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Detection of Nucleated Erythrocytes (NRBC) In Chorionic Villus Sample Supernatant Fluid Using an anti-e Monoclonal Antibody

Sir—J Lee Nelson and colleagues’ report (Feb 21, p 559) presents evidence from PCR experiments that some women with scleroderma who have given birth to sons have male cells in their peripheral blood. Possibly these invaders cause scleroderma, and getting rid of them would cure patients with scleroderma. Their hypothesis that autoimmune diseases are characterised by, indeed caused by, cell lines acquired during pregnancy—either mother-to-child or child-to-mother—is not original. Nor is the finding of male-cell determinants in the peripheral blood of patients with scleroderma by PCR. We have presented the hypothesis and PCR findings. In 1972, I was asked to see a 13-year-old boy with systemic lupus erythematosus and nephritis. Because of Rh erythroblastosis fetalis he had received an exchange transfusion at birth. Had the donor’s cell line persisted and caused his lupus by a graft-versushost reaction? I located the donor and found that our patient’s serum contained antibodies specific for a donor HLA class I antigen. This finding suggested that 13 years after his exchange transfusion the donor’s cell line had persisted and was responsible for his disease. But what of other patients with lupus who did not get an exchange transfusion? Perhaps the mini-exchange from mother or child during pregnancy or delivery had left in them a permanent cell line that produced graft-versus-host disease—ie, lupus. A second such patient was a 25-yearold woman seen in 1988 because she had developed scleroderma and hypertension during the seventh month of pregnancy. Had her child’s cells caused her scleroderma? 3 years after her son was born, we prepared DNA from her peripheral blood and analysed it for Y chromosome elements by PCR. They were found. Apparently, the patient with scleroderma harboured a male cell line from her son. These findings led me to the thought that transplacental cells may be generally responsible for autoimmune 3 Kidson IG, Abbott WM. Low compliance and arterial graft occlusion. Circulation 1978; 58 (suppl 1): I1–I4. 4 Abbott WM, Megerman J, Hasson JE, L’Italien G, Warnock DF. Effect of compliance mismatch on vascular graft patency. J Vasc Surg 1987; 5: 376–82. 5 Lehmann ED, Riley WA, Clarkson P, Gosling RG. Non-invasive assessment of cardiovascular disease in diabetes mellitus. Lancet 1997; 350 (suppl I): 14–19. diseases: both multisystem and organspecific diseases. In addition, the route of cell passage (mother-to-child or child-to-mother) may affect disease expression. It is possible that patients would be cured by getting rid of the foreign cells. If so, it would be a tribute to Ray Owen, who in 1945 set the stage by reporting that cattle twins, known to share blood vessels, share blood groups with their twins and are thus chimeras. The field is fertile. Everyone has a mother. The quantitative PCR test for chimerism used by Nelson et al requires comment/question about the technique. “Results are expressed as the number of male DNA equivalents in 16 mL whole peripheral blood.” In this technique, the purified DNA from 16 mL whole blood is taken up in 2 mL of buffer and 1/200th (10 L) is used in the PCR test. A positive test for a Y amplicon requires at least one Y chromosome as template. Therefore a positive result indicates that at least 200 male cells were present in the whole (16 mL) sample. Yet Nelson and colleagues report that no patient was found to have more than 61 “male cells equivalents” in 16 mL whole blood. Are detected sequences all on the Y chromosome?

Smith-Lemli-Opitz syndrome (SLOS) is caused by an inborn error of cholesterol metabolism that results in deficiency of cholesterol and accumulation of the cholesterol precursor, 7-dehydrocholesterol (DHC) and its isomer, 8-DHC. Affected patients present with dysmorphic facial features, congenital anomalies, and growth and mental retardation. Postnatal treatment with cholesterol supplementation has been shown to improve plasma sterol levels in most patients. The most significant clinical improvement is seen in patients who began treatment at the youngest ages.

