一例ABO新等位基因B112的分子生物学研究及其家系分析

目的 研究1例新的ABO亚型B112的分子机制,并对其家系进行分析.方法 应用单克隆抗体检测先证者红细胞ABO血型抗原,标准A、B、O红细胞检测先证者血清中的ABO抗体.采用聚合酶链反应(po1ymerase chain reaction,PCR)技术扩增先证者ABO基因的第5至7外显子序列,PCR产物经双酶切后直接测序分析第6和7外显子.同时采用基因组单链抽提技术分离先证者的两条单倍型,对分离的单倍型扩增后进行ABO基因测序分析.家系调查采集先证者父母的标本进行ABO血清学实验和ABO基因第6和7外显子测序分析.结果 先证者血清学表型符合B表型特性,直接测序分析显示第6和7外显子有261G/缺失、297A/G、526C/G、559C/T、657C/T、703G/A、796C/A、803G/C、930G/A杂合,推断基因型为BO.基因组单链抽提技术将先证者B和O基因分离后,测序得到两个等位基因为O01和B112.与B101相比,B112第559位C→T导致第187位精氨酸变成半胱氨酸.家系调查显示先证者B112基因从母亲遗传所得,母亲标本ABO血型血清学特性和测序分析结果与先证者完全一致.结论 发现1例559C＞T突变的ABO亚型新等位基因B112,其B抗原表达正常,提示α-1,3-半乳糖基转移酶第187位精氨酸变成半胱氨酸并不影响B转移酶的活性.     

一例CisAB血型样本的血清学和遗传学鉴定

目的 对一份血清学检测为CisAB型的标本进行基因分型,并测序确认.方法 血清学方法采用试管正反定型法;基因分型方法为序列特异性引物聚合酶链反应(PCR-sequence specific primer,PCR-SSP); DNA测序采用双脱氧法.结果 血清学检测结果为:正定型为AB型,反定型检出抗B,抗H+++,自身不凝.PCR-SSP的结果是CisAB01型,家系调查符合CisAB型遗传规律.ABO基因测序结果:第6外显子出现c.261缺失G,297为AA纯合子;第7外显子检出c.467C＞T、c.803G＞C突变,同时发现1个新的杂合突变c.724G＞T.结论 该标本血清学表型为CisAB型,测序结果基因型为ABO*CisAB01与ABO* O01杂合,经提交GenBank确认ABO基因第7外显子c.724G＞T为新突变,序列号为JF304777.     

一种ABx变异型相关的B糖基转移酶基因新突变研究

目的 研究1例ABO血型系统ABx变异型的分子遗传背景.方法 用血型血清学技术鉴定1例ABO血型疑难样本的红细胞表型和唾液血型分泌物质,并用聚合酶链反应分别扩增先证者ABO基因全编码区共7个外显子及侧翼内含子序列,扩增产物经双酶切纯化后直接进行双向测序分析.进一步对存在突变位点的第6～7外显子扩增产物经进行TA克隆和单倍体序列分析.结果 先证者红细胞ABO抗原表达为A强B弱,结合其它血清学特征,鉴定其血型为罕见的ABx变异型.ABO基因全编码区测序发现9处核苷酸杂合位点,分别为第6外显子的297A/G杂合,第7外显子的467C/T、526C/G、657C/T、703G/A、796C/A、803G/C、808T/A、930G/A杂合.单倍体序列分析发现,其中一个等位基因为常见的A102,另一个等位基因除808T＞A突变外,其它变异位点与B101等位基因一致.808T＞A突变可导致B糖基转移酶第270位苯丙氨酸转变为异亮氨酸.结论 B糖基转移酶基因808T＞A突变可能引起酶活性减弱,并进而导致产生Bx变异型.     

