Circulating angiogenic proteins in trisomy 13

American Journal of Obstetrics and Gynecology (2006) 194, 239–45 www.ajog.org Circulating angiogenic proteins in trisomy 13 Yuval Bdolah, MD, MSc,a Glenn E. Palomaki, BS,b,c Yuval Yaron, MD,d Tali Bdolah-Abram, MA,e Marlene Goldman, ScD,a Richard J. Levine, MD, MPH,f Benjamin P. Sachs, MB, BS, DPH,a James E. Haddow, MD,b,c S. Ananth Karumanchi, MDa,* Departments of Medicine and Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MAa; Foundation for Blood Research, Scarborough, MEb; Division of Medical Screening, Department of Pathology, Women and Infants Hospital of Rhode Island, Providence, RI c; Prenatal Diagnosis Unit, Genetic Institute, Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel d; Jerusalem, Israel e; Division of Epidemiology, Statistics and Prevention Research, National Institute of Child Health and Human Development, Department of Health and Human Services, Bethesda, MDf Received for publication March 20, 2005; revised May 17, 2005; accepted June 7, 2005 KEY WORDS Preeclampsia Trisomy 13 Soluble fms-like tyrosine kinase-1 (sFlt-1) Placental growth factor Angiogenesis factor Objective: Women who are carrying a trisomy 13 fetus are more prone to develop preeclampsia. Excess circulating soluble fms-like tyrosine kinase-1 has been implicated recently in the pathogenesis of preeclampsia. Since the fms-like tyrosine kinase-1/soluble fms-like tyrosine kinase-1 gene is located on chromosome 13q12, we hypothesized that the extra copy of this gene in trisomy 13 may lead to excess circulating soluble fms-like tyrosine kinase-1, reduced free placental growth factor level, and increased soluble fms-like tyrosine kinase-1/placental growth factor ratio. This may then contribute to the increased risk of preeclampsia that has been observed in these patients. Our objective was to characterize the maternal circulating angiogenic proteins in trisomy 13 pregnancies. Study design: Maternal serum samples of trisomy 13, 18, 21 and normal karyotype pregnancies were obtained from first and second trimester screening programs. We chose 17 cases of trisomy 13 that were matched for maternal age, freezer storage time, and parity with 85 normal karyotype control samples. Additionally, 20 cases of trisomy 18 and 17 cases of trisomy 21 were included. Cases and control samples were assayed for levels of soluble fms-like tyrosine kinase-1 and placental growth factor by enzyme-linked immunosorbent assay in a blinded fashion. Because of the skewed distributions of soluble fms-like tyrosine kinase-1 and placental growth factor, nonparametric analytic techniques were used, and the results are reported as median and ranges. Results: In early pregnancy trisomy 13 cases and control samples, the median circulating soluble fms-like tyrosine kinase-1/placental growth factor ratios were 17.0 (range, 1.2-61.3) and 6.7 (range, 0.8-62.9), respectively (P = .003). The median soluble fms-like tyrosine kinase-1/placental Supported in part by a fellowship from the American Physicians Fellowship for Medicine in Israel (Y.B) and by National Institutes of Health grant HL 079594 (S.A.K.). Dr Karumanchi is listed as co-inventer on a patent filed by the Beth Israel Deaconess Medical Center for the use of angiogenic proteins in the diagnosis and treatment of preeclampsia. * Reprint requests: S. Ananth Karumanchi, MD, Beth Israel Deaconess Medical Center, 330 Brookline Ave, RW 663B, Boston, MA 02215. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.06.031 240 Bdolah et al growth factor ratios in trisomy 18 and 21 were 4.8 (range, 0.9-53.9) and 5.1 (range, 1.0-18.1), which were not significantly different than the control samples. Furthermore, the differences between trisomy 13 and control samples were more pronounced in the second trimester specimens than in the specimens from the first trimester. Conclusion: These data suggest that alterations in circulating angiogenic factors may be involved intimately in the pathogenesis of preeclampsia in trisomy 13. A larger clinical study that measures these factors longitudinally and correlates them with pregnancy outcomes is needed to further establish the link between trisomy 13, altered angiogenic factors, and preeclampsia. Ó 2006 Mosby, Inc. All rights reserved. Preeclampsia is a multiorgan disorder that complicates 5% to 