To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene-chloroform from 44 maternal (7-41 weeks) plasma. The Fetus-derived Y sequence DYZ-1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00%. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89% at early pregnant stage, 100.00% at medium pregnant stage, and 96.55% at late stage respectively. The final accuracy of 95.45% was obtained in all cases. It was concluded that by means of hydroxybenzene-chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.
DNA/*blood ; DNA Primers ; Fetus ; Maternal-Fetal Exchange ; Pregnancy/*blood ; Prenatal Diagnosis/*methods ; *Sex Determination (Genetics)

The discovery of cell-free fetal DNA (cff-DNA) in maternal plasma offered a new way to noninvasive prenatal diagnosis for single gene disorders. In the past decade, many techniques such as real-time PCR, pyrophosphorolysis-activated polymerization, mass spectrum and digital PCR have been developed for noninvasive prenatal diagnosis. In this review, the author discuss the principles, applications, advantages and disadvantages of these techniques.
DNA ; blood ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; Genetic Diseases, Inborn ; diagnosis ; genetics ; Humans ; Maternal-Fetal Exchange ; Pregnancy ; Prenatal Diagnosis ; methods

OBJECTIVETo analyze cell-free fetal DNA in maternal plasma for prenatal screening of beta-thalassaemia major.

METHODSSix couples undergoing prenatal diagnosis of beta-thalassaemia (gestational age range 23-26 weeks) were enrolled in this study. The husbands were all carriers of the CD17 (A-->T) mutation, and the wives carried another beta-thalassaemia mutation. The allele-specific primers and two fluorescent cycling probes were synthesized for the detection of the CD17 (A-->T) mutation, using FAM and HEX fluorescence labeling, respectively. The cell-free fetal DNA in the maternal plasma was detected using real-time PCR, and the fetal genotype was confirmed by cord blood conventional prenatal diagnosis.

RESULTSIn the 6 pregnancies, FAM and HEX fluorescent signals were detected in 3 maternal plasma samples; in the other 3 samples, only FAM fluorescent signals were detected, suggesting the absence of paternally derived CD17 (A-->T) mutation.

CONCLUSIONExamination of cell-free fetal DNA in maternal plasma using real-time PCR and cycling probe technology can be effective means for prenatal screening of beta-thalassaemia major.

Adult ; DNA ; blood ; DNA Mutational Analysis ; DNA Probes ; Female ; Fetal Diseases ; blood ; diagnosis ; genetics ; Heterozygote ; Humans ; Maternal-Fetal Exchange ; Point Mutation ; Pregnancy ; Prenatal Diagnosis ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; beta-Thalassemia ; blood ; diagnosis ; genetics

Neonatal alloimmune neutropenia (NAN) is an uncommon disease of the newborn provoked by the maternal production of neutrophil-specific alloantibodies, whereby neutrophil IgG antibodies cross the placenta and induce the destruction of fetal neutrophils. Affected newborns are usually identified by the occurrence of bacterial infections. The most frequent antigens involved in NAN are the human neutrophil antigen-1a (HNA-1a), HNA-1b, and HNA-2a. We report a neonate who was delivered at 36 weeks and had a severe neutropenia but who responded well to recombinant human granulocyte colony-stimulating factor (rhG-CSF). Anti-HNA-1a antibody was identified by mixed passive hemagglutination assay in both the sera of the baby and the mother. The baby had HNA-1a and HNA-1b but the mother had only HNA-1b on granulocytes. This is the first Korean report of NAN in which the specificity of the causative antibody was identified.
Pregnancy ; Neutrophils/immunology ; Neutropenia/drug therapy/etiology/genetics/*immunology ; Maternal-Fetal Exchange/immunology ; *Isoantigens/genetics ; Isoantibodies/*blood ; Infant, Newborn ; Immunoglobulin G/blood ; Humans ; Granulocyte Colony Stimulating Factor, Recombinant/therapeutic use ; Genotype ; Female ; DNA/genetics ; Base Sequence ; Antibody Specificity ; Adult

