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DOI: 10.1369/jhc.4B6510.2005
Volume 53 (3): 371-373, 2005
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
QF-PCR Examination of Parental and Meiotic Origin of Trisomy 21 in Central and Eastern Europe
Institute of Biology and Medical Genetics, University Hospital Motol, Charles University, Second Medical School, Prague, Czech Republic (MM,MB,AK,MM,MM); Institute of Human Genetics, Charité, Universitaetsmedizin Berlin, Berlin, Germany (AK,KS); Molecular Cytogenetic Laboratory, Institute of Pediatrics and Children Surgery, Ministry of Health, Moscow, Russia (SV); Centre of Clinical Morphology and Genetics, Rostov State Medical University, Ministry of Health, Rostov, Russia (SK); Genetic Department of Institute of Medical Improvement, Kiev, Ukraine (TZ); Interregional Medico-Genetics Centre, Central Hospital Clinic No. 1, Donetsk, Ukraine (SA); Institute of Child Health, Athens, Greece (MP); and Department of Genetics and Microbiology, Charles University, Faculty of Science, Prague, Czech Republic (RK)
Correspondence to: Milan Macek Sr., University Hospital Motol, Institute of Biology and Medical Genetics, Laboratory of Assisted Reproduction and Reproductive Genetics, V Úvalu 84, Prague–5, 150 06, Czech Republic. E-mail: pavel.roubic{at}lfmotol.cuni.cz
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Summary |
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 Top Summary Literature Cited |
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Study of parental/meiotic origin of free trisomy 21 in nuclear families from Russia (70 cases), Ukraine (32 cases), and 22 from Germany revealed maternal nondisjunction in 77.3% (Germany), 93.8% (Ukraine), and 91.4% (Russia), paternal origin in 13.6%, 6.2%, and 8.6%, respectively. Maternal meiosis I errors were found in 84.4% (Ukraine), 77.1% (Russia), paternal origin in 3.1% (Ukraine), 2.9% (Russia). Maternal meiosis II errors occurred in 9.4% and 14.3% and paternal in 3.1% and 5.7% in Ukraine and Russia, respectively. No significant differences were found in maternal/paternal origin among Ukraine, Russia, Germany, and published data from other European regions.
(J Histochem Cytochem 53:371–373, 2005)
Key Words: quantitative fluorescent PCR • trisomy 21 • parental/meiotic origin • Ukraine • Russia
TRISOMY 21 is the most frequent aneuploidy in newborns that originates from nondisjunction at meiosis. Mitosis–postzygotic error incidence occurs in 
5% according to Antonarakis et al. (1993)
. Results from 510 families with DNA polymorphic markers suggest meiotic origin in 94.5% of cases (Antonarakis 1991; Petersen et al. 1992; Yoon et al. 1996). These studies provide evidence that in 72%, nondisjunction occurred in the first maternal meiotic division and in 21% in the second division that is around conception. Antonarakis (1998) disclosed that, in 2.7% of trisomy 21 cases, nondisjunction takes place in the first paternal meiotic division and in 4.3% in the second paternal meiotic division.
Gomez et al. (2000) revealed that, in the Spanish population, trisomy 21 by origin was 80% maternal (90.6% meiosis I, 6.2% meiosis II, 3.1% maternal mosaicism), in 5.6% paternal (50% meiosis I, 50% meiosis II), and in 5.6% mitotic.
Muller et al. (2000) in the French prenatal trisomy 21 study reported 102 informative cases of maternal origin of trisomy 21 in 89.2% and increased paternal origin in 10.8%. In the case of maternal origin, meiosis I was detected in 77% and meiosis II in 23%. In paternal origin of trisomy 21, meiosis I was found in 44% and meiosis II in 56%. Their analysis did not support the hypothesis that an increased proportion of paternal meiotic disorders is caused by the selective loss of paternal origin fetuses.
The meiotic process is very sensitive to the effects of noxious endogenous and exogenous factors, as suggested by the cluster of Down syndrome incidence in Berlin in the first month following the Chernobyl accident (Sperling et al. 1994). Therefore, the impact of the Chernobyl accident on the increased prevalence of Down syndrome in Western Europe during the 1980s merits further investigation (Dolk and Nichols 1999).
Studies of parental and meiotic origin of trisomy 21 and case–control surveillance of these factors are important for further understanding of the pathogenesis of chromosome 21 nondisjunction (Macek et al. 2003).
