Katherine W. Klinger

PCR Quantitation of Fetal Cells in Maternal Blood in Normal and Aneuploid Pregnancies

Fetal cells in maternal blood are a noninvasive source of fetal genetic material for prenatal diagnosis. We determined the number of fetal-cell DNA equivalents present in maternal whole-blood samples to deduce whether this number is affected by fetal karyotype. Peripheral blood samples were obtained from 199 women carrying chromosomally normal fetuses and from 31 women with male aneuploid fetuses. Male fetal-cell DNA-equivalent quantitation was determined by PCR amplification of a Y chromosome-specific sequence and was compared with PCR product amplified from known concentrations of male DNA run simultaneously. The mean number of male fetal-cell DNA equivalents detected in 16-ml blood samples from 90 women bearing a 46,XY fetus was 19 (range 0-91). The mean number of male fetal-cell DNA equivalents detected in 109 women bearing a 46,XX fetus was 2 (range 0-24). The mean number of male fetal-cell DNA equivalents detected when the fetus was male compared with when the fetus was female was highly significant (P = .0001). More fetal cells were detected in maternal blood when the fetus was aneuploid. The mean number of male fetal-cell DNA equivalents detected when the fetal karyotype was 47,XY,+21 was 110 (range 0.1-650), which was significantly higher than the number of male fetal-cell DNA equivalents detected in 46,XY fetuses (P = .0001). Feto-maternal transfusion of nucleated cells appears to be influenced by fetal karyotype. The sixfold elevation of fetal cells observed in maternal blood when the fetus had trisomy 21 indicates that noninvasive cytogenetic diagnosis of trisomy 21 should be feasible.

Laboratory standards and guidelines for population- based cystic fibrosis carrier screening

In 1997, the National Institutes of Health convened a Consensus Development Conference on Cystic Fibrosis (CF).1 The Consensus Conference recommended that genetic screening for CF mutations should be offered to identify carriers among adults with a positive family history of CF, partners of individuals with CF, couples currently planning a pregnancy, and couples seeking prenatal care. A second NIH-sponsored conference that focused on the implementation of the Consensus Conference recommendations was held in 1998.2 Shortly thereafter, the American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG), in conjunction with the National Human Genome Research Institute, formed a Steering Committee to coordinate the implementation of population-based CF carrier screening and to develop “Clinical and Laboratory Provider Guidelines” for (1) provider education; (2) laboratory testing, interpretation, and genetic counseling; and (3) patient education and informed consent. The ACMG charged the Accreditation of Genetic Services Committee, chaired by Dr. Robert Desnick, to establish a Subcommittee on Cystic Fibrosis Carrier Screening (henceforth the “Committee”) to develop recommendations and guidelines for optimal laboratory testing, interpretation, and counseling. The Subcommittee, cochaired by Drs. Wayne Grody and Garry Cutting, met twice yearly since October 1998. The issues considered by the Committee included (1) the target population to be screened (universal vs. limited to certain high-risk ethnic groups); (2) the screening model to be used (couple-based vs. sequential); (3) criteria for and selection of the standard mutation testing panel; (4) potential value and use of an extended testing panel with additional mutations; (5) whether to test for mutations and variants associated with mild or nonclassical phenotypes (such as congenital bilateral absence of the vas deferens); (6) test interpretation, reporting, and genetic counseling; and (7) laboratory quality assurance. The recommendations detailed here have been incorporated into a joint ACMG/ACOG/NIH Steering Committee document entitled “Preconceptual and Prenatal Carrier Screening for Cystic Fibrosis” which will be widely distributed. This document also will include guidelines for providers, patient education, and informed consent. Patient education materials will include two pamphlets, entitled “Cystic Fibrosis Carrier Testing. . . The Decision is Yours” and “Cystic Fibrosis Testing: What Happens if Both My Partner and I are Carriers?” It is important to note that these guidelines were prepared for population CF carrier screening and that different testing and counseling strategies would be employed for the identification of the mutation(s) in patients diagnosed with CF or in relatives of CF patients. Such diagnostic and prenatal mutation analyses should be referred to a genetics center for appropriate testing and counseling.

