SUPT5H mutations associated with elevation of Hb A2 level: Identification of two novel variants and literature review.

ß-thalassemia is one of the most common monogenic disorders in areas of the tropics and subtropics, which represents a major familial and social burden to local people. The elevated Hb A2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of ß-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A2 level. In this study, we identified two novel variants in the SUPT5H gene: a frameshift mutation (SUPT5H: c.3032_3033delTG, p.M1011Mfs*9) and a nonsense mutation (SUPT5H: c.397C > T, p.Arg133*) in two Chinese individuals. Utilizing a combination of phenotype analysis, bioinformatics analysis, and functional analysis, we deduced that these two variants modified the SUPT5H protein's structure, thereby impacting its function and consequently leading to the heightened Hb A2 level phenotype found in the carriers. Furthermore, through a comprehensive literature review, a mutation spectrum was consolidated for SUPT5H, an investigation into the genotype-phenotype correlation was conducted, and factors known to influence Hb A2 levels were identified. Based on this in-depth understanding, clinicians are better equipped to carry out large scale screenings in regions with high prevalence of ß-thalassemia.

A novel gain-of-function PIP4K2A mutation elevates the expression of ß-globin and aggravates the severity of α-thalassemia.

Haemoglobin H (Hb H) disease (intermediate status of α-thalassemia) shows marked phenotypic variability from asymptomatic to severe anaemia. Apart from the combined ß-thalassemia allele ameliorating clinical severity, reports of genetic modifier genes affecting the phenotype of Hb H disease are scarce which bring inconvenience to precise diagnosis and genetic counselling of the patients. Here, we present a novel mutation (c.948C>A, p.S316R) in the PIP4K2A gene in a female Hb H disease patient who displayed moderate anaemia and a relatively high Hb H level. Haematological analysis in her family members revealed that individuals carrying this mutation have upregulated ß-globin expression, leading to a more imbalanced ß/α-globin ratio and more Hb H inclusion bodies in peripheral red blood cells. According to functional experiments, the mutant PIP4K2A protein exhibits enhanced protein stability, increased kinase activity and a stronger regulatory effect on downstream proteins, suggesting a gain-of-function mutation. Moreover, introduction of the S316R mutation into HUDEP-2 cells increased expression of ß-globin, further inhibiting erythroid differentiation and terminal enucleation. Thus, the S316R mutation is a novel genetic factor associated with ß-globin expression, and the PIP4K2A gene is a new potential modifier gene affecting the α-thalassemia phenotype.

A novel splicing mutation of ANK1 is associated with phenotypic heterogeneity of hereditary spherocytosis in a Chinese family.

Hereditary spherocytosis (HS) is a common hematological genetic disorder that results in anemia, jaundice and splenomegaly. It is caused by mutations in the ANK1, SPTA, SPTB, SLC4A1 and EPB42 genes, which encode red blood cell membrane and skeletal proteins. Patients show high heterogeneity in phenotype and genotype and the genotype-phenotype correlation still requires clarification. Here, a novel splicing mutation (ANK1: c.4391-2 A>C) was identified by whole-exome sequencing (WES) and Sanger sequencing in a Chinese boy who exhibited a moderately severe HS phenotype. However, his father exhibited a mild phenotype, despite carrying the same HS-causing mutation. The function of the mutant ANK1 protein was analyzed by both bioinformatics and experimental analysis. The mutant protein (p.N1463Kfs*4) showed a different 3D-structure and altered subcellular localization, when compared with the wild-type ANK1 protein. These changes disrupted the normal cell membrane structure and resulted in spheroidized red blood cells. Amplification of cDNA from the son and his father revealed a difference in expression of the abnormal transcript produced by the splicing mutation. We proposed that the lower expression of the mutant allele may have contributed to the relatively mild symptoms of the father. Our study verified ANK1 c. c.4391-2 A>C as a novel pathogenic mutation that causes HS. We have also provided new insights into the interpretation of phenotypic variability within families, which could greatly improve the clinical diagnosis and genetic counseling of HS.

Investigation of the mechanism of copy number variations involving the α-globin gene cluster on chromosome 16: two case reports and literature review.