7% of all pregnancies and is associated with significant morbidity and mortality rates for the mother and fetus. In most cases, preeclampsia resolves completely after the delivery of the placenta,1,2 which suggests that the placenta plays a central role in its pathogenesis. Although most preeclampsia occurs in healthy nulliparous women, risk factors include previous preeclampsia, multiple gestation, pre-existing hypertension, diabetes mellitus, or the presence of a trisomy 13 fetus. Trisomy 13 is a chromosomal aberration that occurs in approximately 1 in 5000 live births3 and accounts for 6 in 100 spontaneous abortions.4 Affected newborn infants have multiple severe malformations. Most fetuses who have this anomaly are aborted spontaneously during the first 2 trimesters of pregnancy, and many prenatally diagnosed pregnancies are terminated. One half of the newborn infants die within a month, and only 5% to 10% survive beyond the first year.3,5 Second and third trimester trisomy 13 pregnancies are more prone to the development of preeclampsia. Tuohy and James6 estimated that the incidence of preeclampsia in pregnancies that were complicated by trisomy 13 was significantly higher than the incidence in normal karyotype control subjects (24.0%-44.0% vs 2.0%-8.0%). Numerous other studies report trisomy 13 pregnancies that are linked with preeclampsia.7-14 Hence, it has been hypothesized that gene products that are coded for on chromosome 13 may play a pathogenic role in preeclampsia.6,8,9,11,13 We recently presented evidence that excess secretion of a naturally occurring anti-angiogenic molecule of placental origin, which is referred to as soluble fms-like tyrosine kinase-1 (sFlt-1; also referred to as sVEGFR1), may contribute to the pathogenesis of the maternal syndrome in preeclampsia.15 sFlt-1 acts by antagonizing 2 proangiogenic molecules, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Several recent studies have confirmed our findings.16-20 Increased sFlt-1 is associated with decreased circulating free PlGF and free VEGF at the time of clinical preeclampsia. Furthermore, alterations in sFlt-1 and free PlGF antedate the onset of clinical symptoms.21 sFlt-1 was noted to be elevated 5 weeks before clinical symptoms, whereas PlGF levels are lower in patients destined to have clinical preeclampsia, even as early as the first trimester.22 In these patients, PlGF concentrations were lower at times when sFlt-1 concentrations were normal; thus, besides reflecting levels of sFlt-1 later in gestation, they also reflect a susceptibility to the development of preeclampsia. The ratio of sFlt-1/PlGF is a more biologically reliable index of the circulating angiogenic state than either of the markers alone and was found to be a better predictor of preeclampsia risk.23 Collectively, these data suggest that alterations in circulating angiogenic factors play a pivotal role in the pathogenesis of the preeclampsia maternal syndrome.24,25 sFlt-1, a splice variant of the Flt-1 gene is encoded on the long arm of chromosome 13, specifically in the 13q12 region. Hence, we hypothesized that the extra copy of the placental Flt-1 gene in women who carry trisomy 13 fetuses may lead to increased serum levels of circulating sFlt-1 in these patients. We further hypothesized that the increased circulating sFlt-1 in women who carry trisomy 13 fetuses will be associated with decreased free PlGF and that the alteration in the circulating angiogenic state may be responsible for the increased risk of preeclampsia that is observed in these patients. The aim of this study was to examine the serum concentrations of sFlt-1 and PlGF in pregnancies with chromosomal abnormalities (trisomy 13, 18, and 21) compared with pregnancies with normal karyotypes and to demonstrate that alterations of sFlt-1 and PlGF are specific for trisomy 13 pregnancies. Archived first trimester and second trimester specimens that were obtained as part of an early gestation fetal malformation screening program were used for this study. Material and methods Maternal serum specimens Maternal serum specimens from pregnancies with trisomy 13, 18, or 21 and those pregnancies from normal karyotypes were obtained from the archived serum bank of the Foundation for Blood Research (Scarborough, Maine). All serum specimens were collected before knowledge