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry

A long-sought goal of medical genetics has been development of prenatal diagnostic procedures that do not endanger the conceptus. Reliable and universal screening for cytogenetic disorders would require analysis of fetal cells isolated from the maternal circulation. This would be applicable to all pregnant women, irrespective of their ages or histories. In the current study fetal nucleated erythrocytes were flow sorted on the basis of four parameters: cell size, cell granularity, transferrin receptor, and glycophorin-A cell surface molecule. By polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific deoxyribonucleic acid sequences, male fetuses were correctly identified among flow-sorted samples in 12 of 12 (100%) pregnancies; female fetuses were correctly identified in 5 of 6 (83%) pregnancies. We also achieved the prenatal diagnosis of fetal aneuploidies by use of flow-sorted nucleated fetal erythrocytes and in situ hybridization with chromosome-specific deoxyribonucleic acid probes: one case of trisomy 21 that was detected in maternal blood taken 1 week after chorionic villus sampling and one case of trisomy 18 that was detected in maternal blood taken immediately before chorionic villus sampling. Although our results are promising, additional data on the background sensitivity and specificity of in situ hybridization in flow-sorted fetal cells will be necessary to minimize subjective interpretation and permit clinical application.

Long-lasting arrest of murine polycystic kidney disease with CDK inhibitor roscovitine

Polycystic kidney diseases (PKDs) are primarily characterized by the growth of fluid-filled cysts in renal tubules leading to end-stage renal disease. Mutations in the PKD1 or PKD2 genes lead to autosomal dominant PKD (ADPKD), a slowly developing adult form. Autosomal recessive polycystic kidney disease results from mutations in the PKHD1 gene, affects newborn infants and progresses very rapidly. No effective treatment is currently available for PKD. A previously unrecognized site of subcellular localization was recently discovered for all proteins known to be disrupted in PKD: primary cilia. Because ciliary functions seem to be involved in cell cycle regulation, disruption of proteins associated with cilia or centrioles may directly affect the cell cycle and proliferation, resulting in cystic disease. We therefore reasoned that the dysregulated cell cycle may be the most proximal cause of cystogenesis, and that intervention targeted at this point could provide significant therapeutic benefit for PKD. Here we show that treatment with the cyclin-dependent kinase (CDK) inhibitor (R)-roscovitine does indeed yield effective arrest of cystic disease in jck and cpk mouse models of PKD. Continuous daily administration of the drug is not required to achieve efficacy; pulse treatment provides a robust, long-lasting effect, indicating potential clinical benefits for a lifelong therapy. Molecular studies of the mechanism of action reveal effective cell-cycle arrest, transcriptional inhibition and attenuation of apoptosis. We found that roscovitine is active against cysts originating from different parts of the nephron, a desirable feature for the treatment of ADPKD, in which cysts form in multiple nephron segments. Our results indicate that inhibition of CDK is a new and effective approach to the treatment of PKD.

Alterations in vascular gene expression in invasive breast carcinoma.

The molecular signature that defines tumor microvasculature will likely provide clues as to how vascular-dependent tumor proliferation is regulated. Using purified endothelial cells, we generated a database of gene expression changes accompanying vascular proliferation in invasive breast cancer. In contrast to normal mammary vasculature, invasive breast cancer vasculature expresses extracellular matrix and surface proteins characteristic of proliferating and migrating endothelial cells. We define and validate the up-regulated expression of VE-cadherin and osteonectin in breast tumor vasculature. In contrast to other tumor types, invasive breast cancer vasculature induced a high expression level of specific transcription factors, including SNAIL1 and HEYL, that may drive gene expression changes necessary for breast tumor neovascularization. We demonstrate the expression of HEYL in tumor endothelial cells and additionally establish the ability of HEYL to both induce proliferation and attenuate programmed cell death of primary endothelial cells in vitro. We also establish that an additional intracellular protein and previously defined metastasis-associated gene, PRL3, appears to be expressed predominately in the vasculature of invasive breast cancers and is able to enhance the migration of endothelial cells in vitro. Together, our results provide unique insights into vascular regulation in breast tumors and suggest specific roles for genes in driving tumor angiogenesis.

