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Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To analyze the gene mutation types and frequence of thalassemia patients in Jingzhou area.
OBJECTIVE
To analyze the gene mutation types and frequence of thalassemia patients in Jingzhou area.
METHODS
A total of 721 suspected thalassemia patients who were visited in Jingzhou Central Hospital from June 2019 to June 2022 were selected as the research objects. There were 204 males and 517 females. PCR-reverse dot hybridization method was used to analyze the types and frequencies of 23 common α or β thalassemia gene mutations.
RESULTS
Among the 721 patients with suspected thalassemia, 228 cases were positive for α or β thalassemia gene, with a total positive rate of 31.62%, including 87 cases of α-thalassemia, accounting for 38.16%, and 140 cases of β-thalassemia, accounting for 61.40%. There was 1 case of α β complex thalassemia, accounting for 0.44%. A total of 4 types of α-thalassemia gene mutations were detected, all of which were deletion types, including αα/-- (64/87, 73.56%), αα/-α (14/87, 16.09%), -- /-α (7/87, 8.05%), αα/-α (2/87, 2.30%). Among 140 patients with β-thalassemia, 138 were pure heterozygotes, and the genotypes of (63/140, 45.00%), (34/140, 24.29%), (18/140, 12.86%) and (10/140, 7.14%) accounted for 89.29% of all mutations (125/140), 2 cases of double heterozygosity (2/140, 1.43%) were found, no homozygous β-thalassemia were detected; 1 case of αβ complex thalassemia with genotype -α/ was found. The incidence of difference types of thalassemia was statistically significant (χ=194.250, < 0.001). The percentage of positive thalassemia genes was not significantly difference between male and female suspected patients (χ=0.199, =0.655).
CONCLUSION
The α-thalassemia gene mutation in Jingzhou area is dominated by αα/--, and the mutation is more common in β-thalassemia, and α β complex thalassemia is relatively rare, which can provide a reference for the formulation of prevention and treatment measures for thalassemia in Jingzhou area.
Topics: Humans; Male; Female; Mutation; alpha-Thalassemia; beta-Thalassemia; China; Heterozygote; alpha-Globins
PubMed: 38926976
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.028 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to...
OBJECTIVE
To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to provide scientific evidence for the prevention and control strategies of thalassemia.
METHODS
Four hundred and fifty-eight children suspected with thalassemia in Wuzhou were enrolled from March 2017 to June 2022. The level of HbA2 was detected using Bio-Rad VARIANT II Hb analysis system. The deletion of α-thalassemia was measured with gap-PCR assay, and the point mutation of α- and β-thalassemia was tested with DNA reverse dot blot hybridization assay. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of HbA2 for children's thalassemia.
RESULTS
A total of 304 thalassemia carriers were detected in 458 children, accounting for 66.38%. One hundred and seventy-five cases were defined to be α-thalassemia, with the main type of --/αα (54.86%). Thirty-six cases were defined to be intermediate α-thalassemia, with the main type of -α/-- (9.72%). In 108 cases with β-thalassemia, / was the main type, accounting for 49.07%, followed by / (14.81%). Seven cases were moderate/severe β-thalassemia (predominantly / and //). Twenty-one genotypes of α- and β-thalassemia were found in the children. There was significant difference of HbA2 level between the children with different types of thalassemia and healthy controls (all < 0.001). ROC curve analysis showed that the sensitivities of HbA2 for α-thalassemia, β-thalassemia and αβ-thalassemia were 74.3%, 82.4% and 85.7%, with the optimal cut-off values of 2.60%, 3.60% and 3.70%, respectively, the specificities were 64.3%, 96.1% and 96.8%, and the area under the curve were 0.690, 0.887 and 0.916, respectively.
CONCLUSION
The thalassemia genotypes of children in Wuzhou are diverse. It is necessary to further strengthen the prevention and control measure of thalassemia to reduce birth defects and improve birth quality.
Topics: Humans; Genotype; China; Child; alpha-Thalassemia; beta-Thalassemia; Hemoglobin A2; Point Mutation; Male
PubMed: 38926975
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.027 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To analyze the prognostic value of del(1p32) in patients with newly diagnosed multiple myeloma (MM).
OBJECTIVE
To analyze the prognostic value of del(1p32) in patients with newly diagnosed multiple myeloma (MM).
