遗传病二代测序临床检测全流程规范化共识探讨(2)——样品采集处理及检测

二代测序技术(next generation sequencing,NGS)具有极高的检测通量,相对低的检测成本,高度的准确性(accuracy)和精准性(precision),是遗传病临床检测的有力工具之一。NGS实验室的检测流程是否规范,将直接影响NGS数据的稳定性、可靠性和有效性,将决定其是否能被用于遗传病的临床辅助诊断或筛查。因此.作为遗传病基因检测的重要环节之一,NGS实验室中流程的规范化与标准化非常重要。2019年5月,在第二届基因检测联盟会议上,针对如何规范NGS检测流程,从事遗传病临床诊治、实验室检测以及第三方基因检测机构的专家进行了全面充分的讨论,旨在规范基于NGS的基因检测流程,对检测流程中的前、中、后三个阶段(包括样本采集/接收/保存、NGS建库、上机测序及数据质控)的操作与实施提出了专业性的指导意见,以规范NGS技术在遗传病基因检测领域中的应用。本文根据此次研讨会上各行业专家的讨论,总结并发布NGS实验室检测流程的规范共识,以促进NGS实验室流程的规范化和标准化,推进我国NGS实验室在遗传病基因检测领域的快速和专业化发展。     

甲泼尼龙对大鼠急性脊髓损伤后髓鞘碱性蛋白的作用研究

目的探讨大剂量甲泼尼龙(MP)对急性脊髓损伤(ASCI)后大鼠脊髓组织和血清中的髓鞘碱性蛋白(MBP)的影响。方法 60只雄性SD大鼠,随机分成A组(正常对照组)、B组(实验对照组)、C组(治疗组)三组,每组20只。A组单纯全椎板切除,不损伤脊髓;B、C组采用Allen's法制作T10脊髓损伤模型,C组首剂量以30 mg/kg甲泼尼龙尾静脉注射,余剂量以5.4 mg·kg-1·h-1,每4 h静脉给药一次,24 h给完,A、B组以生理盐水代替甲泼尼龙尾静脉注射,剂量同C组。术后24 h、48 h对A、B、C三组大鼠后肢运动功能行BBB评分;再在各时间点取受损节段脊髓行HE染色及免疫组化染色观察形态学变化和MBP在脊髓组织中的分布特点;同时应用RT-PCR方法测定各组相应时间点脊髓组织中MBP m RNA表达量;取血清应用ELISA方法检测各组中血清MBP含量。结果 C组大鼠在损伤后24 h、48 h后肢运动功能恢复程度显著高于B组,差异有显著性(P<0.01)。B、C组血清中的MBP蛋白浓度较A组均明显升高,而C组血清的MBP蛋白浓度显著低于B组,差异有显著性(P<0.01);C组脊髓组织中MBP表达高于B组,而低于A组,差异有显著性(P<0.01);C组MBP m RNA表达显著高于B组(P<0.01),而低于A组,差异有显著性(P<0.05)。结论大鼠急性脊髓损伤后血清中MBP显著升高,脊髓组织中MBP表达会降低,早期应用大剂量MP对脊髓急性损伤具有明显的保护作用。     

极早产儿输血相关性坏死性小肠结肠炎危险因素分析

目的 探讨极早产儿发生输血相关性坏死性小肠结肠炎(TA-NEC)的危险因素。方法 选择2013年4月至2021年4月新生儿重症监护室收治的接受输注红细胞的极早产儿为研究对象。符合TA-NEC组纳入标准的极早产儿为TA-NEC组;按1:2比例匹配同期同性别、胎龄(±3d)、出生体重(±200g)、输血日龄(±3d)的非NEC极早产儿作为对照组。比较两组间临床特点差异,探讨TA-NEC发生的危险因素。结果 共纳入204例极早产儿,男138例、女66例,平均胎龄(29.0±1.5)周,中位出生体重1 100.0(951.0～1 200.0)g。TA-NEC组68例,对照组136例。多因素条件logistic回归分析结果显示,宫内窘迫、绒毛膜羊膜炎、晚发型败血症是极早产儿发生TA-NEC的独立危险因素(P<0.05),完全经口喂养是其独立保护因素(P<0.05)。结论 患有宫内窘迫、绒毛膜羊膜炎和/或晚发型败血症的极早产儿在输注红细胞后48 h内更容易发生NEC。预防围生期缺氧和败血症,在安全前提下完成到完全经口喂养的过渡,对降低极早产儿TA-NEC的发生率有积极作用。     

Ⅱ型先天性厚甲症一家系KRT基因突变检测

目的:检测先天性厚甲症一家系中KRT6b和KRT17基因突变位点。方法:提取先证者、其父母(母亲为患者,父亲正常人)及100名正常对照者外周静脉血DNA,PCR技术扩增KRT6b和KRT17基因编码序列,Sanger测序法对PCR扩增产物进行测序。结果:先症者及其母亲在KRT17基因1号外显子上存在错义突变(c.275AG),KRT6b基因不存在任何突变。先证者父亲及100名正常对照者中未检测到任何突变。结论:此家系患者是由于KRT17基因突变(c.275AG,p.Asn92Ser)所致。     

Phase I study of abagovomab in patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer.

PURPOSE: This open-label study assessed the safety and immunogenicity of two doses and two routes of the anti-idiotypic monoclonal antibody abagovomab (formerly ACA125) in patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer. EXPERIMENTAL DESIGN: Eligible patients from the three participating institutions were any stage at diagnosis, had relapsed, and had complete or partial response to additional chemotherapy. Patients were randomized to receive abagovomab at 2.0 versus 0.2 mg and i.m. versus s.c. for four immunizations every 2 weeks and then monthly for two additional immunizations. Planned evaluation included interval physical examinations and laboratory assessments with immune assessment, including HLA typing, human anti-mouse antibody, ELISA, and enzyme-linked immunospot. Patients were required to remain on study until week 10 (the first post-baseline Ab3 determination) to be considered for immunologic assessment. The primary end points were safety and immunogenicity primarily determined by Ab3 response. RESULTS: Forty-two patients received at least one vaccination and were eligible for safety analysis. Thirty-three patients were available for Ab3 analysis (removed for progression of disease, 6; withdrawal of consent, 2; unrelated adverse event, 1). The most common adverse events were self-limited pain at injection site, myalgia, and fever. No hematologic or nonhematologic toxicity grade>2 related to immunization was seen. Ab3 was detectable in all patients (median, 236,794 ng/mL); none of route of administration (P=0.6268), dose (P=0.4602), or cohort (P=0.4944) was statistically significant in terms of effect on maximum post-baseline Ab3 titer. Human anti-mouse antibody was not detectable at baseline but was present in all patients at week 16 (range, 488-45,000 ng/mL). CONCLUSIONS: Immunization with abagovomab is well tolerated and induced robust Ab3 responses at the two doses and routes tested. A phase III randomized study with abagovomab (2.0 mg s.c.) is warranted.     

Cyclophosphamide, hexamethylmelamine, doxorubicin, and cisplatin (CHAD) as second-line chemotherapy for ovarian adenocarcinoma

Twenty women with recurrent ovarian adenocarcinoma received a monthly four-drug combination of cyclophosphamide, hexamethylmelamine, doxorubicin, and cisplatin as second-line chemotherapy. There were no objective responses to this regimen. This is in contrast to the 49% response rate reported by Kane et al using these four drugs and the 63% response rate reported by Vogl et al using three of these drugs as second-line chemotherapy. The differences in the three regimens are reviewed; however, we could not identify reasons sufficient to account for the disparity in response rates.