Fetal cells in maternal blood.

Fetal lymphocytes, trophoblasts, and nucleated red blood cells have each been separated from maternal blood by methods such as flow cytometry, magnetic cell sorting, and charge flow separation. The frequency of fetal cells among circulating maternal mononuclear cells remains to be ascertained. Current estimates range from about 10-5 to 10-7, but the numbers may be increased in women carrying aneuploid fetuses. Fetal cells separated from maternal blood have been studied by methods such as polymerase chain reaction and fluorescence in situ hybridization. Among fetal conditions so far identified are sex; human leukocyte antigen and Rh blood types; trisomy 13, 18 and 21; triploidy; and sickle cell anemia and thalassemia. Thus, fetal cell separation might one day be used for screening of the common aneuploidies and, ultimately, for prenatal diagnosis. Individual fetal erythroid precursors have been cultured after separation in some laboratories. Culturing and karyotyping of separated fetal cells might enable diagnosis of a spectrum of chromosomal and genetic disorders. Further development will be required, however, before regular clinical application of these methodologies.

46,XY monozygotic twins with discordant sex phenotype.

OBJECTIVE: To analyze male and female sex differentiation in monozygotic twins. DESIGN: Retrospective study. SETTING: Multiple academic centers. PATIENT(S): A pair of monozygotic twins. INTERVENTION(S): Skin and blood samples were obtained for DNA analysis and karyotyping. MAIN OUTCOME MEASURE(S): Mutation within the SRY gene was analyzed by the polymerase chain reaction-single-stranded conformation polymorphism test. Monozygosity was ascertained by short tandem repeat analysis. Karyotypes were studied in blood and skin fibroblasts. RESULT(S): SRY was present in both twins, but no mutations were detected in the SRY conserved motif. Monozygosity was confirmed by the use of short tandem repeat analysis in four loci: c-fms, thyroid peroxidase, von Willebrand factor, and tyrosine hydroxylase. The karyotype was 46,XY uniformly in both twins. CONCLUSION(S): Monozygotic twins can develop discordant male and female phenotypes despite the presence of a common karyotype and despite the presence of intact testis-determining genes. In the present case, this could be due to mutation or to mosaicism involving occult 45,X cell lines in the dysgenetic gonads.

Localization of SRY by primed in situ labeling in XX and XY sex reversal.

Primed in situ labeling (PRINS) can be used to localize DNA segments too small to be detected by fluorescence in situ hybridization. By PRINS we identified the SRY gene in two XX males, a woman with XY gonadal dysgenesis, and an azoospermic male with Xp-Yp interchange. Because PRINS has been used generally in the study of repetitive sequences, we modified the technique for study of the single copy 2. 1-kb SRY sequence. SRY signals were identified at band Yp11.31p11.32 in normal XY males and in the woman with XY gonadal dysgenesis. SRY signals were identified on Xp22 in one XX male but not in the other. They were identified in the corresponding region (Xp22) of the der(X) in the azoospermic male with Xp-Yp interchange. SRY signals were not observed in normal XX females. Presence of SRY in DNA samples from the various subjects was confirmed by polymerase chain reaction. We conclude that PRINS is ideal for rapid localization of single copy genes and small DNA segments in general.

Monozygotic twins of opposite sex.

Although discordant karyotypes are known in identical twins, cases involving differences in sex phenotype are rare. We studied identical twins with the 46,XY karyotype - a male with mixed gonadal dysgenesis and a female with "pure" gonadal dysgenesis. The testis-determining SRY gene was present in DNA from both twins but no mutations were detected in the SRY conserved motif. Monozygosity was indicated by short tandem repeat polymorphism analysis. These observations could be attributed to (i) mutation and mosaicism involving "downstream" sex-determining loci, (ii) variable penetrance of genes such as DSS/NR0B1, duplication of which can disrupt the male-determining pathway, or (iii) occurrence of cryptic 45,X gonadal cell lines.

X-linked sex-reversing genes.

Rearrangement of the X chromosome generates fertile XY females in the wood lemming, and a duplication has been discovered in Xp21 in some XY females in the human. This has enabled identification and mapping of a novel sex-reversing gene in the human, double dosage of which blocks development of the testis. Whether or not the human gene is related to the gene that causes sex reversal in the wood lemming remains to be seen. Yet similar genes must exist in all eutherian mammals, according to Ohno's Law.

Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy.

We set out to ascertain the numbers of fetal cells that enter the maternal blood stream during pregnancy. Samples of 15-16 ml of whole blood were collected from 225 women--mostly 10-18 weeks pregnant--and then processed by charge flow separation, a novel method based on free flow electrophoresis in a buffer counterflow gradient. After their recovery in four different separation instruments, nucleated red blood cells (NRBC) were enumerated histologically. In some cases fetal NRBC were identified and enumerated by fluorescence in situ hybridization with probes for the X and Y chromosomes and fetal haemoglobin mRNA. Recoveries were consistent among the four separation instruments: the median numbers of NRBC obtained were 4190, 1590, 2805 and 3860. Our data show that approximately 30 per cent of those cells were fetal. Thus, recent reports on the separation of fetal NRBC by other methods, give underestimates of their frequency in maternal blood.

