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1. 发明公开

EP1325963A1 Method for non-invasive diagnosis of transplantations and transfusions 有权
标题翻译： Verfahren zur nicht-invasiven诊断von移植和输血
公开(公告)号：EP1325963A1
公开(公告)日：2003-07-09
申请号：EP01811266.4
申请日：2001-12-24
申请人： Holzgreve, Wolfgang, Prof. , Hahn, Sinueh, Dr. , GARRITSEN, Hendrikus, Stephanus, Paulus
发明人： Holzgreve, Wolfgang, Prof. , Hahn, Sinueh, Dr. , GARRITSEN, Hendrikus, Stephanus, Paulus
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6881 , C12Q2600/156
摘要： It is known that rejection of transplant tissue is accompanied by tissue damage of the donor graft. Apart from donor specific proteins and genomic DNA, it is known, that by tissue damage mitochondrial DNA (mtDNA) will be released into the circulation of the recipient. Based on this knowledge it is the object of the present invention to suggest a new method or process to measure the quantity of cell free circulatory mtDNA in bodily fluids as an indicator or marker of tissue damage and to use mtDNA polymorphisms to discriminate between donor and recipient tissue damage in a transplant setting. The method according to the invention is a non-invasive diagnostic procedure characterised by the following steps:

A. Before transplantation
d) Obtaining mtDNA containing material from donor and recipient before transplantation.
e) Isolation of mtDNA.
f) Analysing of the hypervariable regions of the mtDNA for informative polymorphisms which can discriminate between donor and recipient.
B. After Transplantation
c) Obtaining cell free bodily fluids from a patient.
d) Mitochondrial DNA extraction using a commercial procedure. Detection and quantification of donor specific mtDNA in the probe of patient using the donor specific polymorphisms.
摘要翻译：已知移植组织的排斥伴随供体移植物的组织损伤。除了供体特异性蛋白质和基因组DNA之外，已知通过组织损伤线粒体DNA（mtDNA）将被释放到受体的循环中。基于这些知识，本发明的目的是提出一种测量体液中无细胞循环mtDNA的量作为组织损伤指标或标志物的新方法或方法，并使用mtDNA多态性来区分给体和受体组织损伤在移植设置。根据本发明的方法是非侵入性诊断程序，其特征在于以下步骤：A.移植前d）在移植前从供体和受体获得含有mtDNA的物质。 e）mtDNA的分离 f）分析可以区分供体和受体的信息多态性的mtDNA的高变区。 B.移植后c）从患者获得无细胞的体液。 d）使用商业程序进行线粒体DNA提取。使用供体特异性多态性检测和定量患者探针中供体特异性mtDNA。

2. 发明公开

EP1329517A1 Method for the non-invasive prenatal diagnosis of chromosomal and/or genetic abnormalities 审中-公开
标题翻译：方法zur non invasiven prenatalen诊断染色体und / oder遗传学Abnormalitäten
公开(公告)号：EP1329517A1
公开(公告)日：2003-07-23
申请号：EP02405033.8
申请日：2002-01-22
申请人： Holzgreve, Wolfgang, Prof. , Hahn, Sinueh, Dr. , Zimmermann, Bernard
发明人： Holzgreve, Wolfgang, Prof. , Hahn, Sinueh, Dr. , Zimmermann, Bernard
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6883 , C12Q1/6827 , C12Q1/6851 , C12Q1/6858 , C12Q2600/156 , C12Q2600/16 , C12Q2561/113 , C12Q2545/101 , C12Q2535/125
摘要： The detection of gross chromosomal abnormalities is a major focus of invasive prenatal diagnosis testing. The object of the present invention is to suggest a new method for the diagnosis of chromosomal and/or genetic abnormalities that is readily amenable to automation and high-throughput screening. This stated object of the invention is achieved by a method, characterised by the following steps:

