Prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood

We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from maternal blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, maternal blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the maternal blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from maternal blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases. Received: 19 December 1997 / Accepted: 14 February 1998     

Enrichment of fetal trophoblasts and nucleated erythrocytes from maternal blood by an immunomagnetic colloid system

The aim of this study was to isolate fetal trophoblasts and nucleated erythrocytes from maternal blood using the immunomagnetic colloid system. About 25 ml of maternal blood was collected from pregnant women between of 14 and 20 weeks gestation. Nucleated erythrocytes (NRBCs) were isolated from 5 ml of maternal blood and a nested polymerase chain reaction for the Y chromosome was used to determine fetal origin. The sensitivity of the fetal gender diagnosis was 80% and the specificity was 86%. Both fetal trophoblasts and NRBCs were isolated from the remaining 20 ml of maternal blood. The fetal gender of the trophoblast-enriched fraction was determined using fluorescence in situ hybridisation (FISH) with dual-colour XY-specific DNA probes. XY-specific signals were observed in 0.38% of cells sorted from all pregnant women carrying male fetuses (n = 10). Simultaneous immunophenotyping for the fetal haemoglobin and FISH using XY probes were used to evaluate the fetal origin of cells enriched with anti-CD71. The mean percentage of male fetal erythroblasts was 0.24% and the number of fetal erythroblasts was estimated to be about 672 in 20 ml of maternal blood. The number of fetal erythroblasts detected in our study was greater than that detected by most other separation techniques. Our study shows that it would be feasible to use the immunomagnetic colloid system for the isolation of both trophoblasts and NRBCs from the same maternal blood sample with relatively good efficiency. Received: 17 December 1998 / Accepted: 9 February 1999     

利用孕妇血浆DNA检测胎儿性别的研究

本文探讨应用孕妇血浆中游离DNA进行无创性产前性别诊断的可行性。用柱分离法提取73例孕妇血浆中DNA,用巢式PCR技术检测其胎儿SRY基因。 结果73位孕妇血浆DNA含量为0.0062~0.3399μg/μL。巢式PCR检测胎儿SRY基因的灵敏度为97.37%（37/38）,假阴性率2.86%(1/35),特异度85.71%（30/35）,假阳性率13.16%(5/38),总符合率91.78%（67/73）。采用孕妇血浆胎儿DNA和巢式PCR技术可以快速简便的进行无创性产前性别诊断,诊断结果的准确率为91.8%,对性连锁遗传病的预防具有重要意义。 Abstract:To investigate the feasibility and possibility of application of fetal DNA from maternal plasma for noninvasive prenatal diagnosis of fetal sex,plasma DNAs in blood samples of 73 pregnant women at the gestational period of 26 to 41 weeks were extracted by column separation and nested polymerase chain reaction were employed to amplify the SRY gene.A comparison was made between the amplification results and the real sex of the fetus after their delivery.The concordance rate of SRY gene amplification results of plasma free DNA with real fetal sex was 91.78% (67/73),the sensitivity rate was 97.37% (37/38),and the specific rate was 85.71% (30/35).The cell-free fetal DNA in maternal blood can be one of the valuable material sources for noninvasive prenatal diagnosis and the method of nested PCR could be useful for fetal sex determination.The specific rate of the test was 91.78%.It is of significance to prevent sex-linked inheritant diseases.     

Analysis of cell-free fetal DNA in plasma and serum of pregnant women.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.     

A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.

A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease.     

Kleihauer抗酸染色法标记母血中的胎儿有核红细胞

以18例孕7~25周的孕妇外周血为材料, 经Percoll不连续密度梯度离心初步富集胎儿有核红细胞。然后用Kleihauer抗酸染色法进行标记, 结果阳性胎儿有核红细胞的胞浆呈深红色, 而母亲的有核红细胞胞浆无色。显微操作法获取单个胎儿有核红细胞, 经全基因组扩增后, 产物进行性别鉴定及STR连锁分析检测, 验证有核红细胞的来源, 并完成9例杜氏肌营养不良(Duchenne muscular dystrophy,DMD)的无创性产前基因诊断。应用Kleihauer抗酸染色法标记胎儿有核红细胞, 它是一种快速、简单、直接的化学染色方法, 更易于推广到临床应用。     

Multiple testing in fetal gender determination from maternal blood by polymerase chain reaction

