Screening and Diagnosis of Rare Thalassemia Variants: Is Third-Generation Sequencing Enough?

CONTEXT.­: Rare thalassemia subtypes are often undiagnosed because conventional testing methods can only identify 23 common types of α- and ß-thalassemia. OBJECTIVE.­: To assess a comprehensive approach for the screening and diagnosis of rare thalassemia. DESIGN.­: The study cohort included 72 individuals with suspected rare thalassemia variants. Screening was conducted by next-generation sequencing (NGS) combined with third-generation sequencing (TGS) and chromosomal microarray analysis (CMA)/copy number variation sequencing. RESULTS.­: Of the 72 individuals with suspected rare thalassemia, 49 had rare α- or ß-gene variants. NGS combined with gap polymerase chain reaction detected a total of 42 cases, resulting in a positive detection rate of 58.3%. Additionally, 4 α-globin genetic deletions were identified by TGS, which increased the variant detection rate by 5.6%. Two samples with a microdeletion of chromosome 16 or 11 were detected by CMA, which increased the detection rate by 2.8%. For one sample, reanalysis of the NGS and TGS data confirmed the presence of the ß41-42/ßN and ßN/ßN mosaic. The HBB:c.315 + 2delT mutation was initially reported in Guangdong Province, China. Two HBB gene mutations (HBB:c.315 + 5G>C and HBB:c.295G>A) and 4 rare HBA gene deletions (-11.1, -α27.6, -α2.4, and -α21.9) were initially identified in the Zhonshan region. The hematologic phenotypes of all rare cases in this study were clarified. CONCLUSIONS.­: Rare thalassemia variants are more common than previously thought. Despite advancements in TGS, there is still no foolproof method for detection of all types of thalassemia. Thus, a comprehensive approach is necessary for accurate screening and diagnosis of rare thalassemia variants.

Heparan sulfate regulates IL-21 bioavailability and signal strength that control germinal center B cell selection and differentiation.

In antibody responses, mutated germinal center B (BGC) cells are positively selected for reentry or differentiation. As the products from GCs, memory B cells and antibody-secreting cells (ASCs) support high-affinity and long-lasting immunity. Positive selection of BGC cells is controlled by signals received through the B cell receptor (BCR) and follicular helper T (TFH) cell-derived signals, in particular costimulation through CD40. Here, we demonstrate that the TFH cell effector cytokine interleukin-21 (IL-21) joins BCR and CD40 in supporting BGC selection and reveal that strong IL-21 signaling prioritizes ASC differentiation in vivo. BGC cells, compared with non-BGC cells, show significantly reduced IL-21 binding and attenuated signaling, which is mediated by low cellular heparan sulfate (HS) sulfation. Mechanistically, N-deacetylase and N-sulfotransferase 1 (Ndst1)-mediated N-sulfation of HS in B cells promotes IL-21 binding and signal strength. Ndst1 is down-regulated in BGC cells and up-regulated in ASC precursors, suggesting selective desensitization to IL-21 in BGC cells. Thus, specialized biochemical regulation of IL-21 bioavailability and signal strength sets a balance between the stringency and efficiency of GC selection.

Pathogen-selective killing by guanylate-binding proteins as a molecular mechanism leading to inflammasome signaling.

Inflammasomes are cytosolic signaling complexes capable of sensing microbial ligands to trigger inflammation and cell death responses. Here, we show that guanylate-binding proteins (GBPs) mediate pathogen-selective inflammasome activation. We show that mouse GBP1 and GBP3 are specifically required for inflammasome activation during infection with the cytosolic bacterium Francisella novicida. We show that the selectivity of mouse GBP1 and GBP3 derives from a region within the N-terminal domain containing charged and hydrophobic amino acids, which binds to and facilitates direct killing of F. novicida and Neisseria meningitidis, but not other bacteria or mammalian cells. This pathogen-selective recognition by this region of mouse GBP1 and GBP3 leads to pathogen membrane rupture and release of intracellular content for inflammasome sensing. Our results imply that GBPs discriminate between pathogens, confer activation of innate immunity, and provide a host-inspired roadmap for the design of synthetic antimicrobial peptides that may be of use against emerging and re-emerging pathogens.

