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Clinical Chemistry / APOLIPOPROTEIN E ALLELE DISTRIBUTION IN TRISOMY 13, 18, AND 21 Apolipoprotein E Allele Distribution in Trisomy 13, 18, and 21 Conceptuses in a Hungarian Population Bálint Nagy, PhD,1 Zoltán Bán, MD,1 Ernö Tóth-Pál, MD,1 Csaba Papp, MD,1 Lou Fintor, MA, MPH,2 and Zoltán Papp, MD, PhD, DSc1 Key Words: Fluorescent polymerase chain reaction; Trisomy 13; Trisomy 18; Trisomy 21; Apolipoprotein E Abstract Reports documented a higher frequency of apolipoprotein E (apoE) allele e4 among mothers of children diagnosed with Down syndrome. We studied the prevalence of apoE alleles among 56 conceptuses with trisomy 13, trisomy 18, or trisomy 21. The presence of the 3 most common apoE alleles (e2, e3, e4) was determined by polymerase chain reaction–restriction fragment length polymorphism, and trisomy status was detected by fluorescent polymerase chain reaction followed by DNA fragment analysis and by conventional cytologic methods. We found no significant difference in the distribution of apoE alleles in the group of trisomy 21 fetuses compared with samples from healthy blood donors. The odds of having trisomy 18 for the apoE e4 group was 3-fold as high as for apoE e3 allele compared with the healthy control group. Furthermore, a statistically significant association was found for those with trisomy 18 and apoE e4, while for those with trisomy 13 and apoE e4, the test showed no significant association. The observed apoE allele e3 frequencies among patients with Down syndrome and healthy control subjects may help explain and support previous work that did not find high rates of atherosclerosis among these persons. The role of apoE alleles in the development of trisomies needs further study. © American Society of Clinical Pathologists Chromosomal abnormalities are the most frequent genetic disorders observed in live births and miscarriages; more than 90% of these cases are attributable to numeric variations of chromosomes 21, 18, and 13.1 These variations usually are diagnosed via in vitro cultures of nucleated cells followed by standard karyotyping procedures or through fluorescence in situ hybridization.2 Fluorescent polymerase chain reaction PCR (F-PCR) recently has been used more commonly for the detection of trisomy conditions.3-5 Intrauterine mortality remains very high among cases of genetic trisomy; in the case of trisomies 13 and 18, only 5% of the cases results live birth, and most of these infants usually die within the first year of life. Among trisomy 21 conceptuses, approximately 15% of the cases result in live birth, and the first-year mortality rate also is quite high. In addition, of the children who survive the first year of life, respiratory infections, cardiac conditions, or leukemia develop in many. With proper treatment, however, many live beyond age 50 years and some well into their 60s. Among patients diagnosed with the nondisjunction of chromosome 21 (trisomy 21) that is commonly referred to as Down syndrome, investigators have documented high mortality rates but also a low incidence of atherosclerosis that persists even through the fifth and sixth decades of life.6 For example, Yla-Herttuala et al7 found a significantly lower number of raised lesions and less calcium in the coronary arteries of 15 persons with Down syndrome. Nevertheless, there is little consensus in the literature about lipid marker levels found in these patients in the absence of genetic studies. Boccini et al6 studied the cholesterol and triglyceride levels in 18 fetuses with trisomy 21 and found higher levels of these lipids in utero compared with those with normal karyotypes. They concluded that increased triglyceride levels might be related Am J Clin Pathol 2000;113:535-538 535 Nagy et al / APOLIPOPROTEIN E ALLELE DISTRIBUTION IN TRISOMY 13, 18, AND 21 to some degree of placental impairment rather than to the genetic abnormality itself. Meanwhile, Brattstrom et al8 found higher cystathionine synthase activity among patients with Down syndrome that they attributed to a higher expression of the gene. However, apolipoprotein E (apoE) is an important factor in lipid metabolism. Synthesized mostly in the liver, apoE alleles are established genetic markers for dyslipidemia and coronary heart disease.9,10 Apolipoprotein E allele frequencies among fetuses with a trisomy condition have not been studied and reported. Materials and Methods DNA Preparation and Cytogenetic Analysis Amniotic fluid specimens were collected from women participating in the Semmelweis University Medical School (Budapest, Hungary) genetic screening program. From January 1997 through December 1998, 56 samples were trisomic. The