF levels in all populations studied. We studied Iraqi-American non-identical twins with markedly elevated HbF who are homozygous for codon 8 (–AA) β0-thalassemia mutation, but are clinically well and transfusion independent. MLPA analysis of the β-globin gene cluster did not show any hereditary persistence of fetal hemoglobin deletions or γ-globin gene quadruplication. Sequencing HBG2 and HBG1 promoters excluded other γ-globin gene promoter non-deletional HPFH mutations. We characterized 12 Turkish and Iraqi patients who are homozygous for codon 8 (–AA) frame-shift β-thalassemia mutation. Among this group, three patients from two Turkish families have a mild disease phenotype, six patients from four Turkish families have a severe phenotype, and three Iraqi patients from three families have a severe phenotype. To assess the contribution of 3 major HbF QTLs to disease severity, we genotyped polymorphisms in these QTL in all 14 patients (Table). Homozygosity for rs7482144 minor alleles (the -158 5' HBG2 Xmn1 C-T SNP) in the HBB gene cluster is present in 13 of 14 patients with the exception being one patient with a severe phenotype. No distinct pattern for the BCL11A sentinel SNP rs766432 minor allele is found between mild and severely affected patients. Homozygosity for rs9399137 minor alleles marking the HBS1L-MYB intergenic region is present in two patients with a mild phenotype, while heterozygosity for minor alleles and homozygosity for major alleles are present in both mild and severe patients. Although the Iraqi-American twins first studied have the most minor alleles (5 of 6 possible minor alleles), the other three patients with mild phenotype have 4, 3 and 2 minor alleles, respectively. Strikingly, one patient with mild phenotype is only homozygous for the rs7482144 minor alleles. Patients with severe phenotypes have 2 to 4 QTL minor alleles. Neither individual QTL genotypes nor the number of QTL minor alleles can distinguish the mild phenotype and severe phenotype in patients with β-thalassemia major of Iraqi and Turkish origin suggesting that other factors contribute to the mild phenotype and high HbF levels in these patients. The entire 14.7 kb intergenic region of between HBG1 and HBD of one twin was sequenced and revealed 19 known SNPs and 4 novel SNPs. The potential roles of these SNPs in regulating γ-globin gene expression are under investigation. We are also investigating whether there are any other potential cis- or trans-acting factors that contribute to mild disease phenotype in these patients using genome-wide SNP arrays, whole genome sequencing and whole exome sequencing. By studying these unusual cases we hope to provide new understanding and insights for elevated HbF production in β-hemoglobinopathies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.