of the karyotype. The samples that were Bdolah et al used for this study were collected between 1998 and 2003. The sample size was calculated on the basis of previous knowledge of the normal sFlt-1 and PlGF values during early pregnancy21 and on the hypothesis that a difference of 50% in the concentrations of sFlt-1 and PlGF may be present in women who carry trisomy 13 fetuses. A total of 17 serum samples from trisomy 13 pregnancies were identified as cases, each of which was matched with 5 samples from normal karyotype pregnancies (control). Matching factors were collection location, parity (primiparous vs multiparous), maternal age (to within 2 years) and length of time in the freezer (within 1 month). Of the 17 trisomy 13 cases, 6 cases were first trimester samples that were matched with 30 control pregnancies, and 11 cases were second trimester samples that were matched with 55 control pregnancies (total, 85 controls). Nine cases were primiparous pregnancies that were matched with 45 control pregnancies; 8 cases were multiparous pregnancies that were matched with 40 control pregnancies. Additionally, samples from 20 trisomy 18 pregnancies and 17 trisomy 21 pregnancies were included. The study was approved by the Foundation for Blood Research Institutional Review Board. sFlt-1 and PlGF serum levels Measurements of sFlt-1 and free PlGF serum concentrations were performed by personnel who were blinded to the outcome of the pregnancy. Enzyme-linked immunosorbent assays for human sFlt-1 and free PlGF were performed in duplicate, as previously described,21 with the use of commercial kits (R&D Systems, Minneapolis, MN). The minimal detectable doses in the assays for sFlt-1 and free PlGF were 5 pg/mL. The short-term coefficients of variation (both within and between plates) for sFlt-1 and PlGF were 18% and 8%, respectively, as determined by multiple blinded measurements of duplicate patient samples. Statistical analysis The outcome variables of this study (sFlt-1, PlGF, and sFlt/PlGF ratio) are presented as median with ranges (in the text) and as box and whisker plots with medians (Figures 1 and 2).26 For between-group comparison, the nonparametric Mann-Whitney test was applied because the distributions of sFlt-1 and PlGF were highly skewed. All tests were 2-tailed, and a probability value of !.05 was considered statistically significant. SPSS software (SPSS Inc, Chicago, IL) was used for analysis. Results The clinical characteristics of the patients used in this study are described in the materials and methods. Serum 241 Figure 1 Box plot with whiskers of the ratio of sFlt-1/PlGF in normal karyotype control subjects (n = 85) and patients with trisomy 13 (n = 17), trisomy 18 (n = 20), and trisomy 21 (n = 17). The bold line represents the median, and the box and whiskers with the interquartile range is presented. The points beyond the whiskers represent outliers. The triple asterisk represents a probability value of .003, as compared with control subjects. sFlt-1 and PlGF concentrations were analyzed in all the control and trisomy specimens. The median sFlt-1 concentrations were 675.4 pg/mL (range, 215.2-2331.9 pg/ mL) for the normal karyotype control specimens, 780.5 pg/mL (range, 255.2-2134.2 pg/mL) for the trisomy 13 specimens, 341.6 pg/mL (range, 150.0-1447.6 pg/mL) for the trisomy 18 specimens, and 919.9 pg/mL (range, 223.41691.3 pg/mL) for the trisomy 21 specimens. Although the median sFlt-1 concentration in the trisomy 13 group was modestly higher than the control group, the data were not statistically significant. sFlt-1 concentrations in the trisomy 18 and 21 were also not altered significantly when compared with control concentrations. The median PlGF concentrations were 113.3 pg/mL (range, 23.5-454.9 pg/mL) for the normal karyotype specimens, 49.5 pg/mL (range, 19.3-249.6 pg/mL) for the trisomy 13 specimens, 56.1 pg/mL (range, 21.6-570.2 pg/mL) for the trisomy 18 specimens, and 151.6 pg/mL (range, 76.1-350.6 pg/mL) for the trisomy 21 specimens. The PlGF decrease in the trisomy 13 specimens was statistically significant when compared with control specimens (P = .03). Trisomy 18 and 21 specimens did not have a significant decrease in PlGF concentrations when compared with control specimens. These data suggest that pregnant women who carry trisomy 13 fetuses, but not other