average age of women with a trisomic fetus was 34.2 years, and the mean gestational age was 15.1 weeks. Institutional review board approval was sought and granted from Semmelweis University, a national maternal health referral center, and informed consent was obtained from all study participants. Amniotic fluid samples of 1.5 mL were centrifuged at 15,000 rpm for 10 minutes to obtain amniocytes, and the supernatant was discarded. Resin, 100 µL (ReadyAmp Genomic Purification System, Promega, Madison, WI), was added to the pellet, and DNA extraction was performed according to the manufacturer’s standard protocol. Four microliters of this DNA solution was used in each PCR reaction. Cytogenetic analysis was performed according to established protocol.11 Fluorescent PCR PCR amplification was performed to detect chromosomal numeric abnormalities using primer pairs D21S11, D21S1411, D21S1412, D13S631, D13S258, D18S851, and D18S51. The forwarding primer was fluorescent dye labeled in each set, and the exact composition of the PCR reaction was identical to that used previously.4,5 Separation and Quantitation of F-PCR Products Four microliters of F-PCR product was mixed with 24 µL of formamide (Amresco, Solon, OH) and 2 µL of Prism Genescan-500 TAMRA size standard (PE Applied Biosystems, Warrington, Great Britain). The mixture was denatured at 95 C for 3 minutes and placed on ice for 5 minutes followed by electrophoretic analysis using POP4 gel (PE Applied Biosystems, Foster City, CA). Amplification products 536 Am J Clin Pathol 2000;113:535-538 were analyzed using Genscan Analysis 2.1 software (PE Applied Biosystems). ApoE Allele Determination With PCR–Restriction Fragment Length Polymorphism ApoE genotyping was performed with 4 µL of isolated DNA (from amniotic fluid) via PCR–restriction fragment length polymorphism (RFLP) according to standard and accepted protocols.9,10,12,13 ApoE alleles were determined after visualization of DNA fragments through HhaI digestion. Blood samples from healthy donors also were collected for DNA extraction and used as controls (n = 253; mean age, 39.9 years). Statistical Analysis Statistical analysis was performed using SPSS software (Statistical Package for the Social Sciences; SPSS, Chicago, IL) to calculate all odd ratios and establish confidence intervals (CIs) at the 95% threshold. Statistically significant associations reported at the P < .05 threshold were determined using the Fisher exact test, and accompanying values were reported as statistically significant only when they were P < .05. Results Trisomy status was detected using conventional cytologic methods along with F-PCR followed by DNA fragment analyses. Results were obtained consistently using both aforementioned methods. In all trisomic samples, apoE alleles were assigned using PCR-RFLP. Among the 56 trisomy cases, 33 patients (59%) were homozygous for apoE e3, whereas among healthy control subjects, 167 (66.0%) were homozygous for e3 ❚Table 1❚. In the trisomy 18 group, which comprised a total of 34 alleles, 7 apoE e4 alleles (20%) and 23 apoE e3 alleles (68%) were detected. To study the potential association between trisomy type and apolipoprotein, alleles were isolated from the samples of 253 healthy blood donors. Of the resulting 506 healthy control alleles, 413 apoE e3 alleles (81.6%) were ❚Table 1❚ Distribution of Apolipoprotein E Genotype in Conceptuses Showing Trisomy Compared With Samples From Healthy Control Subjects* Genotype E 2/2 E 2/3 E 2/4 E 3/3 E 3/3 E 4/4 Trisomy Group (n = 56) 0 (0) 8 (14) 2 (4) 33 (59) 12 (21) 1 (2) Control Group (n = 253) 5 (2.0) 41 (16.2) 2 (0.8) 167 (66.0) 37 (14.6) 1 (0.4) * Data are given as number (percentage). © American Society of Clinical Pathologists Clinical Chemistry / ORIGINAL ARTICLE found. By considering apoE e3 as a common “normal” allele, we computed odds ratios and a 95% CI by comparing each trisomy type with the healthy control group ❚Table 2❚. A statistically significant association between apolipoprotein and trisomy status was determined based on a 95% CI and odds ratio using the Fisher exact test. The only CIs that excluded 1 were the groups that combined apoE e4 and trisomy 13 and the group with trisomy 18 and apoE e4 (Table 2). Thus, the odds of having trisomy 18 for those in the apoE e4 group is 3-fold as high as apoE e3 compared with healthy controls (95% CI, 1.27-7.43). Furthermore, the test showed a statistically significant association for those with trisomy 18 and apoE e4 (P < .05), while for those with trisomy 13 and apoE e4, the test showed at least some significant association (P = .07) Discussion Chromosomal aneuploidy remains one of the major causes of pregnancy wastage in