Frequency of nucleated red blood cells in maternal blood during the different gestational ages.

We wished to determine the time of pregnancy at which optimal numbers of nucleated red blood cells (NRBC) are present in maternal blood. Because 30% of the NRBC in maternal blood are fetal, there are implications for prenatal screening and diagnosis. Samples of whole blood were collected from each of 225 women at various times during pregnancy. The samples were processed by charge flow separation (CFS), the NRBC enumerated, and the numbers compared on a week-to-week basis. To quantify the relationship between week of pregnancy and actual and log-transformed numbers of NRBC recovered, Pearson product moment and Spearman correlation coefficient were estimated for each of four CFS instruments and for the four instruments combined. When the data were analyzed, we found no relationship between stage of pregnancy and numbers of NRBC recovered. Even after logarithmic transformation, variability among the women, estimated by standard deviation, was large and relatively stable across the different stages of pregnancy. The number of NRBC recoverable by CFS appears to be constant between 7 and 25 weeks.

A survey of attitudes about maternal serum screening for fetal chromosome abnormalities in women 35 years of age and older.

We asked two groups of women their opinion on prenatal diagnosis and maternal serum screening (MSS): group 1 comprised women who had undergone a prenatal diagnostic procedure (amniocentesis or chorionic villus sampling) for advanced maternal age (>/=35 years) and group 2 women who had undergone MSS and were 30-34 years old. Women in group 1 were found significantly less likely to choose MSS over prenatal diagnosis than were women in group 2. The sensitivity of MSS and the age-related risk of chromosome abnormalities influenced opinions on whether to choose MSS or prenatal diagnosis. In both groups, the majority stated that they would accept MSS over prenatal diagnosis, if their obstetrician recommended it.

Fetal cells in maternal blood: recovery by charge flow separation.

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses.

Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling.

OBJECTIVE: Our purpose was to determine the risk of fetal mosaicism when placental mosaicism is found on chorionic villus sampling. STUDY DESIGN: We present a case of mosaic trisomy 22 detected on chorionic villus sampling and subsequently found in the fetus. A review of comprehensive chorionic villus sampling studies with emphasis on follow-up for fetal mosaicism was conducted. RESULTS: Among 13 studies reviewed, 469 cases of placental mosaicism are presented; fetal mosaicism was found in 50 (10.7%). Factors associated with fetal mosaicism are (1) mosaicism on mesenchymal core culture and (2) type of chromosome abnormality involved--specifically, marker chromosomes (26.7%) and common autosomal trisomies (19.0%). Amniocentesis predicted fetal genotype in 93% to 100% of cases of placental mosaicism, depending on the cell type in which mosaicism was diagnosed. CONCLUSIONS: Although mosaicism is usually confined to the placenta, the fetus is involved in about 10% cases. Patients should be counseled about this risk and the accuracy of follow-up amniocentesis.

Identification of p53 mutations in endometrial adenocarcinoma by polymerase chain reaction-single-strand conformation polymorphism.

OBJECTIVE: We determined whether mutations in p53 exons 5-6-7-8, as detected in the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) test, might be correlated with stage or grade in endometrial adenocarcinoma. METHODS: We amplified sequences containing exons 5, 6, 7, or 8 in DNA from tumors and controls. Mutation within the amplified sequences was indicated by changes in electrophoretic mobility (band shifts) in the SSCP test. The results were analyzed statistically and compared with the results of other, similar studies. RESULTS: We identified 15 band shifts among 47 endometrial tumors studied (band shifts in two different exons in two cases) and none among 42 controls. Band shifts in exons 5 and 8 were associated uniformly with grade 2 or grade 3 histology. In other studies p53 mutations were correlated with advanced-stage malignancy. CONCLUSION: Further evaluation of particular p53 mutations and their relation to disease course in endometrial adenocarcinoma seems warranted. The PCR-SSCP test seems well-suited to this purpose.

Lack of heritability in ovarian germ cell malignancies.

OBJECTIVE: Our purpose was to determine whether relatives of patients with ovarian germ cell malignancies not associated with sex chromosome abnormalities are at increased risk for similar tumors. STUDY DESIGN: We reviewed pedigrees of 78 presumptive 46,XX patients (ages ranging from newborn to 20 years) with malignant ovarian germ cell tumors, excluding cases of dysgerminoma and gonadoblastoma. A three-generation family history of each proband was reviewed specifically to identify cancer in any family member. RESULTS: Seventy-eight mothers, 87 sisters, 135 aunts, and 156 grandmothers were surveyed. None had a malignant ovarian germ cell neoplasm or other malignant ovarian neoplasm. CONCLUSION: First- and second-degree relatives of probands with ovarian germ cell malignancies do not have an increased risk for similar tumors. These findings were not predicted because of the well-recognized association of hereditary tumors and early age of onset.