Vascular Gene Expression in Nonneoplastic and Malignant Brain

Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression.

Detection of fetal cells with 47,XY,+21 karyotype in maternal peripheral blood

Fetal cells were isolated from the peripheral blood of a pregnant woman at 19 weeks of gestation whose fetus had Down syndrome. An amniocentesis had been performed 2 weeks earlier because of abnormalities detected on an antenatal sonogram. Fetal cells were separated by fluorescence-activated cell sorting using monoclonal antibody to the transferrin receptor (TfR). Fluorescence in situ hybridization studies with probes for chromosomes Y and 21 revealed a small number of 47,XY,+21 cells in the TfR- sorted fraction. Although preliminary, the results of this study suggest the possibility that one day, fetal chromosome aneuploidy will be routinely diagnosed from maternal venous blood samples.

Maternal origin of nucleated erythrocytes in peripheral venous blood of pregnant women

We studied the origin of nucleated red blood cells (NRBC) in peripheral venous blood samples from 40 pregnant women carrying a male fetus, using a technique that allows direct chromosomal analysis by in situ hybridisation on immunologically and morphologically classified cells. Samples from ten nulligravid women were studied as controls. NRBC were enriched by negative magnetic activated cell sorting (miniMACS) using anti-CD45 monoclonal antibody. NRBC were detected by alkaline phosphatase anti-alkaline phosphatase immunostaining using a monoclonal anti-glycophorin A antibody. The origin of the NRBC was determined by fluorescence in situ hybridisation using X and Y specific probes. NRBC were found in 37 of the 40 pregnant women at a range of 1 to 230 per 20 ml of venous blood and in 6 of the 10 controls at a range of 1 to 3 per 20 ml of venous blood. All NRBC detected in the pregnant women were evidently of maternal origin, and in the pregnant women the number of NRBC was significantly higher (P < 0.05) than in the controls. Pregnancy per se seems to induce the appearance of maternal NRBC in the circulation, and it cannot therefore be assumed that NRBC isolated from the maternal blood are of fetal origin on the basis of morphology alone. Discrimination of fetal NRBC must occur for prenatal diagnosis of fetal genetic disorders.

Mutation Detection of PKD1 Identifies a Novel Mutation Common to Three Families with Aneurysms and/or Very-Early-Onset Disease

It is known that several of the most severe complications of autosomal-dominant polycystic kidney disease, such as intracranial aneurysms, cluster in families. There have been no studies reported to date, however, that have attempted to correlate severely affected pedigrees with a particular genotype. Until recently, in fact, mutation detection for most of the PKD1 gene was virtually impossible because of the presence of several highly homologous loci also located on chromosome 16. In this report we describe a cluster of 4 bp in exon 15 that are unique to PKD1. Forward and reverse PKD1-specific primers were designed in this location to amplify regions of the gene from exons 11-21 by use of long-range PCR. The two templates described were used to analyze 35 pedigrees selected for study because they included individuals with either intracranial aneurysms and/or very-early-onset disease. We identified eight novel truncating mutations, two missense mutations not found in a panel of controls, and several informative polymorphisms. Many of the polymorphisms were also present in the homologous loci, supporting the idea that they may serve as a reservoir for genetic variability in the PKD1 gene. Surprisingly, we found that three independently ascertained pedigrees had an identical 2-bp deletion in exon 15. This raises the possibility that particular genotypes may be associated with more-severe disease.