Thalassemia is one of the most common single-gene disorder worldwide. An important genetic cause of thalassemia is copy number variations (CNVs) in the α-globin gene cluster. However, there is no unified summary and discussion on the detailed information and mechanisms of these CNVs. In this study, two novel CNVs, a tandem duplication (αααα159) and deletion (--259), were identified in two Chinese families with thalassemia patients, according to the results of hematologic analysis, routine genetic testing for thalassemia, multiplex ligation-dependent probe amplification (MLPA), next-generation sequencing (NGS) and other molecular methods. Co-inherited with ßCD41-42 mutation and --SEA deletion separately, αααα159 and --259 resulted in a patient with ß-thalassemia intermedia and a lethal fetus with Hb Bart's hydrops fetalis syndrome, respectively. Next, a literature review was performed to summarize all known CNVs involving the α-globin gene cluster. The molecular structure characteristics of these CNVs were analyzed and the possible mechanism was explored. It is the first time to analyze the generation mechanism of genome arrangements in the α-globin gene cluster systematically.

Compound Heterozygosity for KLF1 Mutations Causing Hemolytic Anemia in Children: A Case Report and Literature Review.

BACKGROUND: Anemia is one of the most common diseases affecting children worldwide. Hereditary forms of anemia due to gene mutations are difficult to diagnose because they only rely on clinical manifestations. In regions with high prevalence of thalassemia such as southern China, pediatric patients with a hereditary hemolytic anemia (HHA) phenotype are often diagnosed with ß-thalassemia. However, HHA can be caused by other gene defects. Here, a case previously diagnosed with thalassemia in a local hospital was sent to our laboratory for further genetic diagnosis. Preliminary molecular testing did not identify any mutations in globin genes. METHODS: All blood samples were collected after informed consent had been obtain from the proband's parents. Both clinical and genetic analyses were conducted for the patient and her family members, including clinical data collection and sequencing of the KLF1 gene. Relevant literature was reviewed, including genetically confirmed cases with well-documented clinical summaries. RESULTS: Based on the detailed clinical data for this case, we diagnosed the patient with severe HHA. Sanger sequencing confirmed that there was a mutation on each KLF1 allele in the proband, which is missense mutation c.892G > C (p.Ala298Pro) inherited from father and frameshift mutation c.525_526insCGGCGCC (p.Gly176Argfs∗179) from the mother, respectively. A summary of the KLF1 mutation spectrum and a clarification of genotype-phenotype correlation were performed through a combined analysis of the case and literature studies. CONCLUSION: This study corrected the misdiagnosis and identified the etiology in a Chinese patient with HHA. Identification of the disease-causing gene is important for the treatment and care of the patient and prevention of another affected childbirth in her family. In addition, this study provided insight to better distinguish HHA patients with ß-thalassemia mutations from those with KLF1 mutations.

Pharmacokinetic Characteristics of Steamed Notoginseng by an Efficient LC-MS/MS Method for Simultaneously Quantifying Twenty-three Triterpenoids.

Steamed Panax notoginseng (SNG) has been widely used as a restorative medicine instead of the raw one, but its pharmacokinetic profile is entirely unknown. To address this, we've developed an LC-MS/MS method with high efficiency and sensitivity for simultaneous quantification of 23 triterpenoids (notoginsenosides Fa, Fc, R1, 20( S)-R2, 20( R)-R2, ginsenosides F4, Rb1, Rg1, Rd, Re, Rb2, 20( S)-Rh1, 20( R)-Rh1, Rh4, R k1, R k3, 20( S)-Rg2, 20( S)-Rg3, 20( R)-Rg3, Rg5, C-K, 20( S)-PPT, 20( S)-PPD) from SNG in rat plasma. This validated approach exhibits great linearity, precision, accuracy, recovery, and stability for all analytes. Furthermore, we, for the first time, applied this method to the pharmacokinetic study of SNG and proposed Rb1, Fa, Rd, R k1, Rg5, R k3, Rh4, and 20( S)-PPD to be suitable pharmacokinetic markers of SNG due to their high exposure levels of systemic plasma. Hence, this developed approach would be a powerful tool for future in vivo investigation of various sources of notoginseng-related samples.

Development and validation of a UFLC-MS/MS method for simultaneous quantification of sixty-six saponins and their six aglycones: Application to comparative analysis of red ginseng and white ginseng.