Therefore, the aim of this study was to assess the parental and meiotic origin of free trisomy 21 in nuclear families in different regions of Ukraine and Russia comparing to Central Europe by means of quantitative fluorescent PCR (QF-PCR) to find out the possible differences due to different lifestyles and to load of exogenous noxious agents including Chernobyl radiation accident exposure. Cases with translocation trisomy 21 detected by cytogenetic examination were excluded.
From 208 referred nuclear families from Ukraine and Russia, 87 incomplete families and families with poor DNA quality samples were excluded as well as 19 families due to the non-informativity of the STR markers or inconclusive peak height ratio (PHR) in the range between 1.5 and 1.7 in more than two investigated markers. Finally, 102 nuclear families from Russia and Ukraine and 22 from Germany (Berlin region) were examined by QF-PCR. All families excluded from the current study will be analyzed in the near future with more STR markers.
DNA samples from Moscow, Rostov (Russia), and Kiev (Ukraine) were obtained by classical DNA extraction method. DNA from frozen blood samples or amniotic fluid referred from Donetsk (Ukraine) was extracted by commercial kit (PUREGENE Blood Kit; Gentra Systems, Minneapolis, MN) and QiaAmp DNA mini Kit (QIAGEN GmbH; Hilden, Germany), respectively.
QF-PCR (Pertl et al. 1999) was slightly modified (Macek et al. 2002). Each STR marker was analyzed in singleplex reaction with the exception of the first samples analyzed by sets of primers. Previously used dinucleotide marker D21S167 was eliminated due to its high peak height ratio variability. New STR markers (D21S1432, D21S1446, D21S1270, and D21S2052) were added. The following fluorescently labeled primers for the detection of trisomy 21 were used: D21S11 (Fam), D21S1414 (Fam), D21S1446 (Fam), D21S2052 (Fam), D21S167 (Fam), D21S1411 (Hex), D21S1412 (Hex), D21S1432 (Hex), and D21S1270 (Hex).
PCR was carried out in a total of 15 µl of reaction mix containing 1x reaction buffer PE, 1.5 mM MgCl2 PE, 200 µM each of dNTP, and 1 U Ampli Taq Gold polymerase PE (all from Applied Biosystems, Foster City, CA), 5 pmol of each primer, and 50 ng of DNA. The amplification was performed in PTC–200 DNA Thermal Cycler (MJ Research, Inc., Waltham, MA) started with 12 min of initial denaturation at 94C followed by 30 cycles of denaturation at 94C for 60 sec, annealing at 55–60C for 60 sec, elongation at 72C for 60 sec, and the final extension at 72C for 10 min. The fragment analysis of fluorescently labeled PCR products was performed on ABI Prism 310 Genetic Analyzer (Applied Biosystems) following manufacturers recommendations, and electrophoresis results were analyzed with the GeneScan Analysis Software.
For the assessment of trisomy, the PHR was evaluated. In triallelic trisomies it was 1:1:1. The trisomy diagnosis is safe. It reveals nondisjunction in meiosis I. Parental and meiotic origin is reliably ascertained if parental heterozygosity confined to different electrophoretic localizations is preserved.
If parental heterozygosity was reduced to homozygosity, the error occurred during meiosis II indicating diallelic type of trisomy with PHR 2:1.
Monoallelic and diallelic cases with PHR were in the range of 1.4–1.7, or in the case of non-informativity, more STR markers were used. All diagnoses were confirmed by two repeated examinations.
For parental and meiotic origin detection, QF-PCR analysis of both parents is necessary, and the conclusions must be confirmed by at least two informative STR markers.
For the statistical evaluation of the differences in the prevalence of the parental and meiotic origin of trisomy 21 in Central Europe and different regions of Ukraine and Russia, homogeneity test in contingency tables and 
2 test were used.
Table 1 documents proportion of meiosis I and II and parental origin of nondisjunction in 22 nuclear families from Berlin, 32 from Ukraine, and 70 from Russia. Statistical analysis did not reveal significant differences in prevalence of meiosis I and II disorders in Kiev and Donetsk regions of Ukraine, as well as between Moscow and Rostov regions. No statistically significant differences were disclosed between proportion in meiosis I/II and maternal/paternal origin between Berlin, Ukraine, and Russia nuclear families with trisomy 21.
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Table 2 documents maternal/paternal proportions in meiosis I and meiosis II in the studied regions of Ukraine and Russia. Statistical analysis did not disclose any significant differences between maternal and paternal proportions in meiosis I between cases in Ukraine and Russia. The same was true for maternal/paternal proportion in meiosis II
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These results are completely based on the QF-PCR analysis of the parental and meiotic origin of trisomy 21 with preservation of strict criteria to determine exactly the reliable conclusions. This study confirms that QF-PCR is also a very effective method for population studies of meiotic and parental disorders for aneuploidy analysis with very high throughput of 48 samples per 24 hr by ABI Prism 310 Genetic Analyzer.
