No abstract

Isolating fetal erythroblasts from maternal blood offers a promising noninvasive alternative for prenatal diagnosis. The current immunoenzymatic methods of identifying fetal cells from background maternal cells postenrichment by labeling ␥-globin are problematic. They are nonspecific because maternal cells may produce ␥-globin, give poor hybridization efficiencies with chromosomal fluorescence in situ hybridization (FISH), and do not permit simultaneous visualization of the fetal cell identifier and the FISH signal. We describe a novel technique that allows simultaneous visualization of fetal erythroblast morphology, chromosomal FISH, and ⑀-globin labeled with AMCA (7-amino-4-methylcoumarin-3-acetic acid).AMCA was chosen as the fluorescent label to circumvent the problem of heme autofluorescence because the mean difference in relative fluorescence intensity between fetal erythroblasts stained positive for antiglobin antibody and autofluorescence of unstained cells was greater with AMCA (mean 43.2; 95% confidence interval [CI], 34.6-51.9; SD ‫؍‬ 14.0) as the reporting label compared with fluorescein isothiocyanate (mean 24.2; 95% CI, 16.4-31.9; SD ‫؍‬ 12.4) or phycoerythrin (mean 9.8; 95% CI, 4.8-14.8; SD ‫؍‬ 8.0). Median FISH hybridization efficiency was 97%, comparable to the 98% (n ‫؍‬ 5 paired samples) using Carnoy fixative. One ⑀-positive fetal erythroblast was identified among 10 5 maternal nucleated cells in 6 paired mixture experiments of fetal erythroblasts in maternal blood (P < .001). Male ⑀-positive fetal erythroblasts were clearly distinguishable from adult female ⑀-negative erythroblasts, with no false positives (n ‫؍‬ 1000). The frequency of fetal erythroblasts expressing ⑀-globin declines linearly from 7 to 14 weeks' gestation (y ‫؍‬ ؊15.8 ؋ ؉ 230.8; R 2 ‫؍‬ 0.8; P < .001). We describe a rapid and accurate method to detect simultaneously fetal erythroblast morphology, intracytoplasmic ⑀-globin, and nuclear FISH. IntroductionIsolating fetal nucleated red blood cells (NRBCs) from maternal blood should allow first trimester noninvasive prenatal diagnosis of aneuploidy and monogenic disorders. 1 There are currently 3 steps: enrichment of fetal cells in maternal blood, identification of fetal cells among background maternal cells, and diagnosis using fluorescence in situ hybridization (FISH) or single-cell techniques. Antibody directed against the ␥ chain of fetal hemoglobin is commonly used for both the fetal cell enrichment 2,3 and identification 4 steps. There may be a case for using ␥-globin for sorting, favoring yield over purity, but it is not nearly specific enough for accurate fetal erythroblast identification because of increased maternal fetal hemoglobin production in pregnancy 5 and ␤ thalassemia. 6,7 In contrast, ⑀-or -globin chains appear specific for fetal cells in chorionic villus supernatants 7 but have not been tested in maternal blood. Whereas -globin is occasionally produced in adults with ␣ thalassemia, 8 ⑀-globin has not been found in adult peripheral blood, 9 making it the prefer...

Sequence of alleles present in family Z. Alleles 1 and 2 differ at position +3 of the intron. Allele 3 differs, at intronic position +17, from alleles 1 and 2. The mutation A1244G is present on the background of allele 1. mutation. Finally, this case illustrates the danger, in linkage analysis, of overinterpreting slight phenotypic or pathological features, especially for carrier-status determination in X-linked diseases.

DNA analysis of blood is conventionally performed on cells – plasma and serum are discarded. Free DNA has been demonstrated in serum in cancer and autoimmune disorders and in pregnancy. We investigated possible noninvasive prenatal diagnosis using fetal DNA from maternal plasma and serum in pregnancy. Fetal gender was determined by PCR on DNA from maternal venous blood, serum and plasma of 65 women by boiling with or without phenol/chloroform extraction. When sensitivities were compared for plasma, additional phenol/chloroform extraction proved more sensitive than boiling alone (89 vs. 50%), the observed difference was 50% (CI 19 to 81%). Extracted plasma amplified better than extracted serum (89 vs. 46%), the observed difference being 44% (CI 22 to 66%). In contrast, fetal gender could not reliably be determined from DNA extracted from maternal nucleated blood cells. The size of plasma and serum DNA at 15–17 weeks of gestation was >1,500 bp. This work confirms the presence of fetal DNA in maternal plasma and serum which may be applicable to noninvasive prenatal diagnosis of paternally derived DNA sequences. We conclude that optimal sensitivity requires two methods of DNA extraction and that the use of plasma is preferred to that of serum.

Deletions within 22q11 have been associated with a wide variety of birth defects embraced by the acronym CATCH22 and including the DiGeorge syndrome, Shprintzen syndrome (velocardiofacial syndrome) and congenital heart disease. It is not known how many genes contribute to this phenotype. Previous studies have shown that a balanced translocation disrupts sequences within the shortest region of deletion overlap for DiGeorge syndrome. A P1 clone was isolated which spans this breakpoint and used to isolate a cDNA encoding a transmembrane protein expressed in a wide variety of tissues. This gene (called IDD) is not disrupted by the translocation, but maps within 10 kb of the breakpoint. Mutation analysis of five affected cases with no previously identified chromosome 22 deletion was negative, but a potential protein polymorphism was discovered. No deletions or rearrangements were detected in these patients following analysis with markers closely flanking the breakpoint, data which emphasize that large (i.e. over 1 Mb) interstitial deletions are the rule in DiGeorge syndrome. The proximity of IDD to the balanced translocation breakpoint and its position within the shortest region of deletion overlap indicate that this gene may have a role, along with other genes, in the CATCH22 haploinsufficiency syndromes.