A new and sensitive ultra fast liquid chromatography coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method was developed to evaluate the quality of Red ginseng (RG) and to find out its chemical markers by comparing with multi-batches of RG and white ginseng (WG). This innovative method could quantify sixty-six saponins and their six aglycones including 10 pairs of 20(S) and 20(R) epimers within 35 min simultaneously. All compounds could be determined in individual multiple-reaction monitoring channel without interference, and the optimized method was rapid, accurate, precise, reproducible and efficient. Using the orthogonal partial least squared discriminant analysis, ginsenosides Rg5, Rh4, Rk1, Rs4, F4, and 20(S)-Rg3 were found to be the characteristic components of RG, the six compounds should be suggested as quality control markers to distinguish RG from WG. These findings will be significant for standardizing the processing procedures of RG and ensuring the consistent quality, as well as consequently the efficacy of RG in clinical applications. Results will be helpful in providing crucial chemical profiles of RG.

Characterization of circRNA-Associated-ceRNA Networks in a Senescence-Accelerated Mouse Prone 8 Brain.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Although many researchers have attempted to explain the origins of AD, developing an effective strategy in AD clinical therapy is difficult. Recent studies have revealed a potential link between AD and circRNA-associated-ceRNA networks. However, few genome-wide studies have identified the potential circRNA-associated-ceRNA pairs involved in AD. In this study, we systematically explored the circRNA-associated-ceRNA mechanism in a 7-month-old senescence-accelerated mouse prone 8 (SAMP8) model brain through deep RNA sequencing. We obtained 235 significantly dysregulated circRNA transcripts, 30 significantly dysregulated miRNAs, and 1,202 significantly dysregulated mRNAs. We then constructed the most comprehensive circRNA-associated-ceRNA networks in SAMP8 brain. GO analysis revealed that these networks were involved in regulating the development of AD from various angles, for instance, axon terminus (GO: 0043679) and synapse (GO: 0045202). Following rigorous selection, we discovered that the circRNA-associated-ceRNA networks in this AD mouse model were mainly involved in the regulation of Aß clearance (Hmgb2) and myelin function (Dio2). This research is the first to provide a systematic dissection of circRNA-associated-ceRNA profiling in SAMP8 mouse brain. The selected circRNA-associated-ceRNA networks can profoundly affect the diagnosis and therapy of AD in the future.

Analyses of mRNA Profiling through RNA Sequencing on a SAMP8 Mouse Model in Response to Ginsenoside Rg1 and Rb1 Treatment.

Ginsenoside Rg1 and Rb1 are the major ingredients in two medicines called QiShengLi (Z20027165) and QiShengJing (Z20027164) approved by China. These ingredients are believed to mitigate forgetfulness. Numerous studies have confirmed that GRg1 and GRb1 offer protection against Alzheimer's disease (AD), and our morris water maze (MWM) experiment also indicated that GRg1 and GRb1 may attenuate memory deficits in the 7-month-old SAMP8 mice; however, comprehensive understanding of their roles in AD remains limited. This study systematically explored the mechanism at the genome level of the anti-AD effects of GRg1 and GRb1 in a senescence-accelerated mouse prone 8 (SAMP8) model through deep RNA sequencing. A total of 74,885 mRNA transcripts were obtained. Expression analysis showed that 1,780 mRNA transcripts were differentially expressed in SAMP8 mice compared with the SAMP8+GRg1 mice. Moreover, 1,066 significantly dysregulated mRNA transcripts were identified between SAMP8 and SAMP8+GRb1 mice. Analyses according to gene ontology and the Kyoto Encyclopedia of Genes and Genomes revealed that oral administration of GRg1 and GRb1 improved the learning performance of the SAMP8 mouse model from various aspects, such as nervous system development and mitogen-activated protein kinase signaling pathway. The most probable AD-related transcriptional responses after medication were predicted and discussed in detail. This study is the first to provide a systematic dissection of mRNA profiling in SAMP8 mouse brain in response to GRg1 and GRb1 treatment. We explained their efficacy thoroughly from the source (gene-level explanation). The findings serve as a theoretical basis for the exploration of GRg1 and GRb1 as functional drugs with anti-AD activity.

