Molecular Human Reproduction, Vol. 8, No. 6, 586-588,
June 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive genetics |
Analysis of HLA–DRB1
-A and -B alleles in prenatal diagnosis for determination of maternal contamination in fetal DNA
1 Divisions of Hematology II and 2 Obstetrics and Gynecology IV, University of Bari, Piazza Giulio Cesare 11, Bari 70124, Italy
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Abstract |
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During chorionic villi sampling for prenatal diagnosis with molecular biology techniques, contamination by maternal decidua frequently occurs and can lead to misinterpretation of the test results. To avoid such problems, we present a new method for appraising maternal contamination of fetal DNA, based on genomic typing of the highly variable human leukocyte antigen (HLA) locus–DRB1
, locus A and locus B regions by genetic amplification with sequence-specific primers and PCR. Fetal DNA samples obtained for ß-thalassemia diagnosis were analysed after artificial contamination with increasing maternal DNA concentrations ranging from 0.5 to 10% (0.5, 1, 3, 5 and 10%). The approach was found to be rapid, specific, reproducible and highly sensitive and permits recognition of 1–3% contamination by maternal DNA concentrations. The system currently used for detecting maternal DNA contamination in fetal samples is the analysis of polymorphic loci by variable number of tandem repeats and/or short tandem repeats. We propose that the analysis of HLA alleles may provide a valid alternative or complement to this system. ß thalassemia/DNA maternal contamination/HLA SSP-PCR/prenatal diagnosis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In the first trimester of pregnancy, prenatal diagnosis can be performed by chorionic villi sampling using transabdominal ultrasound guidance. The main problem associated with this method is the risk of contamination by maternal decidua (Cheung et al., 1987
; Ganshirt-Ahlert et al., 1990) due to the inexperience of the operator at the time of sampling or of the laboratory technologist engaged in analysing and separating the fetal material obtained. The presence of maternal cells could lead to misinterpretation of the test results, a serious concern with medical and legal implications. To avoid such problems in laboratories performing prenatal diagnostic procedures based on molecular genetics, various methodologies have been adopted to determine the presence of maternal cell contamination in the fetal tissues (de Martinville et al., 1984; Olson et al., 1986; Williams et al., 1987). These methods generally involve the analysis of polymorphisms to distinguish between maternal and fetal DNA.
In the field of molecular diagnostics, the analysis of polymorphic regions of DNA is currently performed by the assessment of loci with a variable number of tandem repeats (VNTR) or short tandem repeats (STR) (Jeffreys et al., 1985; Wasmuth et al., 1988; Batanian et al., 1990; Decorte et al., 1990). Of the VNTRs, the most commonly used loci are IgJH (chromosome 14) (Decorte et al., 1990), ApoB (chromosome 2) (Decorte et al., 1990), YNZ22 (chromosome 17) (Batanian et al., 1990), DS495 (chromosome 4) (Wasmuth et al., 1988) and D1S80 (chromosome 1) (Budowle et al., 1991). The difficulties involved in the use of these methods are related to their poor specificity; in fact, in VNTR DS180 the level of heterozygosity has been demonstrated to be as high as 80% (Budowle et al., 1991), and 73% in STR D16S282 (Lauthier et al., 1991).
Therefore, in view of the high number of variable human leukocyte antigen (HLA) alleles in the population, the analysis of this region may be more suitable for determining maternal contamination of fetal material obtained for DNA prenatal diagnosis from chorionic villi.
The aim of this study was to present an alternative method which is potentially less labour intensive and more reliable than the analysis of polymorphisms by VNTR/STR. We have used PCR analysis of regions with high variability, such as the HLA regions of class I (A and B) and of class II (DR genes) (Zetterquist and Olerup, 1992; Olerup, 1994; Olerup et al., 1999), as an alternative diagnostic method to those currently in use.
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Materials and methods |
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To evaluate the sensitivity of this novel method for detecting maternal cell contamination, fetal DNA from 10 chorionic villus samples was artificially contaminated in the laboratory with varying percentages of maternal DNA. Serial proportions of 0.5, 1, 3, 5 and 10% maternal contamination were prepared, calculated on the basis of a total quantity of 840 ng DNA necessary for a HLA–DRB1* low resolution genomic typing reaction with the sequence-specific primer (SSP)–PCR technique (GenoVision kit, Wien, Osterreich) (Olerup et al., 1992
).
Extraction of maternal and fetal DNA was performed with the `salting out' method (Miller et al., 1988) using 10 ml of peripheral blood and chorionic villi respectively, withdrawn at week 10 of pregnancy for prenatal diagnosis of ß-thalassemia.