Fetal male DNA can be identified in maternal blood by polymerase chain reaction (PCR) amplification of Y-specific sequences. This technology has not reached a satisfactory accuracy and reproducibility in fetal gender determination because of the very low concentration of fetal cells. Our purpose was to evaluate the possibility of improving the reliability of this test by setting up a repeated amplification system. We amplified, by nested PCR of the Y-specific sequence DYS14, 137 DNA samples extracted from maternal peripheral blood (93 from male-bearing and 44 from female-bearing pregnancies ranging from the 6th to the 36th gestational week). Each maternal DNA sample was tested doubly, in two different PCR sessions, with a total of four amplifications. We obtained discordant results in the four amplifications in 82/137 (60%) samples. The best interpretation of these discordant results was obtained by applying a positivity cutoff of at least two positive amplifications for considering a DNA sample as belonging to a male-bearing pregnancy. We obtained a sensitivity of 83%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 72% in fetal male gender diagnosis. By applying this quadruple testing system, we significantly improved PCR accuracy and predictive values compared with single and double testing of the same samples. We conclude that, for future investigations of fetal DNA retrieved from maternal blood, the application of a quadruple testing system is better than the single PCR test. Received: 18 August 1997 / Accepted: 12 January 1998     

Analysis of HLA-DQ α sequences for prenatal diagnosis in single fetal cells from maternal blood

We have extended a previously developed method that allows prenatal DNA diagnosis of female fetuses through the isolation of single nucleated erythrocytes from maternal blood by developing a method that can distinguish between maternal and fetal nucleated erythrocytes. Nucleated erythrocytes were separated by a density-gradient method and then collected by micromanipulation. Sex was determined after primer extension preamplification (PEP) of the entire genome of a single cell, and human leukocyte antigen (HLA)-DQ α type was determined after further amplification of this gene. The HLA-DQ α genotype of fetal erythrocytes in maternal blood samples and their corresponding paternal and maternal lymphocytes were successfully determined in all cases. The accuracy of the method was determined by using single nucleated erythrocytes from umbilical cord blood from five normal deliveries. This is the first demonstration that the fetal HLA-DQ α gene sequences can be identified in a small aliquot of a single nucleated erythrocyte in maternal blood. We believe that this method ushers in a new era in which the reliability and accuracy of noninvasive prenatal DNA diagnosis from maternal blood is markedly improved. Received: 18 April 1997 / Accepted: 1 October 1997     

Fetal DNA in maternal serum: does it persist after pregnancy?

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.     

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.     

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

Background

Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.     

Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.     

Detection of fetal RHD pseudogene (RHDΨ) and hybrid RHD-CE-Ds from RHD-negative pregnant women with a free DNA fetal kit

Hemolytic disease of the newborn is a clinical condition in which maternal and paternal Rh blood group antigens are incompatible and the mother is negative for the antigen whereas the father is positive. Analysis of fetal cells recovered from maternal plasma can provide a highly sensitive prenatal diagnosis. The fetal RHD gene in plasma DNA is detected by real-time PCR amplification of two different segments of the RHD gene (exons 7 and 10). Each amplicon is revealed with specific probes. We examined 40 female blood samples to verify the specificity of RHD exons (7 and 10) amplified by real-time PCR. Thirty fetuses were predicted to be RHD-positive based on analysis of plasma DNA. Seven fetuses were predicted to be RHD-negative. One fetus was negative for RHD on exon 10, and positive for RHD on exon 7 (early gestation age); two fetuses were RHD-negative on exon 7, and RHD-positive on exon 10 (RHD-CE-D(s) or RHDΨ), indicative of a maternal RHD allele. We conclude that it is necessary to analyze at least two exon regions in the RHD gene.     

改良的PEP方法在无创性产前基因诊断中的应用

应用显微操作技术获取孕妇外周血中的单个有核红细胞,改良的PEP方法扩增单个有核红细胞的全基因组DNA;在此基础上,应用荧光标记聚合酶链反应扩增9个微卫星片段,进行基因型分析判定单个有核红细胞来源。综合性别和DMD基因内的数个STR位点连锁分析进行DMD基因诊断,应用PCR-STR连锁分析进行PKU基因诊断。结果显示,对10例DMD高危胎儿中的6例成功地进行了无创性产前基因诊断。同时对1例PKU也成功地进行了无创性产前基因诊断。改良的PEP方法扩增单个细胞的全基因组可以满足基因诊断的要求,是无创性产前基因诊断中一种极有价值的全基因组扩增的方法。 Abstract：We investigated the feasibility of using improved primer extension preamplificat ion method to diagnose DMD and PKU. The fetal nucleated red blood cells from the peripheral blood of pregnant women were detected and individually retrieved into glass capillary pipettes using a micromanipulator under microscopic observation. The whole genome of a single cell was amplified by improved primer extension preamplification (PEP).Genotypes were analyzed by amplifying the 9 STR fragments using fluorescence?PCR technique and NRBC's(nucleated red blood cell) origin w as determined.We diagnosed DMD prenatally using sex determination and linkage an alysis of several STR sites of dystrophin,and we diagnosed PKU prenatally using PCR?STR linkage analysis.6 of 10 potential DMD patients were diagnosed,includin g 1 male fetal patient,1 potential PKU patient was also diagnosed.The improved P EP method is a very valuable method of amplifying the whole genome of single cel ls,and the products of amplification are enough to the requirements of DNA in no n-invasive prenatal diagnosis.     

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.     

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.     

