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Technical Briefs |
Departments of
1
Chemical Pathology and
2
Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong SAR
3 Department of Paediatrics, The University of Hong Kong, Hong Kong SAR
aaddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Room 38023, 1/F Clinical Sciences Building, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong SAR; fax 852-2194-6171, e-mail loym{at}cuhk.edu.hk
The presence of fetal DNA in maternal plasma has allowed the development of strategies for noninvasive prenatal diagnosis (1). However, because fetal DNA in maternal plasma circulates among a background of maternal DNA, strategies for noninvasive prenatal diagnosis with applications of fetal DNA in maternal plasma have been confined to the detection of autosomal dominant, paternally inherited genetic traits, such as fetal gender (for sex-linked disorders) (1), rhesus D (2)(3), myotonic dystrophy (4), and achondroplasia (5). Using congenital adrenal hyperplasia (CAH) as a model system, we present a strategy for the noninvasive prenatal exclusion of an autosomal recessive condition through the detection of fetal DNA in maternal plasma. The approach described in this study may potentially be applicable to other autosomal recessive conditions.
More than 90% of cases of CAH are a result of deficiency of 21-hydroxylase, an enzyme of the adrenal gland involved in the synthesis of glucocorticoids and mineralocorticoids. 21-Hydroxylase is encoded by CYP21, a MHC class III gene located on chromosome 6p21.3. Most mutations causing 21-hydroxylase deficiency are caused by either gene deletions or gene conversions, whereby deleterious mutations are transferred from the nearby pseudogene, CYP21P, which shares 98% homology with CYP21 (6). Consequent to profound deficiency or the complete absence of activity of 21-hydroxylase, severe forms of CAH manifest as salt-wasting attributable to impaired synthesis of mineralocorticoids and glucocorticoids. In addition, the excess buildup of metabolic precursors causes excessive androgen production, leading to virilization of female fetuses (6). Hence, dexamethasone therapy is customarily prescribed prenatally to prevent in utero virilization of an affected female fetus through suppression of the excessive production of androgens (7). This type of treatment is not indicated for a male fetus. In the antenatal management of CAH, fetal DNA in maternal plasma has been reported to be useful for the stratification of antenatal dexamethasone therapy through noninvasive fetal gender determination (8) because such therapy would be unnecessary for male fetuses. However, it would be ideal to further limit the dexamethasone therapy only to female fetuses known to be affected by CAH because the use of antenatal dexamethasone is not without complications (6). Consequently, this study attempted to develop a noninvasive approach that allows the effective in utero exclusion of CAH. The strategy proposed is derived from the reasoning that the presence of a wild-type paternally inherited CYP21 allele in maternal plasma would infer that the fetus had inherited the nonmutated paternal allele and thus would not manifest CAH.
A set of parents whose first child was diagnosed with the salt-wasting form of CAH attributable to 21-hydroxylase deficiency presented for antenatal care during their second pregnancy. The proband was known to manifest CAH as a result of the inheritance of an exon 8 codon 316 (arginine–stop) mutation of CYP21 (9) from the father and a maternally derived de novo mutation that led to the deletion of CYP21 (data not shown) (10)(11)(12). The parents consented to this study, the aim of which was to develop assays to determine, through analysis of maternal plasma, whether the unborn fetus had inherited the wild-type or mutant CYP21 paternal allele. We propose the use of polymorphic markers within and linked to CYP21 to differentiate the wild-type from the mutant paternal allele.
Blood samples from the couple and the proband were collected into EDTA-containing tubes. Maternal plasma samples were collected at 11 and 17 weeks of gestation. Buffy coat was collected from the blood samples after centrifugation at 1600g for 10 min (Megafuge 1.0R; Heraeus Instruments), whereas maternal plasma was collected after an additional microcentrifugation step at 16 000g for 10 min (Eppendorf Centrifuge 5415D) (13). Buffy-coat DNA was extracted with the Nucleon reagent set (Amersham Life Science), whereas DNA was extracted from maternal plasma according to the Blood and Body Fluid protocol of a QIAamp reagent set (Qiagen).
Analysis of the antenatal maternal-plasma samples did not reveal the presence of SRY sequences (14), suggesting that the mother was carrying a female fetus. Genomic DNA from the buffy-coat samples from the parents and the proband was first used to select informative markers useful for further analysis. It is common experience that molecular analysis of CYP21 is often difficult and misleading because of the presence of CYP21P, the complex rearrangements and conversion between these two genes, and the reported phenomenon of allele-dropout (15). Therefore, to maximize the reliability of the analyses, both extragenic and intragenic polymorphic markers were selected. The family was informative for three sets of markers, including single-nucleotide polymorphisms in intron 2 of CYP21 (16), linked extragenic microsatellite markers D6S273 and D6S299, and the HLA class II DRB1 locus. Identification of the intragenic polymorphic markers was established by PCR amplification with primers specific for CYP21 (16), followed by direct sequencing of intron 2 of CYP21. The microsatellite markers were analyzed by fluorescence PCR and fragment-size analysis by an ABI Prism 377 automated sequencer. The HLA haplotype of each family member was characterized by serologic methods, and the DRB1 locus was selected for further analysis. To facilitate detection of the paternally inherited fetal DRB1 allele in maternal plasma, amplification refractory mutation system primers (17) were designed to amplify the two paternal alleles (DRB1*03 and DRB1*12). The amplification refractory mutation system primers were designed to amplify exon 2 of the DRB1 alleles, and the amplicons were visualized by agarose gel electrophoresis and ethidium bromide staining. Sequences of the DRB1 alleles were obtained from the HLA sequence database (18). The primer sequences and pattern of allelic segregation within the family are shown in Table 1
. The allele of the respective markers not detectable from the proband was inferred to be associated with the nonmutated paternal allele.
