Molecular Diagnosis of Hereditary Spherocytosis By Multi-Gene Target Sequencing in Korea

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3347-3347
Author(s):  
Qute Choi ◽  
Jung Ah Kim ◽  
Kyong Ok Im ◽  
Si Nae Park ◽  
Yoomi Park ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Membrane Protein ◽  
Hemolytic Anemia ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Gene Target ◽  
Protein Encoding ◽  
Encoding Genes

Abstract Background: Hereditary spherocytosis (HS) is the most common cause of hereditary hemolytic anemia. Spherocytes formed by defective membrane proteins are selectively captured in the spleen and destroyed, leading to hemolytic anemia. Current tests used to diagnose HS focus on the detection of hemolysis or indirectly assess protein defects. Direct methods to detect protein defects are complicated and difficult to implement. Recent next-generation sequencing (NGS) methods enable large-scale gene mutation analyses to be used for such diagnoses. In this study, we investigated the patterns of genetic variation associated with HS to determine the molecular mechanisms underlying the condition. Specifically, we analyzed mutations in red blood cell membrane protein-encoding genes in Korean HS patients using NGS. Methods: In total, 60 patients with HS were enrolled in this study. Targeted sequencing of 43 genes (17 membrane protein-encoding genes, 20 enzyme-encoding genes, and 6 additional candidate genes) was performed using the Illumina HiSeq platform and variants were called according to a data-processing pipeline. Results: Of the 60 patients, 50 (83%) had one or more significant variants in a membrane protein gene. A total of 54 significant variants (8 previously reported and 46 novel) were detected in 6 membrane protein-encoding genes, i.e., SPTB, ANK1, SPTA1, SLC4A1, EPB41, and EPB42. The most variants (28) were detected in SPTB. Four significant variants, all of which were previously reported, were detected in genes encoding enzymes (ALDOB, G6PD, GAPDH, and GSR). Additionally, 5 previously reported variants were detected in UGT1A1. These results suggest 35 primer sets that can be used to diagnose HS. Conclusion: This was the first large-scaled genetic study of Korean HS patients. These results clarify the pattern of genetic variation associated with HS in Korean patients. They will enable easier, more rapid diagnosis of HS. Disclosures No relevant conflicts of interest to declare.

Comparison of Clinical Diagnosis for Hereditary Spherocytosis with Molecular Diagnosis By Multi-Gene Target Sequencing in Korea

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1243-1243
Author(s):  
Hyoung Soo Choi ◽  
Qute Choi ◽  
Jung Ah Kim ◽  
Kyong Ok Im ◽  
Si Nae Park ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Molecular Diagnosis ◽  
Large Scale ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Molecular Genetic Analysis ◽  
Illumina Hiseq ◽  
Gene Target ◽  
Protein Encoding ◽  
Encoding Genes

Abstract Background: Hereditary spherocytosis (HS) is the most common cause of hereditary hemolytic anemia. Current tests used to diagnose HS focus on the detection of hemolysis or indirectly assess protein defects. Direct methods to detect protein defects are complicated and difficult to implement. Recent next-generation sequencing (NGS) methods enable large-scale gene mutation analyses to be used for such diagnoses. In this study, we investigated the patterns of genetic variation associated with HS among the patients diagnosed with HS clinically. Specifically, we analyzed mutations in red blood cell membrane protein-encoding genes (17 genes) in context with 5 genes for the differential diagnosis (thalassemia, congenital dyserythropoietic anemia, paroxysmal nocturnal hemoglobinuria) in Korean HS. Methods: In total, 60 patients diagnosed with HS were enrolled in this study. Targeted sequencing of 43 genes (17 membrane protein-encoding genes, 20 enzyme-encoding genes, and 6 additional candidate genes) was performed using the Illumina HiSeq platform and variants were called according to a data-processing pipeline. Results: Of the 60 patients, 50 (83%) had one or more significant variants in a membrane protein encoding genes. A total of 54 significant variants (8 previously reported and 46 novel) were detected in 6 membrane protein-encoding genes; SPTB, ANK1, SPTA1, SLC4A1, EPB41, and EPB42. The most variants (28/60 patients) were detected in SPTB. Interestingly, concurrent mutations of genes encoding enzymes (ALDOB, GAPDH, and GSR) were detected along with mutations of membrane encoding genes. One patient diagnosed with HS harbored mutation of G6PD without mutation of HS related genes. Additionally, UGT1A1 mutations were present in 5 patients. Positive rate of osmotic fragility test was 86% among patients with HS related gene mutations. Conclusion: These results clarify the molecular genetic analysis is required for the accurate diagnosis of HS. About 17% of patients who were clinically diagnosed as HS revealed discrepancy with molecular diagnosis. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Mutation In Erythroid Specific Transcription Factor KLF1 Causes Hereditary Spherocytosis In the Nan (Neonatal Anemia) Hemolytic Anemia Mouse Model

