自动工作站提取基因组DNA方法的建立及其在HLA基因分型中的应用

目的研究和建立从大量全血标本中提取基因组DNA的有效方法,以应用于Luminex HLA流式磁珠基因分型。方法使用自动工作站(瑞士TECAN公司)从600μl全血中提取基因组DNA,提取的DNA样本用紫外分光光度仪测定其浓度和纯度,DNA的完整性用琼脂糖凝胶电泳检测,并统计分析每一DNA样本流式磁珠HLA-A,B和DRB1基因扩增产物经探针分子杂交后的荧光信号强度。结果从600μl全血中提取基因组DNA,含量平均为10.052±0.824μg,样本的A260nm/A280nm值平均为1.821±0.201。琼脂糖电泳法测得DNA的分子量约为21kb,HLA-A,B和DRB1位点PCR产物杂交后的平均荧光信号强度分别为2 183.84±478.12(exon2),2 168.28±338.59(exon3);2 057.27±397.41(exon2),1959.78±283.24(exon3);3 643.38±327.85。结论此方法适用于从大量全血样本中快速提取基因组DNA,所得基因组DNA适用于高通量HLA流式磁珠基因分型等下游的分子生物学实验。     

孕妇外周血胎儿ABO血型基因分型技术的建立

本研究目的是建立孕妇外周血胎儿ABO血型基因分型技术,用于ABO血型不合引起的新生儿溶血病的产前诊断。根据ABO血型基因DNA全序列和mRNA序列设计4对引物,选择20例健康供者血浆,提取血浆中DNA进行扩增,探索最佳的血浆DNA提取及PCR扩增条件,初步建立单人ABO血型基因分型技术。将O型血浆DNA与A型或B型血浆DNA按1：1、2：1、4：1、8：1、10：1、20：1、40：1、100：1进行混合,模拟孕妇外周血胎儿与孕妇自身ABO基因混合状态,建立混合ABO血型基因分型技术。选取14例孕30周以上的孕妇外周血标本,进行胎儿ABO血型基因型鉴定,并对孕妇进行追踪,尽量获取胎儿出生以后的外周血标本进行ABO血型鉴定,以评价孕妇外周血胎儿ABO血型基因分型技术的灵敏度与准确性。结果表明：单人血浆进行准确血型鉴定的最少模板DNA量约为0．625ng,500μl血浆提取的DNA量即可达到PCR扩增要求;当混合血浆中O型DNA所占比例≤10时,可以准确检测出非0基因的存在;14名O型孕妇外周血标本中9例标本扩增出非O型基因,5例未扩增出非0基因;通过血清学方法对5例胎儿出生后外周血进行ABO血型鉴定,其中A型3例,B型1例,O型1例,与其基因分型结果一致,符合率100％。结论：本研究建立的孕妇外周血胎儿ABO血型基因提取、分型技术,可以对妊娠中晚期胎儿ABO血型基因型进行准确鉴定,从而为新生儿溶血病的产前诊断与预防提供指导意见。     

Development of a Genomic DNA Reference Material Panel for Rett Syndrome (MECP2-Related Disorders) Genetic Testing

Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention’s Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.Rett syndrome is a dominant X-linked disorder usually caused by point mutations (approximately 80% in classical and 40% in atypical cases) and deletions or insertions (approximately 15% to 18% in classical and 3% in atypical cases) in the MECP2 gene, although patients with mutations in two other genes, CDKL5 and FOXG1, may also exhibit a Rett-like phenotype.^1,2 MECP2, located on Xq28 and comprising four exons, encodes methyl-CpG binding protein 2 (MeCP2). In males, Rett syndrome is usually lethal, because of abnormal MeCP2 function from the single X chromosome and severe neonatal encephalopathy, although Rett syndrome in males with an XXY karyotype has been reported.³ Duplications in MECP2 have also been observed, and these can cause severe neurodevelopmental disability in males.⁴ The prevalence of Rett syndrome in females is approximately 1:10,000.⁵Girls with classic Rett syndrome (OMIM #312750) exhibit a rapid decline in language and motor skills at approximately 1 year of age. These patients exhibit a loss of acquired purposeful hand use, loss of communication, gait ataxia and apraxia, and stereotypic hand movements. They may also exhibit additional symptoms, including bruxism, seizures, episodic apnea or hyperpnea, abnormal muscle tone, and often acquired microcephaly.^4,6,7Patients with Rett syndrome can present with various phenotypes. Those diagnosed with atypical Rett syndrome may have either more mild or more severe presentation than patients with the classical form. These patients share some of the same symptoms with classical Rett syndrome cases, but must also have some of the additional symptoms listed above.^4,6 Some patients present with only mild learning disabilities (females) or intellectual disability (males).^4,8Molecular diagnosis of Rett syndrome is performed by examination of the patient’s DNA for MECP2 mutations, using a variety of molecular diagnostic methods. Most MECP2 mutations are sporadic, and testing is performed on the proband, although predictive prenatal and preimplantation genetic testing may also be performed. DNA sequence analysis may detect point mutations and small insertions and deletions (indels). Larger deletions and duplications are detected using multiplex ligation-dependent probe amplification (MLPA), quantitative PCR, and array-based comparative genomic hybridization (CGH). To date, there are no US Food and Drug Administration (FDA)–approved assays for Rett syndrome. All testing is performed using laboratory-developed tests.Reference materials are needed by laboratories to comply with regulatory and accreditation requirements for assay development, assay validation, and quality control, and their use is recommended by professional guidelines for clinical laboratories [eg, http://www.acmg.net/Pages/ACMG_Activities/stds-2002/g.htm, last accessed October 16, 2013; Washington State Legislature, http://www.doh.wa.gov/hsqa/fsl/lqa_home.htm, last accessed January 11, 2013; College of American Pathologists http://www.cap.org/apps/cap.portal, last accessed January 11, 2013 (registration required); New York State Clinical Laboratory Evaluation Program, http://www.wadsworth.org/clep, last accessed January 11, 2013).^9–15 Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process. Ideally, these reference materials should closely resemble patient samples containing variants and types of variants common to the disorder and should be thoroughly characterized using methods different from those used in the user’s laboratory.¹⁵ For Rett syndrome, genomic DNA reference materials derived from females (and, if possible, males) containing common point mutations, indels, and larger deletions and duplications should be used. Careful use of a well-characterized and comprehensive set of reference materials helps to assure the proper design and function of a clinical assay. To date, there are no commercially available reference materials for Rett syndrome genetic testing.To address the need for characterized genomic DNA reference materials for Rett syndrome testing, the Centers for Disease Control and Prevention (CDC) based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the genetic testing community and the National Institute of General Medical Sciences (NIGMS) Repository at the Coriell Cell Repositories, have characterized the MECP2 mutations in 35 publicly available cell lines. Twenty-seven of the 35 cell lines were generated as part of this project, using blood collected with informed consent from Rett syndrome patients with variants not previously represented in cell lines at the Coriell Repository. The availability of a renewable source of characterized reference materials for Rett syndrome helps to assure the accuracy of these genetic tests and facilitate research and test development.     

改良盐酸胍法提取脐血DNA 用于HLA基因分型

为了比较经典的盐酸胍(Gu·HCl)抽提DNA法和改良盐酸胍抽提DNA法在提取脐血DNA的效果,探讨这两种方法在脐血组织相溶性抗原(HLA)基因分型中的应用,使用盐酸胍抽提DNA法和改良盐酸胍抽提DNA法提取72例脐血标本的DNA,并分别测定其DNA的浓度和纯度,采用序列特异性引物聚合酶反应方法(PCRSSP)比较这两种方法所提取的DNA.结果表明两种方法提取脐血DNA均获成功;根据对DNA的质量监测发现,用改良盐酸胍抽提DNA在质量上较盐酸胍法好;用于HLA基因分型时,改良盐酸胍抽提DNA法可减少非特异性条带的出现.结论改良盐酸胍抽提DNA法不但操作简便,成本下降,而且在最少血量中能提取出高纯度的DNA,减少非特异性条带的出现.该法完全可以满足脐血库的日常脐血样品DNA的提取以及脐血HLA基因分型的要求.     

