Next-generation sequencing through multigene panel testing for the diagnosis of hereditary epidermolysis bullosa in Chinese population

Epidermolysis bullosa (EB) is a heritable blistering disorder. We performed a next-generation sequencing-based multigene panel test and successfully predicted 100% of the EB types, including, 36 EB simplex (EBS), 13 junctional EB (JEB), 86 dystrophic EB (DEB), and 3 Kindler EB. Chinese JEB and recessive DEB (RDEB) patients have relatively mild phenotypes; for severe type separately accounts for 45.5% and 23.8%, respectively. We identified 96 novel and 49 recurrent pathogenic variants in 11 genes, although we failed to detect the second mutation in one JEB and five RDEB patients. We identified one novel p.E475K mosaic mutation in the clinically normal mother of one out of 13 EBS patients with KRT5 mutations, one recurrent p.G2034R mosaic mutation, and one novel p.G2043R mosaic mutation in the clinically normal relatives of two out of 19 dominant DEB patients. This study shows that next-generation technology could be an effective tool in diagnosing EB.     

维纳托克在肝癌细胞中的抗肿瘤活性及其机制

目的探讨维纳托克(Venetoclax)在HepG2、Hep3B 2个肝癌细胞株的抗癌活性。方法对人肝癌细胞株HepG2、Hep3B细胞株进行体外培养,采用不同浓度的Venetoclax(0、3、10、30μmol/L)处理肝癌细胞,以细胞计数试剂盒(CCK-8)检测细胞活性,0,3,10和30μmol/L Venetoclax作用细胞48 h,锥虫蓝拒染法检测细胞的死亡,0,5μmol/L Venetoclax,作用24 h,流式细胞学检测细胞凋亡率,蛋白质印迹法(Western blot)检测Venetoclax诱导半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3活化,聚腺苷二磷酸核糖聚合酶(PARP)裂解。组间比较采用t检验。结果Venetoclax对HepG2、Hep3B细胞株均有较强活性抑制作用。不同浓度的Venetoclax处理肝癌细胞48、72 h,Venetoclax在HepG2细胞株中取得的半数细胞活性抑制率(IC50)值分别为46.5μmol/L和14.2μmol/L;在Hep3B细胞的IC50值分别为24.6μmol/L和11.2μmol/L。处理24 h,30.0μmol/L的Venetoclax在HepG2和Hep3B细胞株中诱导57%[对照组、实验组凋亡率分别为(4.00±1.00)%、(56.67±8.51)%,t=-10.650,P<0.01]和54%[对照组、实验组凋亡率分别为(5.67±1.53)%、(54.33±9.87)%,t=-8.440,P<0.01]的肝癌细胞发生凋亡,并能诱导肝癌细胞的Caspase-3活化,PARP裂解,差异均有统计学意义。用0、3、10和30μmol/L的Venetoclax处理48 h,在HepG2细胞株中可诱导3%(3.26±1.71)%,12%[(12.36±1.99)%,(t=-12.36,P<0.05)],32%[(32.38±4.57)%,(t=-10.350,P<0.01)]和67%[(66.71±6.29)%,(t=-16.86,P<0.01)]的细胞死亡,差异均有统计学意义;在Hep3B细胞株诱导2%(1.91±0.68)%,16%[(15.72±3.40)%,(t=-6.890,P<0.05)],42%[(42.39±6.86)%,(t=-10.180,P<0.01)]和73%[(73.32±2.69)%,t=-44.490,P<0.01]的细胞死亡,差异均有统计学意义。应用半胱氨酸蛋白酶(Caspase)抑制剂预处理,Venetoclax对HepG2和Hep3B细胞的死亡率分别从56%(56.71±6.29)%降低至11%(10.70±1.43)%,(t=12.350,P<0.01)和68%(68.05±10.16)%至12%(12.7±6.12)%,(t=8.080,P<0.01),差异有统计学意义。结论Venetoclax能够通过诱导Caspase的活化,诱导肝癌细胞发生凋亡,并对肝癌细胞进行杀伤。     

某医学期刊送审论文的统计学问题分析

<正>医学科研论文大多涉及数据收集和统计分析,正确合适的数据统计分析方法,才可能保证科研结果的正确性。虽然论文撰写的国际规范对数据统计学分析有明确的要求,科研人员也学习过医学统计学,但如何正确选择统计分析方法,准确描述统计分析结果,仍然是很多科研人员比较困惑的问题。有些作者在论文中误用、错用统计学方法,直接影响研究结果的可信度,导致论文质量不高或者结论错误[1-3]。本文对某杂志2014年至2015年度送审论文中的统计学问题进行分析,旨在帮助作者避免类似的错误,提升科研论文质量。资料与方法     

Host MyD88 signaling protects against acute graft-versus-host disease after allogeneic bone marrow transplantation

Recent experimental strategies to reduce graft-versus-host disease (GVHD) have focused largely on modifying innate immunity. Toll-like receptor (TLR)-driven myeloid differentiation primary response 88 (MyD88)-dependent signalling pathways that initiate adaptive immune function are also critical for the pathogenesis of GVHD. This study aimed to delineate the role of host MyD88 in the development of acute GVHD following fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT). When myeloablated BALB/c MyD88 knock-out recipients were transplanted with C57BL/6 (B6) donor cells, they developed significantly more severe GVHD than wild-type (WT) BALB/c hosts. The increased morbidity and mortality in MyD88^–/– mice correlated with increased serum levels of lipopolysaccharide and elevated inflammatory cytokines in GVHD target organs. Additionally, MyD88 deficiency in BMT recipients led to increased donor T cell expansion and more donor CD11c⁺ cell intestinal infiltration with apoptotic cells but reduced proliferation of intestinal epithelial cells compared with that in WT BMT recipients. Decreased expression of tight junction mRNA in epithelial cells of MyD88^–/– mice suggested that MyD88 contributes to intestinal integrity. Cox-2 expression in the GVHD-targeted organs of WT mice is increased upon GVHD induction, but this enhanced expression was obviously inhibited by MyD88 deficiency. The present findings demonstrate an unexpected role for host MyD88 in preventing GVHD after allogeneic BMT.     

Using gas chromatography and mass spectrometry to determine 25-hydroxyvitamin D levels for clinical assessment of vitamin D deficiency

Vitamin D is responsible for multiple metabolic functions in humans. Rickets are the most common disease caused by vitamin D deficiency. It is caused by poor calcium intake resulting in poor serum-ionized calcium. The purpose of this study is to develop a rapid, sensitive, and feasible method to determine the 25-hydroxy-vitamin D3 (25(OH)D3) levels in blood samples for clinical assessment. In this study, gas chromatography coupled mass spectrometry with trimethylsilyl derivatization (TMS-GC-MS) is the most suitable protocol for quantitative analyses of 25(OH)D3. Performance of method was evaluated and compared with liquid chromatography and immunoassay. Method validation has been carried out with plasma specimens. The limit of quantitation of TMS-GC-MS method is 1.5 ppb with good linear correlation. Furthermore, the dietary intake and nutritional status of vegetarian and non-vegetarians in Taiwan were assessed by our validated method. As a result, this vitamin D nutrition survey demonstrates that most Taiwanese people have insufficient vitamin D. Due to dietary habits; the male vegans may have the highest risk of vitamin D deficiency.