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3217-3217
Author(s):  
Robert A White ◽  
Daniel P. Heruth ◽  
Troy Hawkins ◽  
Derek Logsdon ◽  
Margaret Gibson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hemolytic Anemia ◽  
Zinc Finger ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Hereditary Spherocytosis ◽  
Competitive Inhibition ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Zinc Finger Domain

Abstract Abstract 3217 The zinc finger protein Erythroid Krüuppel-like factor (EKLF, KLF1) regulates definitive erythropoiesis and terminal differentiation of red blood cells. KLF1 facilitates transcription through high affinity binding to CACCC elements within its erythroid-specific target genes which include genes encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins lead to the hemolytic anemia Hereditary Spherocytosis (HS). We have identified a new HS gene by studying the hemolytic anemia mouse mutant Nan (Neonatal Anemia). Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in Nan mice. The causative nature of the E339D mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1+/− knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild type KLF1. Competitive inhibition may help explain the apparent disconnect between the finding that Nan/+ heterozygous mice are anemic, whereas Klf1+/− heterozygous mice are normal and haplo-sufficient. This is the first direct association of a KLF1mutation with a disease in adult mammals. After examining a small population of HS patients, we also discovered one HS patient with a KLF1 mutation, which resulted in a significant amino acid substitution (T251I) in the activator/repressor domain, 28 amino acid residues upstream of the first zinc finger domain. This HS subject had no known mutations in the exons or intron/exon boundaries of EMS genes (SPTA1, SPTB, ANK1, SLC4A1) which comprise 85% of HS mutations in humans. The lack of a known genetic mutation in EMS genes leaves this patient's KLF1 mutation as the leading candidate defect. The identification of the gene causing the Nan mutation is significant because the Nan mutant has allowed discovery of a new HS gene which may also cause this disease in humans. In addition, the putative dominant/negative competitive inhibition of the Nan mutation makes the Nan mouse an excellent model system to study the function of KLF1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Genetic variation of the λA and λC protein encoding genes of avian reoviruses

2007 ◽  
Vol 83 (3) ◽  
pp. 394-402 ◽  
Author(s):  
Pin Chun Shen ◽  
Yu Fang Chiou ◽  
Hung Jen Liu ◽  
Chih Hong Song ◽  
Yu Pin Su ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Protein Encoding ◽  
Avian Reoviruses ◽  
Encoding Genes

scholarly journals Non-Immune Hemolytic Anemia of Pregnancy: Heterozygous RBC Variants Elude Diagnosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Julie Huang ◽  
Mina Gendy ◽  
Marta Wronska ◽  
Crystal Antoine-Pepeljugoski ◽  
Duc Vo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Hemolytic Anemia ◽  
Blood Cells ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Case Reports ◽  
Unknown Etiology ◽  
Molecular Testing ◽  
Immune Hemolytic Anemia

