高通量测序技术在遗传性耳聋研究中的应用及研究进展

耳聋是一种常见的严重出生缺陷,阐明遗传性耳聋的致病机理不仅能够在临床上辅助诊断,为遗传咨询及耳聋预防提供依据,而且能促进人们更深入地了解耳聋的致病机制,开发新的治疗方法。随着基因组研究技术不断创新,以全基因组测序、全外显子组测序、目标区域测序为代表的高通量测序技术在遗传性耳聋研究中已得到广泛应用。本文总结了近5年全外显子组测序和目标区域测序在遗传性耳聋致病基因研究及临床分子诊断中应用及研究进展,希望能够有助于我国临床耳聋基因诊断技术的发展及诊断水平的提升。     

运用多重PCR-直接测序法检测ABO基因型及其遗传多态性

根据ABO基因座第6和7外显子9个SNP位点设计引物, 复合扩增后直接测序, 根据测序结果判定不同物证检材ABO基因型及其在藏族群体中的多态性分布。成功地检测出经过不同方法处理的血痕、毛发、口腔拭子、骨骼、混合斑等101例腐败、降解及微量检材的ABO基因型, 结果与免疫血清学分型一致, 且该方法具有灵敏度高、特异性好、操作简单、结果准确、客观及能够发现新等位基因等优点。对80名青海藏族无关个体的调查表明, ABO基因型分布符合Hardy-Weinberg平衡, 杂合度H为0.675, 多态信息含量PIC为0.672, 个人识别力DP值为0.874, 非父排除率PE值为0.391, 偶合度I为0.126; 青海藏族ABO等位基因频率O>B>A, 且O等位基因频率高达0.6125。多重PCR-直接测序法检测ABO基因型适用于法医学不同来源的样本, 提高了ABO血型系统的个体识别能力; ABO基因型在青海藏族人群中的分布具有较高多态性, 可用于法医学个体识别及群体遗传学研究。     

自身抗体引起的ABO血型正反定型结果不一致

目的:对因自身抗体引起ABO血型正反定型结果不一致的疑难血标本进行血型鉴定和分析,探讨其原因和解决办法。方法:先用直接抗人球蛋白试验(DAT)和间接抗人球蛋白试验(IAT)确定ABO血型正反定型结果不一致的原因是由于自身抗体的存在,然后做吸收放散试验、抗体筛选试验等排除自身抗体的干扰以便血型的正确判定。结果:ABO血型正反定型结果不一致因自身抗体引起的有10例,其中温抗体7例,冷抗体3例,温抗体同时同种抗体阳性2例,冷抗体同时同种抗体阳性2例。结论:自身抗体会影响ABO血型的正确判定,选用合适的试验进行分析判断以提高血型鉴定的准确性。     

遗传性耳聋分子诊断及梯级检测策略应用

遗传性耳聋是人类最常见的感觉障碍之一，具有高度遗传异质性。目前常用的遗传性耳聋分子诊断方法包括基因芯片、Sanger测序、靶向富集测序和全外显子组测序等，诊断率可达33.5%～56.67%，但还有相当一部分患者不能进行及时有效的分子病因诊断。考虑到患者家庭的经济负担及目前全外显子组/全基因组测序仍相对昂贵，根据患者情况提供包含多种检测手段的梯级诊断策略至关重要。因此，本文对遗传性耳聋分子诊断现状以及梯级检测在遗传性耳聋分子诊断中的应用进行综述，以期为诊断策略的选择提供参考。     

ABO血型综合实验基因分型技术简化及其群体遗传分析拓展

ABO血型是生活中最常见、运用最广泛的遗传性状之一。人类ABO血型由I ^A、I ^B和i 3个复等位基因决定,它们负责编码不同的糖基转移酶,进而决定3种血红细胞表面抗原。ABO血型涉及复等位基因、基因互作、单核苷酸多态(SNP)、基因演化等多个关键知识内容,是理想的遗传学教学案例。本文以ABO血型为研究对象,对遗传学实验进行了创新与整合。首先,在分子遗传学模块中建立了新颖的ABO血型基因分型方法：基于SNP位点设计特异性引物,通过实时定量PCR鉴定基因型; 其次,在群体遗传学模块中创新了基因演化的实验教学方法,开发群体遗传学软件,利用计算机模拟不同条件下ABO血型决定基因频率的演化趋势。这些教学改革举措旨在丰富遗传学实验内容,拓展教学手段,提高学习效率。     