obtaining DNA or RNA with the genetic test locus,
obtaining DNA or RNA of a control locus and
detecting of the chromosomal and/or genetically determined abnormalities by Real-Time Polymerase Chain Reaction (RT-PCR) analysis, which detects specific nucleic acid amplification products as they accumulate in real-time.
In particular the method is further characterised by a multiplex RT-PCR assay in which amplification of both loci can been simultaneously monitored in the same reaction vessel, by a fluorescently labeled oligonucleotide probe, which eliminates the need for post-PCR processing. It is a preferred object of the invention to provide the method for the detection of trisomy 21 and in this way sequence specific primers for this specific analysis has been designed.
摘要翻译：染色体总体异常检测是侵入性产前诊断检测的重点。本发明的目的是提出一种用于诊断易于自动化和高通量筛选的染色体和/或遗传异常的新方法。本发明的上述目的通过一种方法实现，其特征在于以下步骤：用遗传测试基因座获得DNA或RNA，获得对照基因座的DNA或RNA，并通过Real-time检测染色体和/或遗传上确定的异常，时间聚合酶链反应（RT-PCR）分析，检测特定的核酸扩增产物，因为它们实时积累。特别地，该方法的特征还在于通过多重RT-PCR测定法，其中可以通过荧光标记的寡核苷酸探针在同一反应容器中同时监测两个位点的扩增，从而不需要PCR后处理。本发明的优选目的是提供用于检测21三体的方法，并且以这种方式设计了该特定分析的序列特异性引物。

3. 发明授权

EP1325963B1 Method for non-invasive diagnosis of transplantations and transfusions 有权
标题翻译：用于移植和输血的非侵入性诊断方法
公开(公告)号：EP1325963B1
公开(公告)日：2006-09-27
申请号：EP01811266.4
申请日：2001-12-24
申请人： Holzgreve, Wolfgang, Prof. , Hahn, Sinuhe, Dr. , Garritsen, Hendrikus Stephanus Paulus
发明人： Holzgreve, Wolfgang, Prof. , Hahn, Sinuhe, Dr. , Garritsen, Hendrikus Stephanus Paulus
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6881 , C12Q2600/156

4. 发明公开

EP1524321A1 Non-invasive detection of fetal genetic traits 有权转让
标题翻译： Nicht invasiver Nachweisfötalergenetischer Merkmale
公开(公告)号：EP1524321A1
公开(公告)日：2005-04-20
申请号：EP03405742.2
申请日：2003-10-16
申请人： Hahn, Sinuhe, Dr. , Zimmermann, Bernhard , Holzgreve, Wolfgang, Prof. , Li, Ying
发明人： Hahn, Sinuhe, Dr. , Zimmermann, Bernhard , Holzgreve, Wolfgang, Prof. , Li, Ying
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6883 , C12Q1/6806 , C12Q2600/156 , C12Q2565/125
摘要： Blood plasma of a pregnant woman contains both fetal and maternal circulatory extracellular DNA, the latter forming the major part (generally > 90%) thereof. The majority of the circulatory extracellular fetal DNA has now been found to have a small size of approximately 500 base pairs or less whereas the majority of the circulatory extracellular maternal DNA has a size greater than approximately 500 base pairs. Separation of circulatory extracellular DNA fragments which are smaller than approximately 500 base pairs provides a possibility to enrich for fetal DNA sequences from the vast bulk of circulatory extracellular maternal DNA.
There is thus provided a sample of the blood plasma or serum (which preferably is substantially cell-free) of a pregnant woman in which extracellular DNA comprising 500 base pairs or less is enriched; the manufacture of such sample by subjecting a sample of the blood plasma or serum of a pregnant woman to a size separation so as to enrich extracellular DNA comprising 500 base pairs or less; and the use of such sample for the non-invasive detection of fetal genetic traits.
摘要翻译：孕妇的血浆含有胎儿和母体的循环细胞外DNA，后者形成主要部分（通常> 90％）。现在已经发现大多数循环细胞外胎儿DNA具有约500个碱基对或更小的小尺寸，而大多数循环细胞外母体DNA具有大于约500个碱基对的大小。小于约500碱基对的循环细胞外DNA片段的分离提供了从大量循环细胞外母体DNA中丰富胎儿DNA序列的可能性。因此，提供了富含500碱基对或更少的胞外DNA的孕妇血浆或血清（优选基本上不含细胞）的样品; 通过对怀孕妇女的血浆或血清样品进行大小分离以富集包含500个碱基对或更少的细胞外DNA的方式制备这种样品; 以及使用这种样品进行胎儿遗传性状的非侵入性检测。