The analysis of these preliminary data documents that prevalence of maternal meiosis disorders in Ukraine (93.8%) and Russia (91.3%) does not differ from the data ascertaining maternal origin of extra chromosome 21 (Antonarakis 1991; Petersen et al. 1992; Antonarakis et al. 1993; Yoon et al. 1996). The same follows also from the comparison of the proportions of meiosis I and meiosis II in studied populations from France and Spain. The same is true for the comparison of paternal origin—6.2% from Ukraine and 7.8% from Russia, respectively.
If these observations are confirmed by further analyses of Russian and Ukraine nuclear families with trisomy 21 in more than 150 families, it will suggest that different lifestyles, noxious exogenous factors load, and Chernobyl radiation exposure have no impact on proportions of parental and meiotic origin of trisomy 21 in postnatal and prenatal (second trimester) phase of development.
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Acknowledgments |
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This study was supported by contract of EU Copernicus-2-ICA-2-CT-2000-10012; Czech Ministry of Health 00000064203 and IGA NE-6462-3, NR/7962-3; Czech Ministry of School and Educations No. 111300003 and LN00A079.
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Footnotes |
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Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich-Schiller-University, Jena, Germany, April 17–18, 2004.
Received for publication August 25, 2004; accepted December 15, 2004
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Literature Cited |
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TopSummaryLiterature Cited |
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Antonarakis SE (1991) Parental origin of the extra chromosome in trisomy 21 as indicated by analysis of DNA polymorphisms. Down Syndrome Collaborative Group. N Engl J Med 324:872–876
Antonarakis SE, Avramopoulos D, Blouin JL, Talbot CC Jr, Schinzel AA (1993) Mitotic errors in somatic cells cause trisomy 21 in about 4.5% of cases and are not associated with advanced maternal age. Nat Genet 3:146–150[CrossRef][Medline]
Antonarakis SE (1998) 10 years of Genomics, chromosome 21, and Down syndrome. Genomics 51:1–16[CrossRef][Medline]
Dolk H, Nichols R (1999) Evaluation of the impact of Chernobyl on the prevalence of congenital anomalies in 16 regions of Europe. EUROCAT Working Group. Int J Epidemiol 28:941–948
Gomez D, Solsona E, Guitart M, Baena N, Gabau E, Egozcue J, Caballin MR (2000) Origin of trisomy 21 in Down syndrome cases from a Spanish population registry. Ann Genet 43:23–28[Medline]
Macek M Sr, Krebsova A, Horka I, Diblik J, Matejckova M, Brouckova M, Chudoba D, et al. (2002) Quantitative fluorescent PCR (QFPCR) in the examination of microquantity of fetal cell. In Macek M Sr, Bianchi DW, Cuckle H, eds. Early Prenatal Diagnosis, Fetal Cells and DNA in the Mother, Present State and Perspectives. Prague, The Karolinum Press, 304–328
Macek M Sr, Krebsova A, Brouckova M, Matejckova M, Machatkova M, Diblik J, Sperling K, et al. (2003) Quantitative fluorescent polymerase chain reaction (QFPCR) in the prenatal and postnatal diagnosis of the most frequent aneuploidies. Balkan J Med Gen 6:87–94
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Pertl B, Pieber D, Lercher-Hartlieb A, Orescovic I, Haeusler M, Winter R, Kroisel P, et al. (1999) Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders. Mol Hum Reprod 5:1176–1179
Petersen MB, Frantzen M, Antonarakis SE, Warren AC, Van Broeckhoven C, Chakravarti A, Cox TK, et al. (1992) Comparative study of microsatellite and cytogenetic markers for detecting the origin of the nondisjoined chromosome 21 in Down syndrome. Am J Hum Genet 51:516–525[Medline]
Sperling K, Pelz J, Wegner RD, Dorries A, Gruters A, Mikkelsen M (1994) Significant increase in trisomy 21 in Berlin nine months after the Chernobyl reactor accident: temporal correlation or causal relation? Br Med J 309:158–162
Yoon PW, Freeman SB, Sherman SL, Taft LF, Gu Y, Pettay D, Flanders WD, et al. (1996) Advanced maternal age and the risk of Down syndrome characterized by the meiotic stage of chromosomal error: a population-based study. Am J Hum Genet 58:628–633[Medline]
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