a National Institute of Child Health and Human Development, Bethesda, Md.; b Department of Obstetrics and Gynecology, University of Illinois at Chicago, Ill.; c Departments of Obstetrics and Gynecology and Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex.; d Departments of Pediatrics and Obstetrics and Gynecology, New England Medical Center, Boston, Mass.; e Division of Medical Genetics, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Penn.; f Department of Obstetrics and Gynecology, Wayne State University, Detroit, Mich., USA; g Universitats-Frauenklinik, University of Basel, Switzerland; h Genzyne Genetics, Framingham, Mass., USA Received: November 4, 1998 Accepted: November 4, 1998

Improved Specificity of Nrbc Detection in Cvs Supernatant Fluids Using An Anti-ζ Monoclonal Antibody 70

Neonatal leukocytosis, and more specifically, neutrophilia, are associated with vaginal delivery as opposed to cesarean section. We evaluated the role of labor duration in the expression of neonatal leukocyte cell surface antigens associated with the immune response.

FETAL CELL QUANTITATION IN MATERNAL BLOOD SAMPLES FROM NORMAL AND ANEUPLOID PREGNANCIES. • 838

The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activitated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.

Current monitoring of fetal development includes fetal ultrasonography, chorionic villus sampling or amniocentesis for chromosome analysis, and maternal serum biochemical screening for analytes associated with aneuploidy and open neural tube defects. Over the last 15years, significant advances in noninvasive prenatal diagnosis (NIPD) via cell-free fetal (cff) nucleic acids in maternal plasma have resulted in the ability to determine fetal sex, RhD genotype, and aneuploidy. Cff nucleic acids in the maternal circulation originate primarily from the placenta. This contrasts with cff nucleic acids in amniotic fluid, which derive from the fetus, and are present in significantly higher concentrations than in maternal blood. The fetal origin of cff nucleic acids in the amniotic fluid permits the acquisition of real-time information about fetal development and gene expression. This review seeks to provide a comprehensive summary of the molecular analysis of cff nucleic acids in maternal biofluids to elucidate mechanisms of fetal development, physiology, and pathology. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

1. Kan YW, Dozy AM. Antenatal diagnosis of sickle-cell anemia by DNA analysis of amniotic-fluid cells. Lancet 1978;2:910-912. 2. Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase Int J Mol Biol 1977;1 !3:237-251. 3. Goossens MG, Kan YW. DNA analysis in the diagnosis of hemoglobin disorders. Methods Enzymol 1981 ;76:805-817. 4. Gray G R, Towell ME, Wright V J, Hardwick DF. Thalasse- mic hydrops fetalis in two Chinese-Canadian families. Can Med Assoc J 1972;107:1186-1190. 5. Weatherall D J, Clegg JB, Boon WH. The hemoglobin consti- tution of infants with haemoglobin Bart's hydrops foetalis syndrome. Br J Haematol 1970;18:357-367. 6. Horton BF, ThomPson RB, Dozy AM, et al. Inhomogeneity of hemoglobin. VI. The minor hemoglobin components of cord blood. Blood 1962;20(3):302-313. 7. Tuchinda S, Nagai K, Lehmann H. Oxygen dissociation curve of haemoglobin Portland. FEBS Lett 1975;49:390- 391. 8. Thumasathit B, Nondasuta A, Silpisornkosol S, et al. Hydrops fetalis associated with Bart's hemoglobin in northern Thailand. Trop Pediatr 1968;73(1):132-138. 9. Bryan EM, ChaimongkoI B, Harris DA. Alpha-thalassaemic bydrops fetalis. Arch Dis Child 1981;56:476-478.

Fetal cells, potentially usable for prenatal diagnosis, were sorted from maternal blood samples taken as early as 15 weeks of gestation. Immunogenetic and cytogenic criteria established the fetal origin of the observed cells: Y-chromatin-containing (male) cells were detected in the sorted sample if and only if the newborn proved to be male and carried cell-surface antigens detected by the fluorescent-labeled antibody used for sorting with the fluorescence-activated cell sorter.