5. 发明专利

DE4222573C1 未知
公开(公告)号：DE4222573C1
公开(公告)日：1993-08-12
申请号：DE4222573
申请日：1992-07-09
申请人： AHLERT, DOROTHEE, DR., 4400 MUENSTER, DE , HOLZGREVE, WOLFGANG, PROF. DR., 4400 MUENSTER, DE , GARRITSEN, HENDRIKUS STEPHANUS, DR., 4400 MUENSTER, DE
发明人： AHLERT, DOROTHEE, DR., 4400 MUENSTER, DE , HOLZGREVE, WOLFGANG, PROF. DR., 4400 MUENSTER, DE , GARRITSEN, HENDRIKUS STEPHANUS, DR., 4400 MUENSTER, DE
IPC分类号： A61K39/395 , B03C1/00 , C12Q1/68 , G01N33/50 , G01N33/53 , G01N33/543 , G01N33/577 , G01N33/80
摘要： It has been possible to demonstrate clearly both in umbilical cord blood samples and after the separation of pregnancy blood that the combination of the triple gradient and MACS with anti-CD71 marked cells is a very effective method of enriching nucleated erythrocytes. The proportion of nucleated erythrocytes in the umbilical cord blood was between 72 and 89 % after both enrichment methods. It has been possible to detect nucleated erythrocytes in the posivite fraction according to the triple gradient and MACS in all cases in pregnant women at various gestation stages. The variation in the number of enriched nucleated erythrocytes in various pregnancies very probably reflects individual differences in the foeto-maternal cell ratio at various stages in pregnancy. The process described here makes it possible in normal male blood and that of non-pregnant women to demonstrate no nucleated erythrocytes, by contrast with pregnant women at various stages of gestation. The method is also highly reproducible and suitable for clinical diagnosis. With fluorescence in situ hybridisation it has been possible to detect a foetal trisomia in all three cases investigated. The enrichment of nucleated erythrocytes is thus strong enough to diagnose infantile aneuploids using fluorescence in situ hybridisation. It is therefore obvious that it is possible with this only slightly invasive and relatively simple and economical method to conduct screening examinations for three of the most important trisomias (13, 18 and 21) and monogenic diseases (with the aid of the PCR) with virtually no risk to the patient and the child.

6. 发明专利

ES2274861T3 METODO PARA EL DIAGNOSTICO NO-INVASIVO DE TRANSPLANTES Y TRANSFUSIONES. 未知
公开(公告)号：ES2274861T3
公开(公告)日：2007-06-01
申请号：ES01811266
申请日：2001-12-24
申请人： HOLZGREVE WOLFGANG PROF , HAHN SINUHE DR , GARRITSEN HENDRIKUS STEPHANUS
发明人： HOLZGREVE WOLFGANG PROF , HAHN SINUHE DR , GARRITSEN HENDRIKUS STEPHANUS
IPC分类号： C12Q1/68 , C12Q1/6881
摘要： Método para el diagnóstico no-invasivo de trasplantes y transfusiones caracterizado por a) extracción de ADN mitocondrial (ADNmt) no celular circulante de una muestra de fluidos corporales no celulares procedente del receptor del trasplante o transfusión; b) detección, análisis y cuantificación del ADNmt de la muestra; c) comparación del ADNmt de la muestra con el ADNmt que se obtiene del donante y del receptor antes del trasplante o transfusión; d) utilización de polimorfismos de ADNmt en regiones hipervariables para distinguir entre el daño del tejido del donante y el daño del tejido del receptor en el contexto del trasplante o para determinar la supervivencia de componentes de células sanguíneas en una transfusión.

7. 发明专利

AT340871T 未知
公开(公告)号：AT340871T
公开(公告)日：2006-10-15
申请号：AT01811266
申请日：2001-12-24
申请人： HOLZGREVE WOLFGANG PROF , HAHN SINUHE DR , GARRITSEN HENDRIKUS STEPHANUS
发明人： HOLZGREVE WOLFGANG PROF , HAHN SINUHE DR , GARRITSEN HENDRIKUS STEPHANUS
IPC分类号： C12Q1/68 , C12Q1/6881

8. 发明专利

DE59308101D1 未知
公开(公告)号：DE59308101D1
公开(公告)日：1998-03-12
申请号：DE59308101
申请日：1993-05-11
申请人： AHLERT DOROTHEE DR , HOLZGREVE WOLFGANG PROF DR , GARRITSEN HENDRIKUS STEPHANUS
发明人： AHLERT DOROTHEE DR , HOLZGREVE WOLFGANG PROF DR , GARRITSEN HENDRIKUS STEPHANUS
IPC分类号： A61K39/395 , B03C1/00 , C12Q1/68 , G01N33/50 , G01N33/53 , G01N33/543 , G01N33/577 , G01N33/80

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