The primer set contained 5' and 3' primers for HLA–DR, allowing grouping of the DRB10101 to DRB11001 alleles into the corresponding serological groups DR1 to DR18, as well as primer pairs for recognizing the DRB3, DRB4 and DRB5 groups of alleles. The primer solutions were pre-aliquoted into 0.2 ml PCR tubes. Each tube in the set contained a dried primer solution consisting of a specific primer mix, i.e. allele and group specific primers as well as a control primer pair matching non-allelic sequences.
The optimal DNA concentration was 30 ng/µl. For one DR `low resolution' typing, in a 0.5 ml tube at room temperature, 56 µl of DNA (30 ng/µl) was added to 84 µl of PCR mix with Taq (11 units) and 140 µl of dH2O, and 10 µl of the DNA-PCR mix-H2O mixture were dispensed into each of the 24 wells.
Amplification was carried out with a PE 9700 thermal-cycler; the PCR cycling parameters were: 1 cycle at 94°C for 2 min; 10 cycles at 94°C for 10 s, 65°C for 60 s; and 20 cycles at 94°C for 10 s, 61°C for 50 s and 72°C for 30 s. Subsequently, analysis of the amplified products was performed by 2% agarose gel electrophoresis in a 0.5x Tris–borate–EDTA buffer with staining by ethidium bromide and visualization on a transilluminator.
The preparation and amplification technique for the study of loci HLA-A and -B was as described above for the HLA–DRB loci.
In addition, 100 prenatal diagnoses performed in our laboratory by means of molecular analysis of locus HLA–DRB1-A and -B were retrospectively analysed to ascertain the success rate of this method.
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Results |
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The results of this study of artificially contaminated fetal DNA as part of a procedure for prenatal diagnosis of ß-thalassemia are shown in Figures 1–8
. In Figures 1 and 2, DRB1 low resolution genomic typing of the father and mother respectively demonstrates the presence of the specific PCR products in lanes 9, 13, 16, 22 and 23 (corresponding to DRB1* 07011-0704,11011-1137 of the father) and lanes 5, 6, 15, 16, 17, 22 (corresponding to DRB1* 03011-03016,1301-1336 of the mother). Figure 3 illustrates DRB1 low resolution genomic typing of the non-contaminated fetal DNA after prenatal diagnosis, demonstrating the presence of the specific PCR products in lanes 5, 6, 9, 17, 22 and 23 (corresponding to DRB1* 03011-03016,07011-0704 of the fetus).
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DRB1 genomic typing of the 0.5% artificially contaminated maternal–fetal DNA mixture showing the presence of the specific PCR products in lanes 5, 6, 9, 17, 22 and 23 corresponding to DRB1* of the fetus is illustrated in Figure 4
, while Figure 5 shows DRB1* genomic typing of the 1% artificially contaminated maternal–fetal DNA mixture. Apart from the expected presence of the specific PCR products for the fetus (in lanes 5, 6, 9, 17, 22 and 23), it is noteworthy that a weakly positive signal was observed in lanes 15 and 16, most likely due to maternal contamination as it corresponds to the maternal genotype.
In Figures 6, 7 and 8 regarding the 3, 5 and 10% artificially contaminated samples respectively, the contaminated PCR products in lanes 15 and 16 appear progressively more evident and are finally practically identical to the fetal bands in the 10% contaminated sample.
The retrospective analysis of 100 prenatal diagnoses performed in our laboratory showed that the DRB1 locus provided sufficient information in 83% of cases, while it was necessary to additionally study the HLA-A locus in 14% of cases and the HLA-B locus in 3% of cases.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Our study shows that molecular analysis of locus HLA–DRB1-A or -B is highly sensitive and specific. In fact, it is able to reveal a 1% level of maternal DNA contamination. Moreover, analysis with the SSP–PCR method was demonstrated to be faster and easier to perform than other current methods. Moreover, technical problems can arise in the PCR analysis of VNTR polymorphisms due to the relatively poor amplification of large alleles in the presence of smaller ones (Batanian et al., 1990
). The present method is at the same time less expensive and more sensitive and specific than the analysis of polymorphisms. A large group of alleles can be analysed simultaneously starting from a single PCR preparation with the addition of successive aliquots to tubes containing the specific primer pairs. In contrast, analysis of VNTRs or STRs requires 3–5 probe systems to be set up (D1S80, YNZ22, APO-B, IgJH, DS495, etc.), each involving separate preparation of the amplification reagents. Maternal–fetal contamination is now routinely checked by analysis of the HLA loci in our laboratory.
This method would enable us to cover the whole population of Apulia, S.E. Italy. Other authors (Batanian et al., 1998) have obtained 100% informative results in 30 cases examined with two VNTR (APO-B and YNZ22). This success rate could be attributed to the extreme polymorphism of the VNTRs used by these authors in their population, which is distinct to the Apulian population considered in our study.