trisomies, tend to have modestly higher circulating sFlt-1 and significantly lower PlGF levels. The ratio of sFlt-1/PlGF is a more reliable index of the circulating angiogenic state than either of the markers alone and was found to be a better predictor of preeclampsia risk.23 Hence, we calculated the ratios of sFlt-1/PlGF for all the specimens. The median ratios of 242 Figure 2 Box plot with whiskers of the ratio of sFlt-1/PlGF in the normal karyotype control samples (n = 30 and n = 55) and specimens of trisomy 13 (n = 6 and n = 11) in first and second trimester serum specimens, respectively. The triple asterisk represents a probability value of .007 in the trisomy 13, as compared to control specimens. sFlt-1/PlGF were 6.7 (range, 0.8-62.9) for the normal karyotype control specimens, 17.0 (range, 1.2-61.3) for the trisomy 13 specimens, 4.8 (range, 0.9-53.9) for the trisomy 18 specimens, and 5.1 (range, 1.0-18.1) for the trisomy 21 specimens (Figure 1). The increased ratio that was observed in the trisomy 13 specimens was highly statistically significant (P = .003) when compared with control specimens, although the sFlt-1/PlGF ratio was not significantly altered in the other trisomies. Because this study included a mixture of first trimester and second trimester specimens, we then studied the changes of sFlt-1 and PlGF in the trisomy 13 cases and the control pregnancies on the basis of gestational ages. Although only 6 specimens were collected during the first trimester in the trisomy 13 group, the median sFlt1/PlGF ratio, nevertheless, was modestly elevated (by approximately 23%) compared with the normal karyotype control specimens (19.6 vs 15.9; Figure 2). The ratios of sFlt-1/PlGF were significantly elevated in the second trimester trisomy 13 cases compared with normal karyotype control specimens (9.4 vs 5.1; P = .007; Figure 2). These data suggest that, although there were modest differences in these angiogenic factors in early gestation, they became more apparent at later gestation. These findings are consistent with the dramatic alterations in sFlt-1 and PlGF that are noted in pregnancies that are complicated by preeclampsia during second and early third trimester in the weeks preceding clinical symptoms. Comment We previously hypothesized that alterations in circulating angiogenic factors (sFlt-1, free PlGF, and free Bdolah et al VEGF) may play a pathogenic role in preeclampsia.15 Increased sFlt-1 and decreased free PlGF have been found in women with preeclampsia not only during clinical disease, but even before clinical symptoms.21,22 Women with trisomy 13 pregnancies have a higher rate of preeclampsia, regardless of parity.8,9 Other chromosomal abnormalities, such as trisomy 18 or trisomy 21, have rates that are similar to the general population. As the gene for Flt-1 and sFlt-1 is coded on chromosome 13, we hypothesized that trisomy 13 pregnancies would have a higher basal level of circulating sFlt-1 and would produce more sFlt-1 with advancing gestation as compared with normal karyotype control pregnancies. Furthermore, because high circulating sFlt-1 is associated with a decreased free PlGF, we hypothesized that free PlGF would be decreased in trisomy 13 pregnancies and that the ratio of sFlt-1/PlGF would be increased. In this case-control study, we have compared 17 trisomy 13 cases with 85 control pregnancies, which were closely matched for maternal age, trimester of pregnancy, parity, and freezer storage time (5 control pregnancies for each case). Additional control pregnancies were provided by evaluating 2 other trisomies, 18 and 21. We have demonstrated that circulating sFlt-1 is on average 35% higher (although not statistically significant) and that circulating free PlGF is 32% lower in trisomy 13 pregnancies during early gestation, as compared with normal karyotype control pregnancies. We further demonstrate that the ratio of sFlt-1/PlGF is significantly increased in trisomy 13 cases as compared with normal karyotype control pregnancies and that this difference is more apparent during second trimester than during first trimester. Furthermore, the ratio of sFlt-1/ PlGF is not elevated in other trisomies such as 18 or 21. Finally, the alterations in sFlt-1 and PlGF levels that were observed in trisomy 13 patients during the first and second trimester are similar