Microarray-Based Comparative Genomic Hybridization Using Sex-Matched Reference DNA Provides Greater Sensitivity for Detection of Sex Chromosome Imbalances than Array-Comparative Genomic Hybridization with Sex-Mismatched Reference DNA

In array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs.Array comparative genomic hybridization (array-CGH) is a high resolution genome analysis technique used to detect DNA copy number alterations, ie, segmental genomic gains and losses. The implementation of array-CGH in clinical diagnosis and research is a fundamental step toward the elucidation of the etiology of congenital malformations frequently associated with genomic disorders,¹ mental retardation,² autism and behavioral abnormalities,³ cancer,⁴ and benign human genome copy number variants.^5,6 Array-CGH compares genomic DNAs isolated from test and reference samples that are differentially labeled with red (Cy5) and green (Cy3) fluorescent dyes and competitively hybridized to known mapped segments of human genomic DNA (eg, bacterial artificial chromosomes/P1-derived artificial [BAC/PAC] or oligonucleotide probes) attached to a slide. The fluorescent signal intensity of the two fluorochromes at each spot on the microarray is proportional to the amount of test and reference DNA samples bound to the DNA sequence at a given genomic position. The presence of chromosomal imbalance can be detected and quantified by calculating the ratio of signal intensities of test DNA versus reference DNA. For statistical purposes, these ratios are usually converted to a log₂(Cy5/Cy3) ratio for interpretation and analysis (see Supplemental Table 1 at http://jmd.amjpathol.org). The log₂(Cy5/Cy3) scaling has the consequence of centering the ratios at approximately zero, making DNA copy number losses appear negative, and copy number gains positive. In general, a patient''s test genome may contain 0, 1, 2, 3, or more copies of a genomic interval, when compared with the reference diploid genome. The ability of array-CGH to detect single copy changes (ie, ratios 1:2, 3:2, 0:1, 2:1) has been previously demonstrated.^7,8 In contrast, distinguishing homozygous deletions, triplications, and other amplifications (ie, ratios 0:2, 4:2, 3:1, 5:2, and so forth), as well as ascertainment of DNA copy numbers in samples with a mixed population of abnormal and normal cells, such as in tumor tissue or a blood specimen with mosaicism, remains a challenge.The use of array-CGH is expanding rapidly as a tool for the identification of genomic copy number abnormalities in patients.^{9,10,11,12,13,14,15} Abnormality rates of 10 to 15% are widely reported for heterogeneous patient populations typically studied by cytogenetic methods in the past.^2,16,17 Sex chromosome aneuploidy, microdeletion, and microduplication abnormalities are relatively common, and are of special interest because of their frequent association with congenital anomalies, mental retardation syndromes, infertility, and lethal conditions. Abnormalities involving the X and Y chromosomes can be quite complex, are frequently present in mosaic form, and are associated with a variable phenotype between males and females, the latter complicated by random X-inactivation. Identification of sex chromosome rearrangements is particularly important for adequate genetic counseling.In array-CGH experiments the measurement of DNA copy number of sex chromosomal regions depends heavily on the sex of the patient and reference DNA used. Although the difference in copy number between X and Y chromosomes in array-CGH studies using sex-mismatched reference DNAs serves as a positive experimental control,¹⁸ the sensitivity of various CGH platforms to detect constitutional sex chromosome imbalances when using sex-mismatched as compared with sex-matched references has not been systematically evaluated. This study evaluates the sensitivity of two array-CGH platforms (BAC/PAC and oligonucleotide microarrays) to detect sex chromosome abnormalities using sex-matched versus sex-mismatched reference DNA samples and demonstrates the importance of experimental design for the interpretation of array-CGH results in clinical diagnosis.     