Simultaneous quantification of twenty-one ginsenosides and their three aglycones in rat plasma by a developed UFLC-MS/MS assay: Application to a pharmacokinetic study of red ginseng.

To track the pharmacokinetic features of red ginseng (RG), a rapid and sensitive ultra fast liquid chromatographic coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method was developed for simultaneous quantification of twenty-one ginsenosides and their three aglycones, including 18 prototype compounds (ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Rg5, Rh4, Rk1, Rk3, 20(S)-Rf, 20(S)-Rg2, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh1, 20(R)-Rh1, 20(S)-NG-R2), and 6 metabolites (ginsenosides 20(S)-Rh2 and Rh3, 20(S)-protopanaxadiol (PPD), 20(S)-protopanaxatriol (PPT), 20(R)-PPT, ginseng saponin compound K) of RG in rat plasma after oral administration of RG water extract at a single dose of 4g/kg body weight to rats. All analytes with internal standard (digoxin) were detected by multiple reaction monitoring in negative ionization mode and separated on an ACQUITY UPLC® BEH RP-C18 column (1.7µm, 100×2.1mm). This established method was well validated in terms of linearity, sensitivity, intra- and inter-day precisions, accuracy, recovery, matrix effect, stability, and had a lower limit of quantification at the concentration range of 0.12-8.12ng/mL for all of analytes. This UFLC-MS/MS approach was successfully applied to the pharmacokinetic study for RG water extract in rats. We firstly proposed that Rb1, Rb2, Rc, Rd, Rg1, Rg5, 20(S)-Rg3, 20(S)-Rh2, and 20(S)-PPD measured in rat plasma were suitable pharmacokinetic markers of RG extract in rats due to their high systemic exposure levels. Thus, this specific and reliable method will be useful for future applications to pharmacokinetic studies for various sources of ginsenoside samples and Panax herbs in vivo.

(+)-Catechin ameliorates diabetic nephropathy by trapping methylglyoxal in type 2 diabetic mice.

SCOPE: Accumulation of glycolytic metabolite methylglyoxal (MG) in diabetic kidney is thought to contribute to the pathogenesis of nephropathy, either as a direct toxin or as a precursor for advanced glycation end products (AGEs). Using (+)-catechin (CE), a novel MG trapper, we investigated whether MG trapping is sufficient to prevent the progression of diabetic nephropathy in type 2 diabetic mice. METHODS AND RESULTS: CE markedly trapped exogenous MG in a time- and dose-dependent manner and formed mono-MG-CE and di-MG-CE adducts, which were characterized by HPLC-ESI-Q-TOFMS. In vivo, CE administration for 16 wk significantly ameliorated renal dysfunction in type 2 diabetic db/db mice, partially due to MG trapping, which in turn inhibited AGEs formation and lowered proinflammatory cytokines, including tumor necrosis factor α and IL-1ß. Similarly, the MG trapping and cellular signaling inhibition effects of CE were observed in human endothelium-derived cells under high glucose conditions. CONCLUSION: CE might ameliorate renal dysfunction in diabetic mice as consequences of inhibiting AGEs formation and cutting off inflammatory pathway via MG trapping. Thus, CE may be a potential natural product as an MG scavenger against diabetes-related complications.

Inhibition of advanced glycation end product formation by Pu-erh tea ameliorates progression of experimental diabetic nephropathy.

Accumulation of advanced glycation end products (AGEs) has been implicated in the development of diabetic nephropathy. We investigated the effects of Pu-erh tea on AGE accumulation associated with diabetic nephropathy. Although it did not affect blood glucose levels and insulin sensitivy, Pu-erh tea treatment for 8 weeks attenuated the increases in urinary albumin, serum creatinine, and mesangial matrix in db/db mice. We found that Pu-erh tea prevented diabetes-induced accumulation of AGEs and led to a decreased level of receptor for AGE expression in glomeruli. Both production and clearance of carbonyl compounds, the main precursor of AGE formation, were probably attenuated by Pu-erh tea in vivo independent of glyoxalase I expression. In vitro, HPLC assay demonstrated Pu-erh tea could trap methylglyoxal in a dose-dependent manner. Our study raises the possibility that inhibition of AGE formation by carbonyl trapping is a promising approach to prevent or arrest the progression of diabetic complications.