中国人罕见的cisAB变异型分子机制分析

为研究中国人群ABO血型系统中罕见的cisAB变异型的分子遗传背景, 用血型血清学方法鉴定12例ABO血型疑难样本和116例随机无血缘关系样本, 应用聚合酶链反应和DNA序列分析等方法对样本ABO基因酶活性编码区外显子6、7和侧翼内含子进行突变筛选和检测, 并对不同基因型的扩增片段进行单倍体序列分析。血清学检测发现12个样本均为(A强B弱)或(A弱B强), PCR产物直接序列分析表明这些样本均为cisAB变异型, 并存在4种基因型。通过单倍体序列分析, 从中发现2种cisAB等位基因, 其中cisAB01不仅存在外显子7的467C>T和803G>C变异, 还在第6内含子存在未经报道的163T>C和179C>T变异; 另一种等位基因是在B101基础上保留了A101的803G位点, 编码一条176G、235S、266M和268G组合的多肽链。经检索, 未发现该组合的ABO等位基因, 该等位基因已被国际血型抗原基因突变数据库命名为cisAB05等位基因。文章系统研究了中国人群cisAB变异型分子机制, 发现了一种新的cisAB05等位基因, 同时根据内含子序列推测cisAB01等位基因可能是通过A101和B101等位基因间的同源交换而形成的。     

高通量测序技术在罕见病分子诊疗中的 应用及临床实例分析

罕见病病种繁多,且表型复杂多样,不仅仅体现在疾病间的不同,同一种疾病的不同患者在表型上也可能大相径庭。这种普遍存 在的遗传异质性和临床异质性,使罕见病的诊疗极具挑战。近年来,在后人类基因组计划时代,各种测序技术快速发展,使得大规模测 序如疾病目标基因集测序、全外显子组测序、全基因组测序等成为了现实。高通量测序技术可实现对多个靶基因进行高通量平行测序, 有效节约了成本与时间,越来越广泛地应用到临床疾病分子诊疗领域。分析传统测序技术与高通量测序技术的优缺点,介绍罕见病诊疗 中常用的高通量测序策略,并结合临床实例,综述高通量测序技术在罕见病诊疗中的应用。     

高通量测序筛查多巴反应性肌张力障碍家系突变基因

快速发展的高通量测序技术使遗传病家系突变基因的筛查鉴定成为可能,本研究通过全外显子测序、生物信息分析和PCR-测序技术筛选一个多巴反应性肌张力障碍(DRD)家系中可能的致病突变位点,从1 005个低频位点中选出在3个疾病个体至少有一个杂合突变的9个候选SNP,再通过DRD家系12个个体PCR扩增测序验证,新发现突变基因SLC18A1可能是该DRD家系的致病基因。此外,PCR和Sanger测序发现高通量外显子测序和信息分析的SNP分型准确性可高达96.3%,是一种遗传病家系突变基因筛查鉴定高效、准确的方法。     

重大生殖疾病的分子机制研究

重大生殖疾病是一组严重影响生殖健康,病因和临床表现高度异质的疾病群,其中女性常见的有多囊卵巢综合征(PCOS)、卵巢早衰(POF)等,遗传因素在疾病的发生发展中起到重要作用。近年来,依靠高通量技术,如全基因组关联分析、全外显子组测序等,我国在重大生殖疾病的遗传学研究中取得了重要进展,主要包括对PCOS和POF大样本散发病例及其对照进行全基因组关联研究、家系患者的全外显子组或全基因组测序研究等,鉴定出多个遗传易感位点,获得大量候选基因的信息。虽然这些研究结果为解析疾病提供了大量线索,但是也提出了更多挑战。如何深入研究这些易感位点在疾病中的致病机制,及其在复杂疾病诊治中的转化应用成为日后的工作重点,现就相关分子机制研究进展做一简要论述。     