humans. While increased maternal age has been established as a risk factor for chromosomal abnormalities, the biologic mechanism of this phenomenon remains unknown.14 Trisomies 21, 18, and 13 are the most frequently observed aneuploidy conditions among newborns. Lately, apoE and its alleles have become the focus of investigations.14-17 Avramopoulos et al14 found a higher frequency of the apoE e4 allele in young mothers of children with Down syndrome with a meiosis II error. They suggested that this allele is a risk factor,14 while the biologic role of apoE in oocytes remains to be studied. Hansen et al18 studied the frequency of apoE alleles in a combined cohort of samples and found higher e2 allele frequencies among mothers of trisomy 18 conceptuses with meiosis I errors. They combined samples from different countries, but it is important to note that ethnic differences exist in the distribution of apoE alleles. There are similarities in Alzheimer disease and Down syndrome. Hymen et al19 studied the epidemiologic differences associated with the apoE genotype. In both groups of patients, increased beta-amyloid protein deposition was observed in the cerebral lesions and cerebral blood vessels. They found a higher frequency of apoE e4 alleles in patients with Alzheimer disease, while in patients with Down syndrome, the frequency did not differ from that of the healthy control group. The mechanism of the deposition of senile plaques seems to be different. In trisomy 21, there are large plaques reflecting increased betaamyloid production, probably due to the higher activity of the amyloid gene, which is located in the chromosome 21q22 region. This region seems to be critical in the pathogenesis of the Down syndrome phenotype.19 We studied the frequency of apoE alleles among conceptuses with trisomy 21, 18, or 13 in ethnically homogeneous Hungarians. We examined DNA samples of fetuses around the 15th week of gestation so we could include the cases that might not reach full-term gestation period. A healthy population from same region was used as the control group. We applied 2 methods for the detection of aneuploidies, the conventional cytogenetic and F-PCR followed by DNA fragment analysis. PCR-RFLP was used for apoE genotyping; DNA fragments were separated on polyacrylamide gels and visualized with silver staining. We found a higher frequency of apoE e4 allele in the trisomy 13 and trisomy 18 groups. In the trisomy 21 group, we did not observe statistically significant differences in the distribution of apoE alleles. This finding is in accordance with those of previous studies on smaller numbers of patients with Down syndrome.20,21 The biologic role of apoE in the oocyte and the role of the e4 allele in the development of aneuploidies remain to be studied. In the case of trisomy 13 and 18, the microcirculation theory could have a role in the pathogenesis according to Avramopoulos et al.14 As e4 allele carriers have higher plasma cholesterol levels, which is related strongly to atherosclerosis, it could lead to an oxygen deficit inside the follicle and, in turn, diminish the size of the spindle with consequent nondisjunction of a chromosome.14 For trisomy 13 and 18, our results support this explanation. Another hypothesized mechanism for chromosomal ❚Table 2❚ Distribution of Apolipoprotein E (ApoE) Alleles for Conceptuses Showing Trisomy 13, 18, or 21 and in Samples From Healthy Control Subjects* Allele ApoE e2 Conceptuses showing trisomy 13 (n = 18) 18 (n = 34) 21 (n = 60) Healthy control alleles (n = 506) * † 1 (6) 4 (12) 5 (8) 52 (10.3) ApoE e4 4 (22) 7 (20)† 5 (8) 41 (8.1) ApoE e3 13 (72) 23 (68) 50 (83) 413 (81.6) Data are given as number (percentage). P < .05. © American Society of Clinical Pathologists Am J Clin Pathol 2000;113:535-538 537 Nagy et al / APOLIPOPROTEIN E ALLELE DISTRIBUTION IN TRISOMY 13, 18, AND 21 segregation is the allele-specific antioxidant activity protecting from oxidative cell death, with E2 being the most protective followed by E3 then E4.16,18,22 The 2-hit model also is in use, and apoE could represent an example of disruption of the meiotic process due to the isoform-specific binding to microtubule-associated proteins and possible interference with microtubule stability and function.18,23,24 It is difficult to decide which is the most appropriate of the aforementioned theories. We conclude that the frequency of the e4 allele is higher in the case of trisomies 13 and 18. We found no difference in the case of trisomy 21 compared with the healthy Hungarian population. This observation helps explain and support the findings of the studies that did not find significantly higher rates of atherosclerosis among patients with Down