to the changes that have been reported in pregnancies that subsequently developed preeclampsia.21,22 These data suggest that the increased sFlt-1 and decreased free PlGF levels that are noted in trisomy 13 patients may contribute to an increased risk of preeclampsia that was reported previously in these patients. We have hypothesized that the abnormal placentation of preeclampsia leads to the elaboration of antiangiogenic factors, which in turn induces the hypertensive syndrome.15,21 Therefore, it makes sense that abnormalities in angiogenic factors in women who are at risk for preeclampsia are more apparent with increasing gestation. An interesting result is that PlGF was significantly higher in the trisomy 21 samples than in the control samples, which is consistent with some of the published literature,27,28 but which contradicts other references.29,30 The average trisomy 21 ratio sFlt-1/PlGF was lower than the equivalent control value, although it did not reach significance (P = .09). Bdolah et al The notion that preeclampsia is related to a gene or a group of genes is long-lived, yet the cause of preeclampsia remains largely unknown, despite intensive research. A large interest in genetic factors that predispose to preeclampsia is seen in the literature,31-37 as recently reviewed by Lachmeijer et al.34 In fact, the genetic contribution to preeclampsia pathogenesis was noted long ago.38 Based on polymorphisms and mutations of maternal genes, numerous candidates have been published as potential ‘‘preeclampsia-genes’’: genes that are involved in hemodynamic changes of pregnancy (such as the angiotensinogen39,40 and endothelial nitric oxide synthase genes), genes that are involved in thrombophilia (such as the 5,10–methylene tetrahydrofolate reductase and factor V Leiden genes), genes that are involved in oxidative stress and genes that are involved in immunogenetics (such as histocompatibility leukocyte antigen).33 Certain pregnancy complications that have excess trophoblastic tissue (such as multifetal pregnancy, hydatidiform mole, and triploidy) have also been long known to predispose to preeclampsia. The fact that pregnancies with excess fetal genetic material are more susceptible to preeclampsia may emphasize the fetal genomic contribution to preeclampsia. Several reports have documented the linkage between trisomy 13 and preeclampsia. In a case-control study of 25 women who gave birth to trisomy 13 infants, Tuohy and James6 showed that, depending on the definition of preeclampsia, the incidence of this disorder in trisomy 13 pregnancies was much higher than the incidence in normal control pregnancies (24.0%-44.0% vs 2.0%8.0%). Moreover, the incidence of preeclampsia in trisomy 13 was much higher than in another chromosomal aberration, such as trisomy 18 (24.0%-44.0% vs 2.6%-18.4%). Boyd et al8 noted that 5 of 14 women who gave birth to babies with trisomy 13 had signs of preeclampsia, although among the 28 control subjects who were matched for parity and age, none of the women were preeclamptic. Six women with pregnancies that were affected by other trisomies (4 by trisomy 18 and 2 by trisomy 21) did not experience preeclampsia. Bower et al7 found that the incidence of trisomy 13 was 2.3 per 10,000 births in the presence of preeclampsia and 0.5 per 10,000 in pregnancies with no preeclampsia. Several additional reports describe trisomy 13 cases that were associated with preeclampsia.10,13,14,41 These observations prompted investigators to hypothesize that gene products that are encoded on chromosome 13 played a pathogenic role in the development of preeclampsia.6,8,11,13 Several candidate factors that are encoded by chromosome 13 (such as clotting factors X and VII) and collagen type 4 were hypothesized to play a role in preeclampsia; however, none of these factors has been shown to be important.6 In a very interesting case report, Feinberg et al11 described a 30-year-old woman (gravida 3, para 1) with 243 severe early onset preeclampsia at 31 weeks of pregnancy. When delivered, the infant was found to be grossly malformed, and his karyotype revealed a 13q translocation (46, xx-13Ct(13q; 13q)), which resulted in a trisomic state for the long arm of chromosome 13. Biopsy specimens of the placental bed showed an absence of the trophoblastic invasion into the maternal