基因组DNA高通量提取技术及其在骨髓库捐献者样本HLA基因分型中的应用

本研究旨在建立从全血EDTA抗凝样本中高通量提取基因组DNA的有效方法,以常规应用于Luminex rSSO HLA流式磁珠基因分型。使用2ml深孔板和TECAN DNA全自动工作站,从288份骨髓捐献者样本中提取基因组DNA提取的DNA样本用紫外分光光度仪测定其浓度和纯度,并采用琼脂糖凝胶电泳检测DNA的完整性;DNA样本用One lambda rSSO HLA—A、B和DRB1基因分型试剂盒进行PCR扩增、分子杂交和Luminex流式磁珠分析仪检测,统计分析每一DNA样本HLA—A、B和DRB1基因扩增产物经DNA探针分子杂交后的阳性磁珠和阴性磁珠的荧光信号强度。结果表明：从400μl全血中提取了基因组DNA,288份样本的DNA产量平均为3．217±0．715μg,A260／A280值平均为1．710±0．103,A260-A320／A280-A320值平均为1．761±0．151;用琼脂糖凝胶电泳法测得DNA的分子量均大于15kb;每一样本mA—A、-B和-DRB1杂交后的阳性磁珠的荧光信号强度均〉600RFU,而阴性磁珠的荧光信号强度〈50RFU。结论：本方法适用于从大量全血样本中快速提取基因组DNA,所得的基因组DNA适用于高通量中华骨髓捐献者样本的HLA流式磁珠基因分型等下游的移植免疫学与分子生物学实验。     

Development of Genomic DNA Reference Materials for Genetic Testing of Disorders Common in People of Ashkenazi Jewish Descent

Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research.Many ethnic groups have genetic disorders that are over-represented due to founder effects. Examples include cystic fibrosis and α-1-antitrypsin deficiency in European Caucasians,^1,2 and α or β thalassemia in groups living in equatorial regions with endemic malaria (Medical Genetics Information Resource, http://www.genetests.org, last accessed May 4, 2009).³Ashkenazi Jewish (AJ) individuals are the descendents of those belonging to the Hebrew ethnic and religious group that settled in Eastern Europe in the early Middle Ages. Several autosomal recessive disorders are more common in the AJ population than in the general population 4^,5,6 An estimated one in 4.8 AJ individuals is a carrier of one of these diseases,⁸ most of which are severe and cause significant morbidity and mortality. Treatment to reduce symptoms and prolong life is available for some of these disorders, and novel treatments and therapies, including enzyme replacement therapy, have recently become available or are in development.

Table 1

Alleles Included in Clinical Ashkenazi Jewish Testing Panels

Aspects of a Computer-assisted System for Calling Blood Donors from a Specialized Panel

A system is described for locating within a region people who have special blood-group antibodies in their serum, identifying those who may be able to donate blood, and obtaining donations of such blood. This system is oriented to collecting relatively large amounts of blood containing anti-Rh_o(D) to meet present requirements for anti-D immunoglobulin without resorting to artificial immunization, and is extended to include people who have other antibodies which may be required for blood typing. A computer is used to perform much of the intricate clerical work. Certain problems relating to the use of a computer within the context of blood transfusion are identified and discussed, with special reference to systems analysis, donor identification, and blood-group terminology.     

孕妇血浆游离DNA检测在母婴ABO血型不合诊断中的临床研究

目的 探索一种利用孕妇血浆中游离DNA检测胎儿ABO血型用于母婴ABO血型不合的无创性产前诊断方法.方法 用硅胶膜柱提取的方法从50例13～37孕周可疑母婴ABO血型不合的孕妇血浆中提取胎儿DNA,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法检测胎儿ABO血型.结果 50例样本中,检出32例,检出率64%(32/50),出生后血清学证实28例诊断正确,诊断正确率87.5%(28/32).结论 利用孕妇血浆中游离DNA检测胎儿ABO血型可行,对诊断和预防因母婴ABO血型不合所致溶血病的发生具有重要意义.     

从肝藏血论治产后失眠

产后失眠是临床常见的产后病。肝藏血,为睡眠活动提供物质基础。分娩出血、流汗、用劲都可能导致产后肝血亏损。肝血亏虚,神魂失养,则夜寐难安。肝血亏虚为产后失眠的病理基础,因此调补肝血法为治疗产后失眠症的重要治法。