卡式微柱凝胶法在ABO、RhD血型鉴定中的应用价值

目的:探讨微柱法在ABO、RhD血型鉴定中的应用价值。方法:抽取2012年3月至2013年3月我站检验科血液筛查标本共1081份作为研究对象,在metis全自动血型仪进行初筛的基础上,同时进行卡式微柱凝胶法ABO、RhD血型鉴定,据定型结果配血,统计输血结局并作为评价依据。结果:筛查RhD阴性者6份占0.56%,阳性者1075份占99.44%;所有血液输用后均未产生输血反应。结论:ABO、RhD血型鉴定是保障输血安全、抑制溶血性疾病的必要措施,而微柱法具有操作简便、价格低廉、科学准确的优点,适合血站的血型鉴定和临床的推广。     

Red cell genotyping and the future of pretransfusion testing

The ABO blood group, based on molecular biological detection technology, has the advantages of simple operation, high sensitivity, and standardized result interpretation, and is not affected by sample immunological characteristics. However, clinically, performance verification, clinical application scope, quality management, abnormal result processing, and other issues associated with the ABO blood group molecular detection technology are relatively complex, and there is a lack of unified norms and standards. Therefore, from the perspective of the whole process of ABO molecular biology detection, this study aims to provide standardized opinions on important links affecting the detection results, common problems encountered in the detection process, and the assessment and treatment of abnormal results. Finally, a Chinese expert consensus on molecular biological technology based on genotyping and sequencing detection was put forward, which standardizes the detection process, improves the accuracy of results, and promotes the development of technology and broader clinical application.     

Serology and gene sequence analysis of rare subgroup A3 blood group

To investigate the serological phenotypic characteristics and possible mechanism of subgroup A₃, a blood donor's ABO phenotypes were detected by the conventional microcolumn gel method and classic tube method. N-acetylgalactosaminyl transferase activity was detected by the non-radioactive phosphate coupling method. ABO subtype genotyping was determined by PCR-SSP and exons 1-7 of ABO gene were analyzed by Sanger sequencing. The donor's blood type was subgroup A₃ as evaluated by serological test. There was no N-acetylgalactosaminyl transferase activity in the red blood cells and weak N-acetylgalactosaminyl transferase activity in the plasma. The ABO blood group genotyping result was ABO*AO1, and the gene sequencing result was confirmed as A221/O01. Sequencing results showed two mutations, 467C>T and 607G>A in exon 7 in ABO*A allele. In conclusion, it is suggested that the ABO blood group of the donor be subgroup A₃, which may be induced by mutations 467C>T and 607G>A, and led to a decrease in N-acetylgalactosaminyl transferase activity and resulted in weakened A antigen.     

Advances in ABO gene expression regulation

The ABO blood group system is vital to blood transfusion and organ transplantation. ABO antigens are the most important of all blood group antigens in clinical practice, and are not only present in red blood cells and platelets, but also in most secretions and epithelial tissues. ABO antigens are known to undergo drastic changes during the development, differentiation, and maturation of normal cells. Profound changes have also been documented in pathological processes such as tumorigenesis. To elucidate the molecular basis of how ABO genes are controlled in cell type specific expressions, such as normal cell differentiation or in cancer cells lacking A/B antigens, it is essential to understand the regulatory mechanisms of ABO gene expression. In this review, current knowledge concerning the regulatory mechanisms of ABO gene expression was summarized.     