ABO基因278C>T突变导致Bw12一例

目的 对1例ABO疑难血型样本进行血清型和基因型鉴定.方法 用血清学方法对标本进行ABO血清型检测,根据血清型结果,选取ABO基因亚型检测试剂盒,用聚合酶链反应-序列特异性引物法进行基因亚型鉴定,用直接测序法对ABO基因的第6、7外显子进行序列测定.结果 血清学结果显示,该样本的红细胞上具有高凝集强度的A抗原和中等凝集强度的B抗原,样本血浆中含有弱凝集强度的抗-B抗体,初步判定为ABw型;B亚型基因分型试剂的结果显示,该样本ABO基因为ABw12型;直接测序结果显示,该标本ABO基因第6外显子序列为278CT、297GA,第7外显子序列为467CT、526CG、657CT、703GA、796CA、803GC、930GA杂合,即在基因型A102/B101的基础上,该序列nt278位发生了C＞T杂合突变,经与血型抗原基因变异资料库的数据比对,确定突变的等位基因为Bw12,基因型为A102/Bw12.结论 中国人群中发现A102/Bw12基因型.     

ABO亚型ABw07一家系的分子生物学研究

目的 研究一个ABO亚型ABw07家系的分子机制.方法 用单克隆抗体检测先证者红细胞ABO血型抗原.标准A、B、O红细胞检测先证者血清中的ABO抗体,采用聚合酶链反应技术扩增先证者ABO基因的第6和7外显子序列,PCR产物经酶切后直接测序分析.同时PCR产物经TOPO TA克隆到质粒载体中获得单链,对所得克隆进行ABO基因双向测序分析.家系调查采集先证者父母和姐姐的标本进行血型血清学实验和ABO基因第6和7外显子直接测序分析.结果 先证者红细胞有A、B抗原,同时血清中存在抗B抗体.直接测序分析发现第261位无缺失,第297位A/G、467C/T、526C/G、657C/T、703G/A、796C/A、803G/C、930G/A、1055C/A、1096A/G杂合,可推断为A102Bw07基因型.克隆测序得到两个等位基因A102和Bw07.与B101相比,Bw07第1055位G→A,导致1个氨基酸改变:第352位氨基酸精氨酸变成谷氨酰胺.家系调查显示先证者Bw07基因从母亲遗传所得,母亲血液标本ABO血型血清学特性和测序分析结果与先证者完全一致.结论 α-1,3-半乳糖基转移酶基因(B基因)第1055位G→A突变导致产生Bw07表型,其血清中可含有抗B抗体.     

一例ABO血型重组等位基因的分子特性

目的分析一例ABO血型重组等位基因的分子特性。方法ABO表型鉴定采用试管法。ABO基因和FUT1基因编码区序列检测采用PCR测序法。利用等位基因特异性引物扩增测序技术鉴别先证者ABO等位基因。先证者及其母亲ABO基因全长序列测定采用二代测序方法。结果先证者红细胞与抗H不凝集,FUT1基因为c.551_552del AG纯合,判定先证者为类孟买型。先证者ABO基因双链测序结果为c.261G/del、467C>T、c.526C>G、c.657C>T、c.703G>A、c.796C>A、c.803G>C、c.930G>A杂合。单链测序结果显示先证者有一个ABO*A1.02等位基因,另一个为ABO*O.01.01和ABO*B.01重组形成的等位基因。二代测序数据显示可能重组的位置在核苷酸c.375-269到c.526之间,家系分析显示先证者重组等位基因遗传自母亲。结论ABO血型等位基因存在重组现象。发现了1例ABO*O.01.01和ABO*B.01重组形成的新等位基因。     

一例ABO亚型ABx09的分子生物学研究和鉴定

目的 研究1例ABO亚型ABx09的分子机制.方法 应用单克隆抗体检测先证者红细胞ABO血型抗原,标准A、B、O红细胞检测先证者血清中的ABO抗体,采用聚合酶链反应(polymerase chain reaction,PCR)技术分别扩增先证者ABO基因的第1～7外显子序列,PCR产物经酶切后直接测序分析.第5～7外显子扩增产物经TOPO TA克隆到质粒载体中获得单链,对所得克隆进行ABO基因第5～7外显子双向测序分析.家系调查采集先证者父母的标本进行血型血清学实验和ABO基因第6和7外显子直接测序分析.结果 先证者红细胞有A、B抗原,同时血清中存在抗B抗体.直接测序分析发现第6外显子第261位无缺失、第297位AG,第7外显子467CT、526CG、657CT、703GA、796CA、803GC、889GA、930GA杂合,可指定为A102Bx09基因型.克隆测序得到两个等位基因A102和Bx09.与B101序列相比,Bx09第889位G→A,导致第297位谷氨酸变成赖氨酸.家系调查显示先证者Bx09等位基因从母亲遗传所得.结论 α-1,3-半乳糖基转移酶基因第889位G→A突变导致产生Bx09表型,其血清中可含有抗B抗体.     