应用胎儿特异性抗体HbF（γ链）标记法无创性 产前基因诊断DMD

以孕8~26周孕妇外周血为材料,经过Percoll密度梯度离心初步富集,胎儿细胞特异性抗体—HbF标记、识别胎儿有核红细胞,母体和胎儿有核红细胞的精确区分是以胎儿和成人血红蛋白的组成差异为基础的。胎儿细胞胞浆黄染,而具有成人血红蛋白的母体细胞没有颜色。显微操作法获取全部阳性细胞后,以其全基因组扩增（PEP）的产物为模板,进行性别检测、DMD基因的多重PCR检测和STR连锁分析。结果,20名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC。并完成7例DMD的产前基因诊断。HbF抗体标记法能有效识别胎儿有核红细胞,是无创性产前基因诊断中很有应用前景的标记方法。 Abstract： Maternal blood was obtained at 8-26weeks of gestation.After discontinuous density gradient centrifugation with Percoll ,HbF antibody was used to identify fetal NRBC.The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb).Fetal cells appear yellow cytoplasmic staining,while adult cells colorless. NRBCs were collected by micromanipulation andwhole genome amplification was performed. DMD was prenatally diagnosed by using the combination of sex determination,multiplex PCR and linkage analysis of several STR sites of dystrophin. NRBCs stained with HbF were found in all of 20 maternal blood samples with gestations, and 7 fetuses with risk of DMD were diagnosed. We concluded that HbF antibody could identify fetal NRBC efficaciously,and this is a kind of more prospective application method.     

Detection of Y STR markers of male fetal dna in maternal circulation

BACKGROUND:

Circulating fetal cells and cell free DNA in the maternal blood has been shown to help in prenatal diagnosis of genetic disorders without relying on invasive procedures leading to significant risk of pregnancy loss.

AIM:

The current study was undertaken to detect the male fetal population using Y STR markers DYS 19, DYS 385 and DYS 392 and also to study the extent of persistence of fetal DNA in the mother following delivery.

MATERIALS AND METHODS:

Blinded study was conducted on 50 mothers delivering male and female babies. Cellular and cell free DNA was extracted from maternal and fetal cord blood and amplified for Y STR markers by PCR.

RESULTS:

The amplification sensitivity of Y specific STR, DYS19 was 100% (22/22) in the male fetal DNA samples. The incidence of other STRs, i.e., DYS385 and DYS392 were 91% (20/22) each. Analysis of results revealed that thirteen of the twenty six women had detectable male fetal DNA at the time of delivery. However fetal DNA was not detectable twenty four hours after delivery.

CONCLUSION:

Preliminary results show that the separation of fetal cell-free DNA in the maternal circulation is a good low-cost approach for the future development of novel strategies to provide non-invasive techniques for early prenatal diagnosis.     

Real-time PCR for prenatal and preimplantation genetic diagnosis of monogenic diseases

The provision of prenatal diagnosis requires the highest standards in laboratory practice to ensure an accurate result. In preimplantation genetic diagnosis protocols additionally have to address the need to achieve an accurate result from 1 to 2 cells within a limited time. Emerging protocols of "non-invasive" prenatal diagnosis, which are based on analysis of free fetal DNA in the circulation of the pregnant mother, also have to achieve a result from a limited quantity of fetal DNA against a high background of maternal free DNA. Real-time PCR uses fluorescent probes or dyes and dedicated instruments to monitor the accumulation of amplicons produced throughout the progress of a PCR reaction. Real-time PCR can be used for quantitative or qualitative evaluation of PCR products and is ideally suited for analysis of nucleotide sequence variations (point mutations) and gene dosage changes (locus deletions or insertions/duplications) that cause human monogenic diseases. Real-time PCR offers a means for more rapid and potentially higher throughput assays, without compromising accuracy and has several advantages over end-point PCR analysis, including the elimination of post-PCR processing steps and a wide dynamic range of detection with a high degree of sensitivity. This review will focus on real-time PCR protocols that are suitable for genotyping monogenic diseases with particular emphasis on applications to prenatal diagnosis, non-invasive prenatal diagnosis and preimplantation genetic diagnosis.     

Combined analysis of the MspI and XbaI polymorphisms in intron 22 of the factor VIII gene for detection of hemophilia A in a Korean population

To determine the usefulness of MspI/int22h-1 (intron 22 homologous region-1) polymorphism of the factor VIII gene for molecular genetic diagnosis of hemophilia A in the Korean population, MspI/intron 22 and XbaI/intron 22 polymorphisms were analyzed in 101 unrelated Korean families with severe hemophilia A. The expected heterozygosity rates of MspI/int22h-1 and XbaI/int22h-1 polymorphisms were 49.5 and 43.6%, respectively; these polymorphisms were not in complete linkage disequilibrium. Combined analysis using both polymorphisms provided an informative rate of 66.3%. These results suggest that PCR analysis of the MspI/int22h-1 polymorphism of the factor VIII gene would be useful for carrier detection and prenatal diagnosis of hemophilia A in the Korean population.