METHODS
The clinical data of 341 newly diagnosed MM attended in Jiangsu Province Hospital were retrospective analyzed. Clinical characteristic combined with genetic features, especially del(1p32), were analyzed for survival and prognostic of patients.
RESULTS
Among the 341 patients with newly diagnosed MM, 24(7.0%) patients were del(1p32) positive. The progression-free survival (PFS) and overall survival (OS) were significantly shorter in MM patients with del(1p32) than those without del(1p32) (PFS: < 0.001;OS: < 0.001). The COX proportional-hazards model showed that del (1p32) was an independent risk factor for PFS and OS of patients with MM. The patients with both 1q21 gain/amplification and del(1p32), as "double-hit chromosome 1", have worse prognosis than those with only 1q21 gain/amplification or only del(1p32) (PFS: < 0.001; OS: < 0.001).
CONCLUSION
Del(1p32) is an independent risk factor for PFS and OS of patients with MM. Del(1p32) detection should be widely used in the prognostic analysis for newly diagnosed MM patients.
Topics: Humans; Multiple Myeloma; Prognosis; Retrospective Studies; Chromosomes, Human, Pair 1; Risk Factors; Chromosome Deletion; Proportional Hazards Models; Male; Female; Middle Aged
PubMed: 38926965
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.017 -
Scientific Reports Jun 2024Muscular dystrophy is a group of genetic disorders that lead to muscle wasting and loss of muscle function. Identifying genetic modifiers that alleviate symptoms or...
Muscular dystrophy is a group of genetic disorders that lead to muscle wasting and loss of muscle function. Identifying genetic modifiers that alleviate symptoms or enhance the severity of a primary disease helps to understand mechanisms behind disease pathology and facilitates discovery of molecular targets for therapy. Several muscular dystrophies are caused by genetic defects in the components of the dystrophin-glycoprotein adhesion complex (DGC). Thrombospondin-4 overexpression has been shown to mitigate dystrophic disease in mouse models for Duchenne muscular dystrophy (dystrophin deficiency) and limb-girdle muscular dystrophy type 2F (LGMD2F, δ-sarcoglycan deficiency), while deletion of the thrombospondin-4 gene exacerbated the diseases. Hence, thrombospondin-4 has been considered a candidate molecule for therapy of muscular dystrophies involving the DGC. We have investigated whether thrombospondin-4 could act as a genetic modifier for other DGC-associated diseases: limb-girdle muscular dystrophy type 2E (LGMD2E, β-sarcoglycan deficiency) and laminin α2 chain-deficient muscular dystrophy (LAMA2-RD). Deletion of the thrombospondin-4 gene in mouse models for LGMD2E and LAMA2-RD, respectively, did not result in worsening of the dystrophic phenotype. Loss of thrombospondin-4 did not enhance sarcolemma damage and did not impair trafficking of transmembrane receptors integrin α7β1 and dystroglycan in double knockout muscles. Our results suggest that thrombospondin-4 might not be a relevant therapeutic target for all muscular dystrophies involving the DGC. This data also demonstrates that molecular pathology between very similar diseases like LGMD2E and 2F can differ significantly.
Topics: Animals; Laminin; Sarcoglycans; Mice; Thrombospondins; Mice, Knockout; Disease Models, Animal; Muscle, Skeletal; Gene Deletion; Muscular Dystrophies; Muscular Dystrophy, Animal
PubMed: 38926599
DOI: 10.1038/s41598-024-65473-8 -
Methods in Molecular Biology (Clifton,... 2024MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of...
MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of target genes. In that regard, their utility has been demonstrated as a strategy to improve the cellular phenotypes important in the biomanufacturing of recombinant proteins. Common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, both of which require the introduction and expression of extra genetic material in the cell. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate CHO cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in influencing CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We show that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures, making it a potentially interesting target to improve bioprocess performance of CHO cells.
Topics: Animals; CHO Cells; Cricetulus; MicroRNAs; Recombinant Proteins; CRISPR-Cas Systems; Gene Editing; Gene Deletion
PubMed: 38926286
DOI: 10.1007/978-1-0716-3878-1_18 -
The British Journal of Dermatology Jun 2024Primary cutaneous acral CD8+ T-cell lymphoproliferative disorder (TLPD) is a rare and indolent lymphoma entity. Although known for years, the molecular pathogenesis is...
BACKGROUND
Primary cutaneous acral CD8+ T-cell lymphoproliferative disorder (TLPD) is a rare and indolent lymphoma entity. Although known for years, the molecular pathogenesis is still unknown.
OBJECTIVES
In order to better understand the molecular pathogenesis of cutaneous acral CD8+ TLPD and to identify further discriminatory markers to discern this lymphoma subtype from further CD8+ cutaneous lymphomas, we analysed five cases of cutaneous acral CD8+ TLPD for putative molecular alterations.
METHODS
Somatic alterations were assessed by whole exome and targeted sequencing of paraffin-embedded tissue. Results were evaluated by immunohistochemical staining of respective relevant proteins. CD8+ cutaneous T-cell lymphomas (n = 12) served as control for KIR3DL1-staining.
RESULTS
Copy number variations (CNV) analysis revealed a homozygous deletion of the KIR3DL1 gene in two of the analysed cases. This resulted in loss of KIR3DL1 protein expression which was observed in all cases of cutaneous acral CD8+ TLPD. In contrast, KIR3DL1 expression was more variable in other CD8+ cutaneous T-cell lymphomas with 50% of analysed cases (n = 12) being positive. In addition, one further case of acral CD8+ TLPD harboured a loss of function mutation in the PIK3R1 gene presumably activating the PI3K-AKT pathway.
CONCLUSION
Alterations of KIR3DL1 gene may be of pathogenetic relevance for acral CD8+ TLPD. Loss of KIR3DL1 protein expression may support the diagnosis of this indolent lymphoma entity, albeit not being a subtype-specific discriminative feature.
PubMed: 38924750
DOI: 10.1093/bjd/ljae256 -
Microbial Biotechnology Jun 2024Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the...
Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.
Topics: Beauveria; Gene Knockout Techniques; Animals; Gene Deletion; Fungal Proteins; Insecta; Genetics, Microbial
PubMed: 38923821
DOI: 10.1111/1751-7915.14512 -
Plant Biotechnology Journal Jun 2024Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole-genome resequencing (WGRS) based QTL-seq identified...
Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole-genome resequencing (WGRS) based QTL-seq identified the loci governing earliness in lentil. The genetic analysis for maturity duration provided a good fit to 3:1 segregation (F), indicating earliness as a recessive trait. WGRS of Globe Mutant (late parent), late-flowering, and early-flowering bulks (from RILs) has generated 1124.57, 1052.24 million raw and clean reads, respectively. The QTL-Seq identified three QTLs (LcqDTF3.1, LcqDTF3.2, and LcqDTF3.3) on chromosome 3 having 246244 SNPs and 15577 insertions/deletions (InDels) and 13 flowering pathway genes. Of these, 11 exhibited sequence variations between bulks and validation (qPCR) revealed a significant difference in the expression of nine candidate genes (LcGA20oxG, LcFRI, LcLFY, LcSPL13a, Lcu.2RBY.3g060720, Lcu.2RBY.3g062540, Lcu.2RBY.3g062760, LcELF3a, and LcEMF1). Interestingly, the LcELF3a gene showed significantly higher expression in late-flowering genotype and exhibited substantial involvement in promoting lateness. Subsequently, an InDel marker (I-SP-383.9; LcELF3a gene) developed from LcqDTF3.2 QTL region showed 82.35% PVE (phenotypic variation explained) for earliness. The cloning, sequencing, and comparative analysis of the LcELF3a gene from both parents revealed 23 SNPs and InDels. Interestingly, a 52 bp deletion was recorded in the LcELF3a gene of L4775, predicted to cause premature termination of protein synthesis after 4 missense amino acids beyond the 351st amino acid due to the frameshift during translation. The identified InDel marker holds significant potential for breeding early maturing lentil varieties.
PubMed: 38923713
DOI: 10.1111/pbi.14415 -
Biotechnology and Bioengineering Jun 2024Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the...
Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6 > C conversion in the SMA-iPSCs. The SMN2 T6 > C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.
PubMed: 38923503
DOI: 10.1002/bit.28780 -
Microbial Biotechnology Jun 2024Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss...
Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.
Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.
Topics: Pseudomonas putida; Catabolite Repression; Pyruvate Dehydrogenase Complex; Hydrocarbons, Aromatic; Biodegradation, Environmental; Acetyl Coenzyme A; Pyruvic Acid; Gene Deletion; Metabolic Networks and Pathways
PubMed: 38923400
DOI: 10.1111/1751-7915.14514