A new method for noninvasive prenatal diagnosis of fetal sex was developed by using single-cell PEP-PCR techniques. Micromamipulation techniques were used to obtain single fetal cells from 273 maternal blood samples. The genome of single cells was preamplified by PEP and SRY genes were analyzed by PCR method. The SRY genes of 149 samples were detected by the new method among 153 samples carrying male fetus, while 119 out of 120 samples carrying female fetus were proved negative for SRY genes. The sensitivity and specificity of the new method were 97.39% and 99.17% respectively and the correct rate was 98.17%. The new method has the advantage of high sensitivity and specificity in noninvasive prenatal diagnosis of fetal sex and provides the basis of other researches such as sex-linked inherited diseases.
Chromosomes, Human, Y ; Erythroblasts/chemistry ; Fetus/*cytology ; Genes, sry/genetics ; Genetic Diseases, Inborn/diagnosis ; Maternal-Fetal Exchange/genetics ; Polymerase Chain Reaction/methods ; Pregnancy/*blood ; Prenatal Diagnosis/*methods ; *Sex Determination (Genetics)

A new method for noninvasive prenatal diagnosis of fetal sex was developed by using single-cell PEP-PCR techniques. Micromamipulation techniques were used to obtain single fetal cells from 273 maternal blood samples. The genome of single cells was preamplified by PEP and SRY genes were analyzed by PCR method. The SRY genes of 149 samples were detected by the new method among 153 samples carrying male fetus, while 119 out of 120 samples carrying female fetus were proved negative for SRY genes. The sensitivity and specificity of the new method were 97.39% and 99.17% respectively and the correct rate was 98.17%. The new method has the advantage of high sensitivity and specificity in noninvasive prenatal diagnosis of fetal sex and provides the basis of other researches such as sex-linked inherited diseases.
Adult ; Chromosomes, Human, Y ; Erythroblasts ; chemistry ; Female ; Fetus ; cytology ; Genes, sry ; genetics ; Genetic Diseases, Inborn ; diagnosis ; Humans ; Male ; Maternal-Fetal Exchange ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; blood ; Prenatal Diagnosis ; methods ; Sex Determination Processes

This study looked into a family involving a rare mother-child ABO blood type inconsistency and explored its genetic and molecular basis. In the family, the mother had type AB blood and the father was blood type B and they gave birth to a baby of blood type O. Their blood types were phenotypically identified by using different techniques, including micro-column gel test, immune inhibition test, absorption and elution tests. The sequences of all 7 exons of ABO allele from the core family members were determined by using PCR and clone-based sequencing. The loci of mutated gene were compared against normal human genes. The result showed that the mother's erythrocytes were agglutinable with monoclonal anti-A antibody (2+) and had agglutination reaction with anti-B antibody (4+). The mother's serum registered agglutination action with standard blood type A cells. The findings showed an ABO inconsistency. When domestic antibodies were used, the mother's erythrocytes yielded agglutination reaction with humanized anti-B serum (4+) and anti-B monoclonal antibody but were non-agglutinable with humanized anti-A serum and anti-A monoclonal antibody. Upon absorption and elution, the titer of anit-A antibody was 128 both before and after the absorption test, with no significant difference found between pre- and post-absorption values. Our results confirmed that the mother's allelic gene was type B and contained type A. The father's blood type was type B, and son's blood type was type O. Clone-based sequencing revealed that the mother carried a heterozygous gene of B101.01 (ntA640→G)/O01, which contained an M214→V mutation that could express a weak expression of antigen A, resulting in blood type AB. However, their son did not have the M214→V mutation, which yielded a false ABO-inconsistency between him and his mother. We were led to conclude that type B gene with a M214→V mutation can encode both antigen B and weak antigen B that can lead to false ABO-inconsistencies.
ABO Blood-Group System ; genetics ; immunology ; Adult ; Base Sequence ; DNA Primers ; Female ; Humans ; Maternal-Fetal Exchange ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Pregnancy ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid