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Annals of Clinical & Laboratory Science 34:319-323 (2004)
© 2004 Association of Clinical Scientists

Review

Human Chromosome 21-Specific DNA Markers Are Useful in Prenatal Detection of Down Syndrome

Jui-Der Liou1,*, Da-Chang Chu3,4,*, Po-Jen Cheng2, Shuenn-Dyh Chang2, Chien-Feng Sun3, Ying-Chung Wu3, Wen-Yin Liou4 and D.T.Y. Chiu3,4
1 Department of OB/GYN, Chang Gung Memorial Hospital, Taipei, Taiwan; 2 Department of OB/GYN, Chang Gung Memorial Hospital, Lin-Kou, Taiwan; 3 Department of Clinical Pathology, Chang Gung Memorial Hospital, Lin-Kou, Taiwan; 4 Graduate Institute of Medical Biotechnology, School of Medical Technology, Chang Gung University, Tao Yuan, Taiwan.

Address correspondence to Da-Chang Chu PhD, Graduate Institute of Medical Biotechnology, School of Medical Technology, Chang Gung University, Tao Yuan, 333, Taiwan, ROC; tel 886 3 211 8800 x5086; fax 886 3 211 8512; e-mail: dcchu{at}mail.cgu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Trisomy 21 is the most common chromosomal aberration in live births. In this study we employed human chromosome 21-specific short tandem repeat (STR) DNA markers to determine the numbers of chromosome 21 present in fetal cells. Forty amniotic fluid samples from pregnancies complicated with fetal Down syndrome and 98 samples from euploid pregnancies were analyzed for D21S11 and interferon-{alpha} receptor (IFNAR) gene intervening sequence. Fluorescent dye-labeled primers were used in PCR amplification of these 2 markers. The PCR amplicon was analyzed with an automatic DNA sequence analyzer. The results showed that 35 of 40 fetal Down syndrome samples analyzed for IFNAR showed 3 distinct peaks, while 24 of 30 cases analyzed for D21S11 showed 3 distinct peaks. Two Down syndrome samples showed two uneven peaks. By analyzing 98 euploid pregnancies as controls, the ratios of area under the peaks were determined to be 1.31 ± 0.22 and 1.96 ± 0.18 (mean ± SD) for the euploid pregnancies and pregnancies complicated by fetal Down syndrome with 2 peaks, respectively. Our data showed that altogether 39 of 40 (97.5%) Down syndrome cases were correctly identified based on either the 3-peak pattern in one or more of the DNA markers or the relative peak area ratio calculation. In conclusion, polymorphic STR DNA markers are useful for determining the numbers of chromosome 21 in fetal cells. The high sensitivity and automation of the procedures suggest a good prospect for use of this method in prenatal detection of fetal Down syndrome. However, this is a preliminary investigation and a large-scale study is necessary to validate the clinical application of this protocol.

(received 22 February 2004; accepted 1 May 2004)

Keywords: Down syndrome, short tandem repeat markers, fluorescent PCR, interferon-{alpha} receptor gene


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Down syndrome (MIM #190685), also known as trisomy 21, results from an extra copy of chromosome 21 in the cell. With the incidence estimated to be approximately 1 in 1,000 to 2,000 [1], it ranks as a major issue in prenatal medicine. The Multiple Marker Screening Test (MMST) of maternal serum has been used worldwide in the past decades to screen for pregnancies complicated by fetal Down syndrome [2–6]. In cases at high risk based on MMST screening or because of advanced maternal age, genetic amniocentesis is recommended in order to perform fetal karyotyping. About 60 to 70% of fetal Down syndrome cases can be identified by MMST, with a 5% false positive rate [5–7]. The interval between genetic amniocentesis and the completion of karyotyping is a period of anxiety for the family [8]. Therefore, scientists continue to seek nascent marker(s) or protocols in order to detect this aneuploidy more rapidly [9–11].

Human chromosome 21 is the smallest human chromosome, comprising about 1.2% of the human genome [12]. Many polymorphic DNA markers containing short tandem repeats (STR) located on chromosome 21 have been discovered [13]. These polymorphic DNA markers have been applied in genomic studies such as constructing genetic linkage maps of chromosome 21 [14,15]. It was proposed that STR polymorphism could be applied to detect aneuploidies [16–23].