In conclusion, it appears that HLA low resolution genomic typing of class I (A and B) and class II (DRB1) genes can offer a valid alternative to the methods currently used to evaluate maternal–fetal contamination.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The authors are grateful to Ms Paulene Butts for her assistance in the preparation of the manuscript and to M.V.C.Pragnell for revising the English of the manuscript.
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Notes |
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3 To whom correspondence should be addressed. E-mail: ematolog{at}cimedoc.uniba.it
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References |
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Batanian, J.R., Ledbetter, S.A., Wolff, R.K., Nakamura, Y., White, R., Dobyins, W.B. and Ledbetter, D.H. (1990) Rapid diagnosis of Miller–Dieker syndrome and isolated lissencephaly sequence by the polymerase chain reaction. Hum. Genet., 85, 555–559.[Web of Science][Medline]
Batanian, J.R., Ledbetter, D.H. and Fenwick, R.G. (1998) A simple VNTR–PCR method for detecting maternal cell contamination in prenatal diagnosis. Genet. Test, 2, 347–350.[Web of Science][Medline]
Budowle, B., Chakraborty, R., Giusti, A.M., Eisemberg, A.J. and Allen, C. (1991) Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am. J. Hum. Genet., 48, 137–144.[Web of Science][Medline]
Cheung, S.W., Crane, J.P., Beaver, H.A. and Burgess, A.C. (1987) Chromosome mosaicism and maternal cell contamination in chorionic villi. Prenat. Diagn., 7, 535–542.[Web of Science][Medline]
de Martinville, B., Blakemore, K.J., Mahoney, M.J. and Francke, U. (1984) DNA analysis of first-trimester chorionic villus biopsies: test for maternal contamination. Am. J. Hum. Genet., 36, 1357–1368.[Web of Science][Medline]
Decorte, R., Cuppens, H., Marynen, P. and Cassiman, J.J. (1990) Rapid detection of hypervariable regions by the polymerase chain reaction technique. DNA Cell Biol., 9, 461–469.[Web of Science][Medline]
Ganshirt-Ahlert, D., Pohlschmidt, M., Gal, A., Horst, J., Miny, P. and Holzgreve, W. (1990) Transabdominal placental biopsy in the second and third trimesters of pregnancy: what is the risk of maternal contamination in DNA diagnosis? Obstet. Gynecol., 75, 320–323.[Web of Science][Medline]
Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Individual-specific `fingerprints' of human DNA. Nature, 316, 76–79.[Medline]
Lauthier, V., Mariat, D. and Zoroastro, M. (1991) A synthetic probe STR 14C19, detects a new polymorphic locus at 16pter (D16S282). Nucleic Acids Res., 19, 4015.
Miller, S.A., Dykes, D.D. and Polesky, H.F.A. (1988) Simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215–1218.
Olerup, O. (1994) HLA-B27 typing by a group-specific PCR amplification. Tissue Antigens, 43, 253–256.[Web of Science][Medline]
Olerup, O. and Zetterquist, H. (1992) HLA-DR typing by PCR amplification with SSP-PCR in two hours: an alternative to serological DR typing in clinical practice including donor–recipient match in cadaveric transplantation. Tissue Antigens, 39, 225–235.[Web of Science][Medline]
Olerup, O., Daniels, T. and Baxter-Lowe, L.A. (1999) Correct sequence of the A* 3001 allele obtained by PCR–SSP typing and automated nucleotide sequencing. Tissue Antigens, 44, 265–267.
Olson, S., Buckmaster, J., Bissonnette, J. and Magenis, E. (1986) Comparison of maternal and fetal chromosome heteromorphism to monitor maternal cell contamination in chorionic villus samples. Prenat. Diagn., 7, 413–417.[Web of Science]
Wasmuth, J.J., Hewitt, J., Smith, B., Allard, D., Haines, J.L., Skarecky, D., Partlow, E. and Hayden, M.R. (1988) A highly polymorphic locus very tightly linked to the Huntington's disease gene. Nature, 332, 734–736.[Medline]
Williams, J. 3rd, Medearis, A.L., Chu, W.H., Kovacs, G.D. and Kaback, M.M. (1987) Maternal cell contamination in cultured chorionic villi: comparison of chromosome Q-polymorphisms derived from villi, fetal skin, and maternal lymphocytes. Prenat. Diagn., 7, 315–322.[Web of Science][Medline]
Zetterquist, H. and Olerup, O. (1992) Identification of the HLA-DRB1* 07 and DRB1* 09 alleles by PCR amplification with sequence specific primer in 2 hours. Hum. Immunol., 32, 67–74.
Submitted on June 25, 2001; accepted on February 22, 2002.
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