syndrome.7,20,21 It also supports the hypothesis that suggests more cystathione synthetase production (the gene is located on chromosome 21) and as a consequence, lower homocysteine levels in these patients. These preliminary results require additional study to unravel the elusive cause of chromosomal numeric abnormalities. From the 1Molecular Diagnostics Laboratory, First Department of Obstetrics and Gynecology, Semmelweis University Medical School, Budapest, Hungary; and the 2Department of Internal Medicine, The University of Michigan Medical School, Ann Arbor, MI. Mr Fintor is now with McGill University, Montreal, Quebec, Canada. Address reprint requests to Dr Nagy: Liljasaarentie 3A2, 00340 Helsinki, Finland. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. References 1. Cuckle HS, Wald NJ. Screening for Down’s syndrome. In Liford R, ed. Prenatal Diagnosis and Prognosis. Vol 11. London, England: Butterworths;1990:67-92. 20. 2. Adinolfi M, Crolla J. Nonisotopic in situ hybridization: clinical cytogenetics and gene applications. Adv Hum Genet. 1994;22:187-255. 21. 3. Adinolfi M, Pertl B, Sherlock J. Rapid detection of aneuploidies by microsatellite and quantitative fluorescent polymerase chain reaction. Prenat Diagn. 1997;17:1299-1311. 4. Tóth 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. 5. Findlay I, Tóth T, Matthews P, et al. Rapid trisomy diagnosis (21, 18 and 13) using fluorescent PCR and short tandem repeats: application for prenatal diagnosis and preimplantation genetic diagnosis. J Assist Reprod Genet. 1998;15:266-275. 6. Boccini L, Nava S, Fogliani R, et al. Trisomy 21 is associated with hypercholesterolemia during intrauterine life. Am J Obstet Gynecol. 1997;176:540-543. 7. Yla-Herttuala S, Luoma J, Nikkari T, et al. Down’s syndrome 538 Am J Clin Pathol 2000;113:535-538 22. 23. 24. and atherosclerosis. Atherosclerosis. 1989;76:269-272. Brattstrom L, Englund E, Brun A. Does Down syndrome support homocysteine theory of atherosclerosis. Lancet. 1987;1:391-392. Nagy B, Rigó J, Fintor L, et al. Apolipoprotein E alleles in women with severe pre-eclampsia. J Clin Pathol. 1998;51:324325. Nagy B, Karádi I, Fintor L, et al. Apolipoprotein E gene polymorphism frequencies in a sample of healthy Hungarians. Clin Chim Acta. 1999;282:147-150. Wolstolme J, Crocker M, Jonasson J. A study of chromosomal aberrations in amniotic fluid cell cultures. Prenat Diagn. 1988;8:339-353. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:545-548. Clavel C, Durlach A, Durlach V, et al. Rapid and sensitive determination of human apolipoprotein E genotypes by miniaturized SDS-PAGE in non-insulin dependent diabetes mellitus. J Clin Pathol. 1995;48:295-299. Avramopoulos D, Mikkelsen M, Vassilopoulos D, et al. Apolipoprotein allele distribution in parents of Down’s syndrome children. Lancet. 1996;347:862-865. Fisher JM, Harvey JF, Morton NE, et al. Trisomy 18: studies of parent and cell division of origin and the effect of aberrant recombination on non-disjunction. Am J Hum Genet. 1995;56:669-675. Gaulden ME. Maternal age effect: the enigma of Down syndrome and other trisomic conditions. Mutat Res. 1992;296:69-88. Salo MK, Solakivi-Jaakkola T, Kivimaki T, et al. Plasma lipids and lipoproteins in Down’s syndrome. Scand J Clin Lab Invest. 1979;39:485-490. Hansen C, Bugge M, Brandt AC, et al. Apolipoprotein E alleles in mothers of trisomy 18 conceptuses. Clin Genet. 1998;53:321-322. Hyman TB, West LH, Rebeck WG, et al. Quantitative analysis of senile plaques in Alzheimer disease: observation of log-normal size distribution and molecular epidemiology of differences associated with apolipoprotein E genotype and trisomy 21 (Down syndrome). Proc Natl Acad Sci U S A. 1995;92:3583-3590. Saunders MA, Schmader K, Breitner SCJ, et al. Apolipoprotein E e4 allele distribution in late onset Alzheimer’s disease and in other amyloid forming diseases. Lancet. 1994;343:979-980. Pickering-Brown MS, Mann AMD, Bourke PJ, et al. Apolipoprotein E4 and Alzheimer disease pathology in Lewy body disease and in other beta-amyloid–forming disease [letter]. Lancet. 1994;343:1155. Miyata M, Smith JD. Apolipoprotein E allele specific antioxidant activity and effects on cytotoxicity by oxidative insults and beta-amyloid peptides. Nat Genet. 1996;14:55-61. Strittmatter WJ, Saunders AM, Goedert M, et al. Isoformspecific interaction of apolipoprotein E with microtubuleassociated protein tau: implications for Alzheimer disease. Proc Natl Acad Sci U S A. 1994;91:11183-11186. Strittmatter WJ, Saunders AM, Schmechel D, et al. Apolipoprotein E: high avidity binding beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci U S A. 1993;90: 1977-1981. © American Society of Clinical Pathologists