arteries, one of the pathophysiologic hallmarks of preeclampsia. In a later case report, Boyd et al9 reported another trisomy 13 neonate who was born to a mother with severe preeclampsia at 32 weeks of gestation. In this case, the baby was chromosomally unbalanced, being trisomic for part of chromosome 13 (13q1213q32), as a consequence of a maternal balanced translocation between chromosome 13 and 3. Interestingly in both these cases of partial trisomy 13 that presented with preeclampsia, the region of translocation included the Flt-1/sFlt-1 locus. There are other case reports of partial trisomy 13 that do not include the Flt-1/sFlt-1 locus, but those women did not experience preeclampsia. One example is Phadke and Patil42 who report a partial trisomy 13 of q22-q ter (excluding the Flt-1/sFlt-1 locus) in a 9-month-old Indian male baby. They cite no evidence of preeclampsia. The partial trisomies are of particular interest because they provide an opportunity to dissect candidate genes that are involved in the pathogenesis of preeclampsia. A further analysis of the regions of the Flt-1 gene that are necessary for causing preeclampsia that is based on the different types of partial trisomies 13 and their correlation to phenotype has not been done. To our knowledge, this is the first report that presents a linkage between trisomy 13 and a higher maternal serum level of sFlt-1 and decreased serum-free PlGF. Furthermore, the knowledge that the Flt-1/sFlt-1 locus was included in several of the case reports with partial trisomy 13 that were complicated with preeclampsia, and the previous work that links excessive placental sFlt1 production in preeclamptic pregnancies are strong evidence for a pathogenic role for sFlt-1 in preeclampsia. There are several limitations and unanswered questions in this retrospective study. First, pregnancy outcomes (namely the presence or absence of preeclampsia) were unknown because most of the patients with trisomy underwent elective termination or spontaneously aborted. Second, we did not have specimens that were taken from later gestational periods at which point differences between cases and control subjects would have been more pronounced. Nevertheless, the alterations that were seen in these angiogenic factors in the second trimester specimens were more pronounced than those from the first trimester. The high coefficients of variation in particular for sFlt-1 measurements, combined with a small number of trisomy specimens, may have contributed to low power to detect differences. Hence, a large sample size may be needed to confirm our results. Finally, the detailed 244 karyotypes of the trisomy 13 cases were unknown, and we were unable to correlate the circulating sFlt-1 levels with the presence of partial versus complete trisomy 13. It is possible that some of the trisomy 13 cases that were included in this study were partial trisomies that did not involve the 13q locus and therefore may have contributed to modest elevations in the sFlt-1 levels that were noted. Nevertheless, because the incidence of partial trisomies is approximately 25%, it is likely that most of our trisomy 13 cases were complete trisomy 13. In summary, this study suggests that trisomy 13 pregnancies have increased circulating sFlt-1/PlGF ratios compared with trisomy 18 or trisomy 21 or normal karyotype pregnancies. Because similar alterations in circulating angiogenic proteins have been observed previously in pregnancies that are complicated subsequently by preeclampsia, it is likely that the increased risk of preeclampsia that was noted in patients with trisomy 13 may be related directly to the alterations in the angiogenic profile. A larger study that uses serial specimens throughout pregnancy along with pregnancy outcome data is needed in to further understand the role of the circulating angiogenic factors in the pathogenesis of increased preeclampsia risk that was noted in trisomy 13 pregnancies. Acknowledgments We thank Vikas Sukhatme, Frank Epstein, Susan Fisher, and Kee-Hak Lim for helpful discussions. References 1. Walker JJ. Pre-eclampsia. Lancet 2000;356:1260-5. 2. Sibai BM, Caritis S, Hauth J. What we have learned about preeclampsia. Semin Perinatol 2003;27:239-46. 3. Chen M, Yeh GP, Shih JC, Wang BT. Trisomy 13 mosaicism: study of serial cytogenetic changes in a case from early pregnancy to infancy. Prenat Diagn 2004;24:137-43. 