A 25 year old G3P2002 El Salvadorean female, with a prior history of pregnancy related anemia of unknown etiology, presented at 24 weeks gestation with symptomatic anemia (hemoglobin 2.9 g/dL) including dizziness, weakness and fatigue and no active signs of bleeding. Blood work included normal range results for LDH, haptoglobin, indirect and direct Coombs indicating no intravascular or immune driven hemolysis. Peripheral smear showed spherocytosis and stomatocytosis, concerning for an intrinsic RBC defect. Other workup included hemoglobin electrophoresis with slight increase in Hb A2 of 3.3%, flow cytometry negative for a lymphoproliferative disorder or paroxysmal nocturnal hemoglobinuria, no evidence of G6PD deficiency, and a bone marrow biopsy negative for marrow dysplasia, aplasia or HLH. Abdominal ultrasound revealed hypersplenism. The anemia was attributed to a non-immune hemolytic anemia with extra corpuscular RBC destruction in the spleen but without evidence of RBC destruction in the bone marrow or peripheral blood. After a prolonged 10-week hospitalization, the patient received a trial of steroids, 8 IV immunoglobulin infusions with minimal benefit, and total of 22 units of packed red blood cells. She underwent an elective induction and delivery at 34 weeks of pregnancy. During the postpartum period, she continued to have persistent anemia. A partial splenic embolization was attempted, complicated by splenic abscesses resulting in a splenectomy. Post splenectomy, the patient's hemoglobin and hematocrit stabilized to 11.7/37.8 at her three week outpatient visit. Molecular testing for Next Generation Sequencing (NGS) with Laboratory Hereditary Hemolytic Anemia Comprehensive Panel was also performed, revealing four different heterozygous variants. While these mutations individually have not been proven to cause hemolysis, the four alterations together, with the stressor of pregnancy, likely induced a non-immune hemolytic anemia. Non-immune hemolytic anemia is caused by intracorpuscular defects within the red blood cells or extracorpscular by environmental factors. The patient was found with four heterozygous variants in HK1, RPS19, SPTA1 and HBB, implicated in intracorpuscular defects. The HK1 gene, expressed in erythrocytes, encodes hexokinase, and provides red blood cells ATP. HK deficiency is a rare hereditary disorder associated with mild to severe non-spherocytic hemolytic anemia. The RPS19 gene encodes for a ribosomal protein involved in erythropoiesis. Clinically significant mutations in this gene cause Diamond Blackfan anemia. The SPTA1 gene encodes alpha spectrin subunits, which are a part of red cell membrane cytoskeleton and maintains its shape. Mutations in this gene have been implicated in hereditary spherocytosis. One case report described severe non-immune hemolytic anemia in a neonate with hereditary spherocytosis secondary a heterozygous mutation of the SPTA1 gene. Lastly, the HBB gene encodes for hemoglobin beta globin chains where alterations have been associated with hemolytic anemia, sickle cell anemia, and beta thalassemia. Several case reports described heterozygous variants of HBB and association with hemoglobin instability and extravascular hemolysis. Heterozygous mutations in the above genes have been rarely reported in literature to cause non-immune hemolytic anemia. Although unclear, pregnancy appeared to be the inciting factor in our patient with these mutational variants that have a potential role in extravascular hemolysis. While there have been few case reports describing autoimmune hemolytic anemia caused by pregnancy, a non-immune hemolytic anemia from 4 heterozygous variants in RBC genes, as seen in our patient, has not been previously described. Disclosures No relevant conflicts of interest to declare.

The Impact of 3D Chromosomal Topology in Acute Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-15-SCI-15
Author(s):  
Iannis Aifantis
Keyword(s):  
Survival Rate ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Tumor Formation ◽  
Cohesin Complex ◽  
Self Renewal ◽  
Treatment Regimens ◽  
The Impact

Abstract Acute myeloid leukemia (AML) is the most common adult leukemia characterized by excessive proliferation of abnormal myeloid progenitors. AML continues to have a dismal survival rate amongst all subtypes of leukemia (<50% five-year overall survival rate), which can largely be attributed to limited advances in treatment regimens that, for the last decades, have relied on the use of two non-targeted cytotoxic drugs: cytarabine and anthracycline. Large-scale sequencing efforts have shed new light on genetic and epigenetic determinants of AML. Interestingly, these studies identified a frequent co-occurrence of somatic mutation between genes encoding cohesin complex subunits (such as STAG2, SMC1A, RAD21 and SMC3) and well-characterized AML oncogenic triggers, such as FLT3-ITD, TET2, and NPM1. Recent work has demonstrated an important role for the cohesin complex in normal stem/progenitor self-renewal and differentiation, gene regulation, and suppression of myeloproliferative neoplasms and AML, despite the precise mechanisms underlying these functions remaining poorly understood. It is believed that cohesin may suppress tumor formation by regulating chromatin looping at loci critical for self-renewal and myeloid progenitor differentiation. Utilizing established models of murine and human AML, this we will focus on the molecular mechanisms of cohesin-dependent myeloid tumor-suppression, with an emphasis on understanding novel treatment approaches that can exploit these functions. Using established protocols for identifying genome-wide changes in chromatin topology and gene expression, we propose undertaking an extensive characterization of cohesin-regulated chromatin changes driving AML. Furthermore, we investigate the application of targeted agents in cohesin-deficient AML whilst extensively mapping the mechanisms-of-action underlying these specific treatments. Ultimately, our work aims to generate novel, pre-clinical disease models of cohesin-mutated AML with strong mechanistic insights into the tumor-suppressive function of this complex. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased [CO2] Causes Changes in Physiological and Genetic Responses in C4 Crops: A Brief Review

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1567
Author(s):  
Renan Gonçalves da Silva ◽  
Rita de Cássia Alves ◽  
Sonia Marli Zingaretti
Keyword(s):  
Molecular Mechanisms ◽  
Photosynthetic Apparatus ◽  
C4 Plants ◽  
Atmospheric Concentration ◽  
Genetic Changes ◽  
Protein Encoding ◽  
Carbon Dioxide Co2 ◽  
Internal And External Factors ◽  
Genetic Responses ◽  
Encoding Genes