Summary Examination of metaphase chromosomes of cultured leukocytes from six individuals exposed in some degree to spray adhesives compared to II control subjects showed no significant elevations of aberration frequencies. No consistent increase in abnormal chromosome numbers nor in incidence of true breaks, achromatic gaps, or other structural aberrations was revealed by pooling of data obtained. Incidental to the lack of any effect upon human chromosomes due to use of spray adhesives, the following indicates the absence of any relation to birth anomalies. A dysmorphic chromosoinally abnormal child was born to control parents in the study and a stillborn fetus with no congenital anomalies was spontaneously aborted by a subject whose husband had been exposed to spray adhesives.

Specific constitutional chromosome rearrangements have been described in a small number of individuals with two solid childhood tumors, retinoblastoma and Wilms' tumor. On the basis of these observations, a causal relationship between these chromosome abnormalities and tumorigenesis has been postulated. Though a specific constitutional chromosome abnormality has yet to be reported in association with neuroblastoma, another childhood tumor, we now confirm the involvement of a particular chromosome segment in structural abnormalities in cells from this tumor. Deletions or rearrangements of chromosome 1p were found in preparations from four of six neuroblastomas from individuals with normal constitutional karyotypes and in three of four permanent neuroblastoma cell lines. Structural abnormalities resulting in the loss or rearrangement of material from 1p (with the most frequent break point being 1p32 and with all rearrangements involving the apparent loss or rearrangement of material distal to 1p31, always including 1p34 to 1pter), represent the single most common class of chromosome aberrations in neuroblastoma. This suggests that the distal portion of 1p contains at least one gene involved in the development of neuroblastoma.

Two female infants with apparently identical interstitial deletions at bands p13 to p15 of chromosome 7 are presented. They differ in phenotype. The first infant has failure to thrive, retardation in development, normal head circumference with ridged metopic suture, blepharophimosis, epicanthal folds, mild hypotelorism, small low-set ears, and a bifid right toe. The second infant has a normal weight, length, and head circumference, blepharophimosis, epicanthal folds, widely spaced nipples, enlarged clitoris, and very large hands and feet. The two patients' clinical and karyotypic findings are compared with previous reports of structural abnormalities of the short arm of chromosome 7. Of the three cases in the literature, craniosynostosis was present in the two patients with deletion of band 7p14. Our observations, thus, suggest that deletion of bands 7p13 to 7p15, in contrast to more distal deletions at band 7p2, is not associated with craniosynostosis.

Background Recent increases in genomic studies of the developing human fetus and neonate have led to a need for widespread characterization of the functional roles of genes at different developmental stages. The Gene Ontology (GO), a valuable and widely-used resource for characterizing gene function, offers perhaps the most suitable functional annotation system for this purpose. However, due in part to the difficulty of studying molecular genetic effects in humans, even the current collection of comprehensive GO annotations for human genes and gene products often lacks adequate developmental context for scientists wishing to study gene function in the human fetus. Description The Developmental FunctionaL Annotation at Tufts (DFLAT) project aims to improve the quality of analyses of fetal gene expression and regulation by curating human fetal gene functions using both manual and semi-automated GO procedures. Eligible annotations are then contributed to the GO database and included in GO releases of human data. DFLAT has produced a considerable body of functional annotation that we demonstrate provides valuable information about developmental genomics. A collection of gene sets (genes implicated in the same function or biological process), made by combining existing GO annotations with the 13,344 new DFLAT annotations, is available for use in novel analyses. Gene set analyses of expression in several data sets, including amniotic fluid RNA from fetuses with trisomies 21 and 18, umbilical cord blood, and blood from newborns with bronchopulmonary dysplasia, were conducted both with and without the DFLAT annotation. Conclusions Functional analysis of expression data using the DFLAT annotation increases the number of implicated gene sets, reflecting the DFLAT’s improved representation of current knowledge. Blinded literature review supports the validity of newly significant findings obtained with the DFLAT annotations. Newly implicated significant gene sets also suggest specific hypotheses for future research. Overall, the DFLAT project contributes new functional annotation and gene sets likely to enhance our ability to interpret genomic studies of human fetal and neonatal development.