一例ABO疑难血型基因分析

目的对一例血型血清学鉴定困难的患者进行相关基因分析,鉴定血型并分析引起血型鉴定困难的原因。方法采用微柱凝胶法和试管法进行血清学血型鉴定、抗球蛋白试验和抗体筛查,PCR扩增患者ABO基因第6、7外显子区及其侧翼序列并进行Sanger测序、克隆测序,寻找突变位点进行血型鉴定。结果患者血清学血型鉴定正反结果不符,其红细胞与单克隆抗A、B、H抗体反应均呈阴性(-);血清与A、B、O型红细胞均无凝集;患者直接及间接抗球蛋白试验均为阴性;血清抗体筛查为阴性。测序发现,患者ABO血型相关基因存在多个变异位点。其中第6外显子区存在c.261delG,第7外显子区存在c.467CCT、c.526CCG、c.657CT、c.703GGA、c.796CCA、c.803GG。以A101基因作为标准序列进行比较,对照Blood Group Antigen Gene Mutation Database进行ABO等位基因突变分析,判断患者血型为A102/B101。结论患者红细胞上抗原减弱是血型鉴定正反不符的主要原因,DNA基因测序第6、7外显子七个点突变导致了红细胞上抗原减弱。     

一例ABO血型系统B抗原减弱表达的分子机制

目的 研究1例B抗原减弱献血者的血清学特性和抗原减弱的分子机制.方法 应用单克隆抗体检测献血者红细胞ABO血型抗原,标准A、B、O红细胞检测其血清中的ABO抗体,并检测其血清转移酶活性.采用聚合酶链反应技术扩增ABO基因全部外显子序列和5′端非编码区序列并进行测序分析,应用逆转录-PCR和克隆测序技术分析献血者ABO cDNA转录剪接情况,采用重亚硫酸盐转化直接测序法分析ABO基因启动子区CpG岛甲基化水平.结果 献血者血清学表现为B抗原明显减弱,血清中无抗-B抗体,B转移酶活性降低.基因型为B101/O01,全部外显子序列和拼接接受位点碱基无任何突变.5′端非编码区序列多态性符合B101/O01的特征,启动子、增强子、负调控序列和微卫星重复序列未发现异常.献血者存在ABO基因全长cDNA转录本,未发现新的转录剪接体.与正常对照标本比较,在启动子区CpG岛内发现启动子附近有多个特异性半甲基化位点.结论 启动子区CpG岛中的甲基化位点可能是引起B抗原弱表达的原因.     

α1，3半乳糖基转移酶基因721C〉T突变导致Bw亚型

目的研究红细胞ABO血型系统Bw亚型的分子基础. 方法通过标准血型血清学方法明确鉴定2个家庭3例Bw亚型,PCR扩增Bw亚型ABO糖基转移酶基因的增强子、启动子和第1～7外显子及侧翼内含子序列,PCR产物经割胶纯化后直接测序.同时将第6和7外显子克隆到pcDNA3.1(-)质粒,转化DH5α后进行序列分析.采用序列特异性引物-聚合酶链反应方法证实测序所发现的突变.结果直接测序发现3例Bw亚型的基因型为B/O杂合,其中糖基转移酶基因的第261位G杂合缺失,第721位C/T杂合.克隆证实一条染色体上为正常的O等位基因,另一条染色体上B等位基因(α1,3半乳糖基转移酶基因)存在第721位C>T突变,导致多肽链Arg241Trp替换.序列特异性引物-聚合酶链反应检测140份随机样本未发现此突变. 结论α1,3半乳糖基转移酶基因第7外显子721C>T突变可能是Bw亚型分子遗传基础之一.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.