Effect of butylphthalide on serum CRP, PARK7, NT-3 and neurological function in patients with acute cerebral infarction.

OBJECTIVE: The effects of butylphthalide on serum C-reactive protein (CRP), Parkinson disease protein 7 (PARK7), and neurotrophin-3 (NT-3) levels, and neurological function in patients with acute cerebral infarction (ACI) were explored in order to provide a reference for clinical treatment of the disease. METHODS: One hundred and twenty patients with ACI treated in our hospital from September 2016 to June 2018 were selected and randomized into the control group and the study group, with 60 cases in each group. Patients in the control group were treated with conventional therapy, while those in the study group were treated with butylphthalide. Clinical efficacy, serum levels of CRP, PARK7, and NT-3, as well as the scores of National Institutes of Health Stroke Scale (NIHSS), Fugl-Meyer Assessment (FMA), and Barthel Index (BI) before and 2 months after treatment were analyzed and compared between the two groups. RESULTS: The study group had a significantly higher effective rate (93.33%) than the control group (73.33%; P<0.05). Before treatment, differences in serum CRP, PARK7, NT-3, IL-6, IL-8, and IL-10 levels between the study group and the control group were barely notable (P>0.05). After treatment, the study group observed lower serum levels of CRP, PARK7, IL-6, IL-8, and a higher levels of IL-10, NT-3 in comparison with those of the control group (P<0.05). Before treatment, NIHSS, FMA, and BI scores between the two groups did not show significant differences (P>0.05). After treatment, the study group yielded a remarkably lower NIHSS score and higher FMA and BI scores than the control group (P<0.05). CONCLUSION: Butylphthalide is effective in the treatment of ACI. It can effectively facilitate the recovery of neurological and motor functions of patients, enhance their quality of life and improve serum CRP, PARK7, and NT-3 levels, which is worthy of clinical promotion.

Molecular mechanisms activating the NAIP-NLRC4 inflammasome: Implications in infectious disease, autoinflammation, and cancer.

Cytosolic innate immune sensing is a cornerstone of innate immunity in mammalian cells and provides a surveillance system for invading pathogens and endogenous danger signals. The NAIP-NLRC4 inflammasome responds to cytosolic flagellin, and the inner rod and needle proteins of the type 3 secretion system of bacteria. This complex induces caspase-1-dependent proteolytic cleavage of the proinflammatory cytokines IL-1ß and IL-18, and the pore-forming protein gasdermin D, leading to inflammation and pyroptosis, respectively. Localized responses triggered by the NAIP-NLRC4 inflammasome are largely protective against bacterial pathogens, owing to several mechanisms, including the release of inflammatory mediators, liberation of concealed intracellular pathogens for killing by other immune mechanisms, activation of apoptotic caspases, caspase-7, and caspase-8, and expulsion of an entire infected cell from the mammalian host. In contrast, aberrant activation of the NAIP-NLRC4 inflammasome caused by de novo gain-of-function mutations in the gene encoding NLRC4 can lead to macrophage activation syndrome, neonatal enterocolitis, fetal thrombotic vasculopathy, familial cold autoinflammatory syndrome, and even death. Some of these clinical manifestations could be treated by therapeutics targeting inflammasome-associated cytokines. In addition, the NAIP-NLRC4 inflammasome has been implicated in the pathogenesis of colorectal cancer, melanoma, glioma, and breast cancer. However, no consensus has been reached on its function in the development of any cancer types. In this review, we highlight the latest advances in the activation mechanisms and structural assembly of the NAIP-NLRC4 inflammasome, and the functions of this inflammasome in different cell types. We also describe progress toward understanding the role of the NAIP-NLRC4 inflammasome in infectious diseases, autoinflammatory diseases, and cancer.