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After characterization of a panel of polymorphic markers associated with the wild-type paternal allele, attempts were made to detect the paternally inherited fetal allele in maternal plasma with the same analytical methods as described above. Throughout the process of maternal plasma analysis, the two researchers studying the plasma were blinded from the mutational analysis of the family. On analysis of the maternal plasma, four of the five paternal markers not inherited by the proband were detectable in maternal plasma. Results of the maternal plasma analysis are shown in Table 1
. The results inferred the inheritance of the nonmutated paternal allele; thus the autosomal recessive disease in question could be excluded.
The volunteers subsequently gave birth to a healthy girl. Umbilical cord blood was subjected to the same polymorphic marker analyses, and the marker haplotype for the paternally inherited allele was concordant with the maternal plasma analyses. To further confirm the validity of the analyses of fetal DNA in maternal plasma, the mother consented to blood sampling at 24 h postdelivery, and the maternal plasma was subjected to analysis for the paternal polymorphic markers. Postdelivery clearance of fetal DNA from maternal plasma is known to be a rapid process (19), and indeed, we failed to detect the previously detectable paternal polymorphisms in the postdelivery maternal plasma sample.
Rijnders et al. (8) reported the use of fetal DNA in maternal plasma for fetal-sex determination to help stratify the need for antenatal dexamethasone therapy. Fetal-sex determination helps to eliminate unnecessary dexamethasone therapy for male fetuses, and the prenatal exclusion of CAH would further reduce unnecessary therapy for nonaffected female fetuses. Using CAH as a model system, we report here that it is feasible to detect polymorphisms associated with the wild-type paternally inherited allele in maternal plasma and, thus, have demonstrated a potential strategy for the noninvasive prenatal exclusion of an autosomal recessive disease. This approach may potentially be applicable to other autosomal recessive conditions, including cystic fibrosis, ß-thalassemia, and certain HLA-linked conditions such as hemochromatosis.
Acknowledgments
This work was supported by the Direct Grants Scheme of the Chinese University of Hong Kong and the Innovation and Technology Fund (AF/90/99). The work performed by the laboratory of P.T.C. was supported by the Committee on Research and Conference Grants of the University of Hong Kong.
References
The following articles in journals at HighWire Press have cited this article:
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  C. F. Wright and H. Burton The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis Hum. Reprod. Update, October 22, 2008; (2008) dmn047v1. [Abstract] [Full Text] [PDF]
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 K.C. A. Chan, A. B.Y. Hui, N. Wong, T. K. Lau, T. N. Leung, K.-W. Lo, and Y.M. D. Lo Investigation of the Genomic Representation of Plasma DNA in Pregnant Women by Comparative Genomic Hybridization Analysis: A Feasibility Study Clin. Chem., December 1, 2005; 51(12): 2398 - 2401. [Full Text] [PDF]
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F. Z. Bischoff, D. E. Lewis, and J. L. Simpson Cell-free fetal DNA in maternal blood: kinetics, source and structure Hum. Reprod. Update, January 1, 2005; 11(1): 59 - 67. [Abstract] [Full Text] [PDF]
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Y. Li, B. Zimmermann, C. Rusterholz, A. Kang, W. Holzgreve, and S. Hahn Size Separation of Circulatory DNA in Maternal Plasma Permits Ready Detection of Fetal DNA Polymorphisms Clin. Chem., June 1, 2004; 50(6): 1002 - 1011. [Abstract] [Full Text] [PDF]
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 H Masuzaki, K Miura, K-i Yoshiura, S Yoshimura, N Niikawa, and T Ishimaru Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism J. Med. Genet., April 1, 2004; 41(4): 289 - 292. [Full Text] [PDF]
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K.C. A. Chan, J. Zhang, A. B.Y. Hui, N. Wong, T. K. Lau, T. N. Leung, K.-W. Lo, D. W.S. Huang, and Y.M. D. Lo Size Distributions of Maternal and Fetal DNA in Maternal Plasma Clin. Chem., January 1, 2004; 50(1): 88 - 92. [Abstract] [Full Text] [PDF]
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D. W. Bianchi Prenatal Exclusion of Recessively Inherited Disorders: Should Maternal Plasma Analysis Precede Invasive Techniques? Clin. Chem., May 1, 2002; 48(5): 689 - 690. [Full Text] [PDF]
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