SRVX, a sex reversing locus in Xp21.2-->p22.11.

Duplication within Xp21 causes female or intersexual development in human embryos with an XY chromosome complement. We have mapped the responsible gene, SRVX (sex reversal X), in XY-sex-reversed maternal half siblings who had inherited the duplication from their mother. The limited size of the duplication in our cases, relative to its extent in other similar cases, allows assignment of the SRVX locus to Xp21.2-->p22.11. We infer that SRVX is part of a pathway of sex-determining genes that includes SRY and SRA1, the latter recently assigned to chromosome 17q. If mutation of SRA1 or SRVX can reverse the sex of the XY fetus, this would explain why mutation within SRY is found only sporadically in women with XY gonadal dysgenesis.

Inability to detect fetal metaphases in flow-sorted lymphocyte cultures based on maternal-fetal HLA differences.

Separation of fetal cells from maternal blood could provide a means for prenatal diagnosis that would not endanger the fetus. In this pursuit, we attempted cytogenetic analysis of candidate fetal cells flow sorted on the basis of parental HLA disparity. Metaphases showing 46,XY or aneuploidy and concordant with prenatal diagnostic studies (i.e., amniocentesis, chorionic villus sampling) would presumably be fetal in origin. Blood samples were obtained from 78 pregnant women and their partners. Among 18 HLA informative cases in which metaphases were recovered, 15 involved fetuses that were 46,XY or aneuploid. From these 15 cases, 2,483 metaphases were analyzed. All metaphases were 46,XX. Cytogenetic analysis of flow-sorted fetal cells thus probably will need to emphasize not metaphase analysis but in situ hybridization with chromosome-specific probes.

Variations in genome mass.

1. Genome size varies considerably among vertebrates, ranging from less than 1 pg to more than 200 pg; the amount of DNA differing among individuals in a population can equal the amount in the entire structural gene complement. 2. Recent technological advances permit evaluation of genome size variation at several levels including sub-chromosomal, chromosomal and cellular. 3. Genome size variation may also be viewed from taxonomic levels, and across evolutionary time frames. 4. As sources of genome size variation are identified and studied, the conundrum of the C-value paradox (lack of correlations among genome size, genomic complexity and phylogenetic status of organisms) may prove to be more apparent than real. 5. For example, the limited and relatively constant genome size of avians may be related to the physiological constraints of flight.

Molecular genetics of sex determination in channel catfish: studies on SRY, ZFY, Bkm, and human telomeric repeats.

In amniotes, the banded krait minor (Bkm) minisatellite (GATA), the human telometric sequence (TTAGGG)7, and the Y-specific genes, ZFY and SRY, are associated with a particular sex. These sequences were studied in the channel catfish, Ictalurus punctatus. However, none was sex-specific in catfish; homologs of each were present in males and females. Our data suggest that components of mammalian sex-determining systems may be widespread and shared among the vertebrates in general. Whether those components are involved in sex determination in lower vertebrates or merely represent evolutionary precursors of sex-determining factors in amniotes remains to be determined.

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.

On the evolution of genome size of birds.

We measured genome size (nuclear DNA content) by fluorescence flow cytometry in 55 species of birds representing 12 different orders. Similar studies were performed in approximately 100 species by laboratories using absorption cytophotometry of Feulgen-stained nuclei. Although there have been apparent discrepancies in the assigned values for the species used as a reference, the values obtained in the different laboratories are generally in agreement. When the data are standardized in relation to a diploid (2C) value of 2.5 picograms (pg) of DNA for the domestic chicken (Gallus gallus domesticus), the mean for DNA content in 135 species representing 17 orders is 2.82 +/- 0.33 (SD) pg with a range of 2.0-3.8 pg. Thus the genome size of birds is the most conservative of any vertebrate class and, all values considered, is smaller and more uniform in size than previous estimates would indicate. This could be explained by a previously unexplored hypothesis: that the genome of birds has evolved from a small ancestral genome that was reduced before emergence of the protoavian.

Studies on the phylogenetic conservation of the SRY gene.

A probe from a conserved motif of the SRY gene (sex-determining region Y), a prime candidate for the human testis-determinant, was hybridized to DNA from 23 species representing 5 vertebrate classes. Hybridization occurred in species with male or female heterogamety, in species with and without sex chromosomes and in those with temperature sex determination. Sex-specific signals were observed only in mammals. Conservation of sequences homologous with SRY through 400 million years of vertebrate evolution would indicate persistence of function. However, if SRY is the primary sex determinant in mammals, it is not clear that it has a similar function, or even one that is sex-related, in nonmammals.