Molecular characterization of ABO blood group frequencies in pre-Columbian Peruvian highlanders

The majority of Native Americans nearly exclusively belong to group O of the ABO blood group system. Several hypotheses have been formulated to explain this observation, primarily differing by the presumption that the observed patterns of ABO diversity are due to the processes of the initial peopling of the Americas or due to subsequent events, especially the demographic consequences in the wake of European contact. A promising strategy to reveal possible diachronic ABO frequency changes is the molecular genetic analysis of relevant genetic markers in precontact populations. A previous study by Halverson and Bolnick [Am J Phys Anthropol 137 (2008) 342‐347] already accomplished this for indigenous North American populations. Here we present the first study to analyze ABO blood types from pre‐Columbian individuals from South America using molecular genetic methods and comparing them to several extant South American, North American, and Siberian populations. We tried to determine ABO blood types for 59 individuals from the southern Peruvian highlands dating to ～650 to 1250 AD using a newly developed multiplex PCR/SBE assay coamplifying the fragments relevant for blood type determination and three highly discriminating autosomal STRs. Analysis was successful for 31 individuals and revealed that all are exclusively in the O group, predominantly carrying the O02 (01v) allele. No significant difference could be observed between the ancient and modern Native American populations, while all significantly differed from the extant Siberian populations, supporting the suggestion that low ABO diversity results from founder effects during the initial peopling of the Americas. Am J Phys Anthropol 149:242–249, 2012. © 2012 Wiley Periodicals, Inc.     

Serological and molecular analysis of ABO and Rh blood group chimeras

The serological examination, blood transfusion strategies and the molecular analysis to blood group chimera were conducted to demonstrate existent of chimera in blood group. The blood grouping of ABO or/and RhD, newborn red blood cells separated by capillary centrifugation. Aabsorption tests and DTT treated agglutination erythrocyte tests were implemented in four patients. Further molecular biological research was conducted on one patient''s sample. The results showed that for patient 1: ABO blood group was AB/B chimera, Rh blood cells contained the RhCE chimera gene; Patient 2: Rh blood cells contained the RhD chimera gene; Patient 3: ABO blood group was AB/B chimera, Rh blood cells contained the RhD chimera gene; Patient 4: ABO blood group was O/B chimera, Rh blood cells contained the RhCE chimera gene. The study suggests that the individuals categorized as chimeras are likely to be more common than existing literature reports. According to the serological tests, in the absence of a history of recent blood transfusion or disease to cause reduced antigen, the phenomena of hybrid aggregation of the ABO and Rh blood system were the main feature. In terms of transfusion strategy, the selection of ABO and Rh blood groups should be depended on the group of cells with more antigens.     

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.     

ABO、MN血型及其与测量体征关系的初步探讨

本文提供了男、女两性共404人ABO、MN血型各表现型的35个体质特征测量数据,对血型和测量体征的关系进行了初步讨论。统计分析表明,不同血型群体的某些单个体征平均值之间有显著差异。对所调查的35个体征作整体分析,可见男性MN系统和女性ABO、MN系统各血型群体平均值之间差异显著。在MN血型系统,MN型群体的测量体征平均值低于M型和N型与MN基因型的杂合状态有密切关系。本文还分析了4个民族的血型资料,表明MN血型在ABO血型系统是随机分布的。     

应用改良流式法检测猕猴和食蟹猴体内血型抗体水平的分布情况

绝大部分灵长类动物存在与人类相似的ABO血型系统,该研究采用改良流式法(flow cytometry method,FCM)检测猕猴及食蟹猴血清中血型抗体水平的分布情况。以流式细胞术为基础,使用商品化人源红细胞为靶细胞,并通过加入特异性荧光标记的抗人IgM或IgG二抗,对收集的实验用猕猴及食蟹猴的血清样本进行检测,以人类健康受试者的血清样本为对照,比较两者血型抗体水平的差异。结果显示:预先用人O型浓缩红细胞吸附猴血清中所含种属间非特异性抗体后,FCM法能够准确检测其血型抗体水平及分型,并且发现猴血清中天然血型抗体的水平明显低于健康人(P<0.05)。由此得出:通过预处理清除非特异性抗体的干扰后,FCM法同样适用于灵长类动物血清中血型抗体的检测,也为构建灵长类动物模拟人ABO血型不合器官移植模型提供了技术保障和实验数据。