In this study, we used fluorescence dye incorporated PCR to amplify 2 highly heterogeneous markers containing tetranucleotide repeats, located on the long arms of chromosome 21, to identify the numbers of chromosome 21 present in amniocytes from pregnancies complicated with fetal Down syndrome and from euploid pregnancies, to see if this protocol might be useful for clinical application.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Study subjects.  Forty amniotic fluid samples from pregnancies complicated with fetal Down syndrome and 98 samples from euploid pregnancies were collected in the Department of OB/GYN, Chang Gung Memorial Hospital. The patients were referred to the hospital because of advanced maternal age or abnormal MMST results. Amniotic fluid samples were obtained by ultrasound-guided amniocentesis for the purpose of karyotyping. Trisomy 21 karyotypes of all samples were confirmed cytogenetically. Small amounts of the amniotic fluid samples (about 3 ml, intended for culture backup) were utilized in this study, after the routine cytogenetic tests had been completed.

Genomic DNA extraction.  Genomic DNA was extracted from each sample of amniocytes using a commercial kit (Puregene, Gentra Systems, Minneapolis, MN), following the manufacturer’s directions.

PCR amplification of D21S11 and human interferon-{alpha} receptor (IFNAR) gene DNA markers.  The DNA extracted from amniocytes was used to amplify the short tandem repeat sequence within intron 5 of the IFNAR gene and the D21S11 locus on human chromosome 21, using the polymerase chain reaction (PCR) as described previously [24,25] with slight modifications. In brief, PCR amplifications were performed in a total reaction volume of 50 µl containing up to 50 ng of DNA, 200 nM each of dATP, dTTP, dCTP, and dGTP, 200 nM each of the primers (only the 5' end of forward primers were fluorescent dye-labeled), 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 1 x buffer (1.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, 0.001% gelatin, pH 8.3). The mixture was cycled 35 times at 95°C, 1 min (denaturation), 55°C for IFNAR, and 56°C for D21S11, respectively, 1 min (annealing), and 72°C, 2 min (extension) on a programmable thermocycler (model 9600, Perkin-Elmer, Norwalk, CT). An extra 7 min at 72°C was added to the last cycle to complete the extension.

Length polymorphism analysis of PCR amplicon.  Four µl of each PCR amplicon was mixed with 9 µl of loading buffer (6x loading dye:formamide = 1:5) and 0.55 µl of Rox-500 size standard (Applied Biosystems). The mixture was denatured at 95°C for 2 min; then 1.6 µl of each PCR product was resolved in a 6% denaturing polyacrylamide gel (Long Ranger, FMC Rockland, ME) and recorded by an automatic sequence analyzer (Applied Biosystems, model 377). The gel image was analyzed with computer software (GeneScan 2.1, Applied Biosystems). Euploid amniotic fluid samples (n = 98) were analyzed likewise as controls.

For samples with 2 peaks, the areas under the peaks were determined with GeneScan 2.1 software (Applied Biosystems). Relative ratios of the two peaks were calculated by dividing the larger peak area by the smaller one.

Statistical analysis.  To determine if Down syndrome samples with 2 uneven peaks, that is, nondisjunction that occurred during meiosis II, could be readily differentiated from euploid pregnancies with typical 2 equal-sized peaks, a statistical analysis using the Mann-Whitney U test was conducted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
For Down syndrome cases, if nondisjunction occurred during meiosis I in gametogenesis, the 3 chromosome 21s should be from different parental origins. Therefore, a 3-peak pattern would most likely be expected for these STR markers (Fig. 1AGo). The 3 peaks represent 3 chromosome 21s with 3 different tetranucleotide repeat numbers. If nondisjunction occurred during meiosis II, 2 of the 3 chromosome 21s were from the same parent and only 2 peaks should appear, owing to the identical repeat numbers in 2 of the 3 chromosomes. However, if a peak resulted from the same STR numbers, it should be, theoretically, approximately 2-fold larger than the other peak, due to the double quantity of PCR amplicon (Fig. 1BGo).



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  		Fig 1. Typical peak patterns of short tandem repeat (STR) analysis. A: 3 peaks seen in most Down syndrome cases; B: 2 uneven peaks; C: 1 peak; and D: 2 equal peaks seen in most euploid subjects (see text).

 
Our data revealed that 35 of 40 fetal Down syndrome samples analyzed for IFNAR showed 3 distinct peaks, each band representing an individual chromosome 21. Likewise, 24 of 30 cases analyzed for D21S11 marker showed 3 distinct peaks. It was realized that if either of the STR markers revealed 3-peak pattern, there were 3 chromosome 21s present. Further analyzing other markers would be redundant. Altogether 37 of 40 (92.5%) Down syndrome cases showed 3 distinct peaks for one or both markers, and 2 Down syndrome cases showed two uneven peaks. For most pregnancies complicated with trisomy 21, one of the following types of peaks was seen: (a) 3 separate peaks, and (b) 2 separate peaks, with one bigger than the other (Fig. 1A,BGo). However, one Down syndrome case only had one peak for the IFNAR marker (Fig. 1CGo). Because the DNA sample was depleted, this case was not analyzed for the D21S11 marker. Two equal peaks, each representing a chromosome 21, were anticipated for the euploid pregnancies. Occasionally, both chromosome 21’s STR numbers were the same and only one peak showed. Ninety-eight amniotic fluid samples from euploid pregnancies were analyzed by this protocol; 65 cases had 2 peaks (Fig. 1DGo) in the IFNAR marker, (each peak represents a single chromosome 21 with a unique STR number) and 33 cases had only 1 peak.

To test whether the 2-peak area ratio is approximately 2:1 in 2 cases of Down syndrome resulting from meiosis II nondisjunction, the relative ratios of the peak areas for 65 euploid pregnancies with two peaks as well as the 2 Down syndrome cases with 2 peaks were calculated. Data revealed that for the euploid pregnancies and Down syndrome cases the peak area ratios were 1.31 ± 0.22 and 1.96 ± 0.18 (mean ± SD), respectively (Fig. 2Go). Statistical analysis using Mann-Whitney U test showed that the peak area ratios of the Down syndrome cases were significantly higher than those of the euploid pregnancies with two peaks (p = 0.0012).



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  		Fig 2. Box-and-whisker plot of the 2-peak ratios of STR assays in Down Syndrome (DS, n = 2) and euploid pregnancies (n = 65). A box encloses the middle 50% and a horizontal line inside the box indicates the median. The "+" inside the box shows the mean. Whiskers extend from the quartiles to the lower and upper 1.5 interquartiles. A point above the range is individually marked with an open square.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Most DNA markers with STR consist of length polymorphism of di-, tri-, or tetranucleotide repeats [12]. With the advent of PCR techniques [26], genotyping of STR has become rapid and practical. PCR amplification of dinucleotide repeat polymorphism was difficult to interpret due to the "stuttering" effect. Tri- or tetra-nucleotide repeat polymorphisms, however, stuttered less and their electrophoretic patterns were more unequivocal [20]. Therefore, 2 chromosome 21-specific STR markers with tetranucleotide repeats were tested in this study.

Theoretically, equal peak areas should be observed from the 3 peaks of Down syndrome cases. But minute decreases of peak sizes usually appeared between the 3 peaks. The peak representing the amplicon with the largest repeat number showed the smallest peak size, and vice versa. This could be explained by the efficiency of the PCR amplification of the region containing various length of STR. That is, the allele containing largest number of repeats tended to amplify most slowly, even though the discrepancies were small.

For euploid pregnancies, two peaks were expected in most cases. Data indicated that 65 cases showed two peaks and the relative ratios under the peaks were 1.31 ± 0.22 (mean ± SD), while that of the two-peak pattern cases of the fetal Down syndrome was 1.96 ± 0.18 (mean ± SD), which was very close to the anticipated number, 2 to 1. Therefore, if 2-peak patterns were present, relative peak area ratios should be calculated to differentiate pregnancies complicated with Down syndrome from euploid ones.

Among the cytogenetically confirmed Down syndrome cases, one sample showed only one large peak. Although the possibility of the same repeat numbers in the STR marker on all 3 chromosomes 21 was very low, this possibility could not be completely excluded. It was also conceivable that this case resulted from failed amplification of the other alleles. Unfortunately, the DNA sample was depleted during rechecking and data for the other STR marker in this subject were unattainable. In this case, cytogenetic results provided the ultimate diagnostic evidence.

Molecular cytogenetic methods, such as fluorescent in situ hybridization (FISH), have been proposed for detecting abnormal numbers of chromosomes [27]. However, FISH involves tedious manual laboratory procedures, expensive fluorescence microscopic equipment, and limited sample numbers can be examined simultaneously. An assay can take days to complete. In contrast, STR analysis can be performed with automation, many cases can be analyzed simultaneously, and results can be interpreted immediately after electrophoresis. The whole test can be done within a few hr. Therefore, the STR analysis seems more appropriate for handling large numbers of samples.

Regarding trisomy 21 cases with 2 uneven peaks, it is assumed that 2 possibilities exist: (a) the nondisjunction of chromosome 21 could have occurred during meiosis II so that 2 of the 3 chromosome 21 had identical numbers of STRs, or (b) meiosis II nondisjunction did not occur but the STR numbers were actually the same in 2 of the 3 chromosome 21s. Although the latter assumption is less likely (the probability in the D21S11 marker is estimated to be 0.1 x 0.1 x 0.9 = 0.009 [24], and that in the IFNAR marker is estimated to be 0.17 x 0.17 x 0.83 = 0.024). Such trisomy cases with 2-peaks can be distinguished from those of euploid pregnancies by comparing their peak area ratios.

In conclusion, we have evaluated a molecular protocol for rapid and sensitive determination of the number of chromosome 21 in fetal amniocytes. Taking into consideration the sensitivity of the method and the possibility of automation, it may prove useful for antenatal detection of Down syndrome. However, this is a preliminary investigation and a large-scale study is necessary to validate the clinical application of this protocol.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This project was supported by research grants from Chang Gung Memorial Hospital (CMRP 808, 686) and the National Science Council, Taiwan (NSC-90-2314-B-182-093, NSC90-2314-B-182A-153). We thank Dr Larry Boots and Dr Paula Cosper for support and assistance with this project.


   Footnotes
 
* The first two authors contributed equally to this study. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Motulsky V. Human Genetics, Problems and Approaches. 3rd Ed. Springer, New York, 1997, pp 44–45.
  2. Cuckle HS. Wald NJ. Lindenbaum RH. Maternal serum alpha-fetoprotein measurement: a screening test for Down syndrome. Lancet 1984;1: 926–929.[Medline]
  3. Bogart MH, Pandian MR, Jones OW. Abnormal maternal serum chorionic gonadotropin levels in pregnancies with fetal chromosome abnormalities. Prenat Diag 1987;7: 623–630.[Medline]
  4. Canick JA, Knight GJ, Palomaki GE, et al. Low second trimester maternal serum unconjugated oestriol in pregnancies with Down’s syndrome. Brit J Obstet Gynaecol 1988;95:330–333.[Medline]
  5. Haddow JE, Palomaki GE, Knight GJ, et al. Prenatal screening for Down’s syndrome with use of maternal serum markers. N Engl J Med 1992;327:588–593.[Medline]
  6. Wenstrom KD, Williamson RA, Grant SS, et al. Evaluation of multiple-marker screening for Down syndrome in a statewide population. Am J Obstet Gynecol 1993;169:793–797.[Medline]
  7. Cheng EY, Luthy DA, Zebelman AM, et al. A prospective evaluation of a second-trimester screening test for fetal Down syndrome using maternal serum alpha-fetoprotein, hCG, and unconjugated estriol. Obstet Gynecol 1993; 81:727–7.
  8. Pertl B, Pieber D, Lercher-Hartlieb A, et al. Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders. Mol Hum Reprod 1999;5:1176–1179.[Abstract/Free Full Text]
  9. Wenstrom KD, Chu DC, Owen J, et al. Maternal serum alpha-fetoprotein and dimeric inhibin A detect aneuploidies other than Down syndrome. Am J Obstet Gynecol 1998;179:966–970.[Medline]
  10. Chu DC, Hsu C, Wenstrom KD, et al. Insulin-like growth factor binding protein-3 in the detection of fetal Down syndrome pregnancies. Obstet Gynecol 1998;91:192–195.[Medline]
  11. Wenstrom KD, Owen J, Chu DC, et al. Prospective evaluation of free beta-subunit of human chorionic gonadotropin and dimeric inhibin A for aneuploidy detection. Am J Obstet Gynecol 1999;181: 887–892.[Medline]
  12. Morton NE. Parameters of human genome. Proc Natl Acad Sci USA 1991;88:7474–7476.[Abstract/Free Full Text]
  13. McInnis MG, Chakravarti A, Blaschak J, et al. A linkage map of human chromosome 21:43 PCR markers at average intervals of 2.5 cM. Genomics 1993;16:562–571.[Medline]
  14. Warren AC, Slaugenhaupt SA, Lewis JG, et al. A genetic linkage map of 17 markers on human chromosome 21. Genomics 1989;4:579–591.[Medline]
  15. Petersen MB, Slaugenhaupt SA, Lewis JG, et al. A genetic linkage map of 27 markers on human chromosome 21. Genomics 1991;9:407–419.[Medline]
  16. Adinolfi M, Sherlock J, Pertl B. Rapid detection of selected aneuploidies by quantitative fluorescent PCR. Bioassays 1995;17:661–664.[Medline]
  17. Toth T, Findlay I, Papp C, et al. Prenatal detection of trisomy 21 and 18 from amniotic fluid by quantitative fluorescent polymerase chain reaction. J Med Genet 1998; 35:126–129.[Abstract/Free Full Text]
  18. Ahlbom BE, Goetz P, Korenberg JR, et al. Molecular analysis of chromosome 21 in a patient with a phenotype of Down syndrome and apparently normal karyotype. Am J Med Genet 1996;63:566–572.[Medline]
  19. Findlay I, Toth T, Matthews P, et al. Rapid trisomy diagnosis (21, 18, and 13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis. J Assist Reprod Genet 1998;15:266–275.[Medline]
  20. Kalaitsidaki M, Cox T, Chakravarti A, et al. Cloning and linkage mapping of three polymorphic tetranucleotide (TAAA)n repeats on human chromosome 21. Genomics 1992;14:1071–1075.[Medline]
  21. Warren AC, McInnis MG, Kalaitsidaki M, et al. D21S210: a highly polymorphic (GT)n marker closely linked to the beta-amyloid protein precursor (APP) gene. Hum Genet 1993;91: 87–88.[Medline]
  22. Garofano L, Pizzamiglio M, Vecchio C, et al. Italian population data on thirteen short tandem repeat loci: HUMTH01, D21S11, D18S51, HUMVWFA31, HUMFIBRA, D8S1179, HUMTPOX, HUMCSF1PO, D16S539, D7S820, D13S317, D5S818, D3S1358. Forensic Sci Intl 1998;97: 53–60.
  23. Ko TM, Hwa HL, Tseng LH, Et al. Fluorescence microsatellite analysis to study the parental origin of the supernumerary chromosome in Down’s syndrome. Intl J Gynaecol Obstet. 1998;61:149–153.
  24. McInnis MG, Lutfalla G, Slaugenhaupt S, et al. Linkage mapping of highly informative DNA polymorphisms within the human interferon-alpha receptor gene on chromosome 21. Genomics 1991;11:573–576.[Medline]
  25. Sharma V, Litt M: Tetranucleotide repeat polymorphism at the D21S11 locus. Hum Mol Genet 1992;1:67.[Free Full Text]
  26. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for sickle cell anemia. Science 1985;230: 1350–1354.[Abstract/Free Full Text]
  27. Eiben B, Trawicki W, Hammans W, et al. A prospective comparative study on fluorescence in situ hybridization (FISH) of uncultured amniocytes and standard karyotype analysis. Prenat Diag 1998;18:901–906.[Medline]




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