Climate change not only worries government representatives and organizations, but also attracts the attention of the scientific community in different contexts. In agriculture specifically, the cultivation and productivity of crops such as sugarcane, maize, and sorghum are influenced by several environmental factors. The effects of high atmospheric concentration of carbon dioxide ([CO2]) have been the subject of research investigating the growth and development of C4 plants. Therefore, this brief review presents some of the physiological and genetic changes in economically important C4 plants following exposure periods of increased [CO2] levels. In the short term, with high [CO2], C4 plants change photosynthetic metabolism and carbohydrate production. The photosynthetic apparatus is initially improved, and some responses, such as stomatal conductance and transpiration rate, are normally maintained throughout the exposure. Protein-encoding genes related to photosynthesis, such as the enzyme phosphoenolpyruvate carboxylase, to sucrose accumulation and to biomass growth and are differentially regulated by [CO2] increase and can variably participate owing to the C4 species and/or other internal and external factors interfering in plant development. Despite the consensus among some studies, mainly on physiological changes, further studies are still necessary to identify the molecular mechanisms modulated under this condition. In addition, considering future scenarios, the combined effects of high environmental and [CO2] stresses need to be investigated so that the responses of maize, sugarcane, and sorghum are better understood.

scholarly journals Genetic variability of Chlamydophila abortus strains assessed by PCR-RFLP analysis of polymorphic membrane protein-encoding genes

2011 ◽  
Vol 151 (3-4) ◽  
pp. 284-290 ◽  
Author(s):  
Michelle Sait ◽  
Ewan M. Clark ◽  
Nicholas Wheelhouse ◽  
Lucy Spalding ◽  
Morag Livingstone ◽  
...  
Keyword(s):  
Genetic Variability ◽  
Membrane Protein ◽  
Rflp Analysis ◽  
Chlamydophila Abortus ◽  
Protein Encoding ◽  
Pcr Rflp ◽  
Encoding Genes

scholarly journals Global scale transcriptome analysis reveal differentially expressed genes during early somatic embryogenesis in Dimocarpus longan Lour

2019 ◽  
Author(s):  
Yukun Chen ◽  
Xiaoping Xu ◽  
Zhuanxia Liu ◽  
Zihao Zhang ◽  
XuHan Xu ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Molecular Markers ◽  
Fatty Acid ◽  
Transcriptome Analysis ◽  
Embryogenic Callus ◽  
Molecular Mechanisms ◽  
Fatty Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Protein Encoding ◽  
Encoding Genes

Abstract Background Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to the totipotent embryonic stem cells and generate embryos in vitro. Longan SE has been established and wildly used as a model system for studying embryogenesis in woody plants, and some SE-related genes had been characterized. In spite of that, a comprehensive overview of SE at a molecular level is still absent. With the aim of understanding the molecular mechanisms underlying SE in longan, we examined the transcriptome changes by using Illumina HiSeq platform from the four distinct developmental stages, including non-embryogenic callus (NEC), embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE). Results RNA-seq of the four samples generated a total of 243.78 million high quality reads, approximately 81.5% of the data were mapped to the reference genome. The cDNA libraries of NEC, EC, ICpEC and GE, generated 22743, 19745, 21144, 21102 expressed transcripts and 1935, 1710, 1816, 1732 novel transcripts, and 2645, 366, 505, 588 unique genes, respectively. Comparative transcriptome analysis revealed the important role of auxin and cytokinin during longan SE. The transcripts profiling of flavonoid and fatty acid biosynthesis related genes suggested that flavonoids were mainly accumulated in NEC, while fatty acid accumulated in early SE. In addition, the extracelluar protein encoding genes LTP, CHI, GLP, AGP, EP1 were related to longan SE. Transcript profiling combined with qRT-PCR performed on selected genes confirmed that 27 SE molecular markers (LEC1, LEC1-like, PDF1.3, GH3.6, AGL80, PIN1, BBM, WOX9, WOX2, ABI3, et al.) and 28 NEC markers (LEA5, CNOT3, DC2.15, PR1-1, NsLTP2, DIR1, PIP1, PIP2.1, TIP2-1, POD-P7 and POD5 et al.) were characterized as potential molecular markers for longan early SE, respectively. Conclusion Our transcriptome reveals the transcription regulation of auxin, cytokinin and other hormones signaling pathway, flavonoids biosynthesis, fatty acid biosynthesis, extracelluar protein encoding genes, and other SE-related genes during early SE. Furthermore, we characterizes the potential molecular markers to distinguish NEC and early SE of longan. The present work provides new insights into future functional studies, as a means of studying the molecular mechanisms in SE.

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