4. Nagaishi M, Yamamoto T, Iinuma K, Shimomura K, Berend SA, Knops J. Chromosome abnormalities identified in 347 spontaneous abortions collected in Japan. J Obstet Gynaecol Res 2004; 30:237-41. 5. Rasmussen SA, Wong LY, Yang Q, May KM, Friedman JM. Population-based analyses of mortality in trisomy 13 and trisomy 18. Pediatrics 2003;111:777-84. 6. Tuohy JF, James DK. Pre-eclampsia and trisomy 13. BJOG 1992;99:891-4. 7. Bower C, Stanley F, Walters BN. Pre-eclampsia and trisomy 13. Lancet 1987;2:1032. 8. Boyd PA, Lindenbaum RH, Redman C. Pre-eclampsia and trisomy 13: a possible association. Lancet 1987;2:425-7. 9. Boyd PA, Maher EJ, Lindenbaum RH, Hoogwerf AM, Redman C, Crocker M. Maternal 3;13 chromosome insertion, with severe pre-eclampsia. Clin Genet 1995;47:17-21. 10. Evers J, Seelen J, Blankenborg G. Severe toxemia, hydramnios and trisomy 13-15. Ned Tijdschr Verloskd Gynaecol 1967;67:395-7. 11. Feinberg RF, Kliman HJ, Cohen AW. Preeclampsia, trisomy 13, and the placental bed. Obstet Gynecol 1991;78:505-8. Bdolah et al 12. Heydanus R, Defoort P, Dhont M. Pre-eclampsia and trisomy 13. Eur J Obstet Gynecol Reprod Biol 1995;60:201-2. 13. Pedersen BW, Gronlund A. [Severe pre-eclampsia and fetal trisomy 13 in a multiparous woman]. Ugeskr Laeger 2003;165:2108-9. 14. Thornton JG, O’Donovan P, Stigter R, Williams J. Pre-eclampsia and trisomy 13. Lancet 1987;2:794. 15. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649-58. 16. Tsatsaris V, Goffin F, Munaut C, Brichant JF, Pignon MR, Noel A, et al. Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences. J Clin Endocrinol Metab 2003;88:5555-63. 17. Sugimoto H, Hamano Y, Charytan D, Cosgrove D, Kieran M, Sudhaker A, et al. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem 2003;278:12605-8. 18. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T, et al. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J Clin Endocrinol Metab 2003;88:2348-51. 19. Chaiworapongsa T, Romero R, Espinoza J, Bujold E, Mee Kim Y, Goncalves LF, et al. Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia: young investigator award. Am J Obstet Gynecol 2004;190:1541-50. 20. Hertig A, Berkane N, Lefevre G, Toumi K, Marti HP, Capeau J, et al. Maternal serum sFlt1 concentration is an early and reliable predictive marker of preeclampsia. Clin Chem 2004;50:1702-3. 21. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672-83. 22. Thadhani R, Mutter WP, Wolf M, Levine RJ, Taylor RN, Sukhatme VP, et al. First trimester placental growth factor and soluble fms-like tyrosine kinase 1 and risk for preeclampsia. J Clin Endocrinol Metab 2004;89:770-5. 23. Levine RJ, Thadhani R, Qian C, Lam C, Lim KH, Yu KF, et al. Urinary placental growth factor and risk of preeclampsia. JAMA 2005;293:77-85. 24. Bdolah Y, Sukhatme VP, Karumanchi SA. Angiogenic imbalance in the pathophysiology of preeclampsia: newer insights. Semin Nephrol 2004;24:548-56. 25. Karumanchi SA, Bdolah Y. Hypoxia and sFlt-1 in preeclampsia: the ‘‘chicken-and-egg’’ question. Endocrinology 2004;145:4835-7. 26. Conover WJ. Practical nonparametric statistics. New York: Wiley & Sons; 1980. 27. Spencer K, Liao AW, Ong CY, Geerts L, Nicolaides KH. First trimester maternal serum placenta growth factor (PIGF) concentrations in pregnancies with fetal trisomy 21 or trisomy 18. Prenat Diagn 2001;21:718-22. 28. Su YN, Hsu JJ, Lee CN, Cheng WF, Kung CC, Hsieh FJ. Raised maternal serum placenta growth factor concentration during the second trimester is associated with Down syndrome. Prenat Diagn 2002;22:8-12. 29. Debieve F, Moiset A, Thomas K, Pampfer S, Hubinont C. Vascular endothelial growth factor and placenta growth factor concentrations in Down’s syndrome and control pregnancies. Mol Hum Reprod 2001;7:765-70. 30. Lambert-Messerlian GM, Canick JA. Placenta growth factor levels in second-trimester maternal serum in Down syndrome pregnancy and in the prediction of preeclampsia. Prenat Diagn 2004;24:876-80. 31. Arngrimsson R, Sigurard ttir S, Frigge ML, Bjarnadttir RI, Jonsson T, Stefansson H, et al. A genome-wide scan reveals a maternal susceptibility locus for pre-eclampsia on chromosome 2p13. Hum Mol Genet 1999;8:1799-805. Bdolah et al 32. Lachmeijer AM, Arngrimsson R, Bastiaans EJ, Frigge ML, Pals G, Sigurdardottir S, et al. A genome-wide scan for preeclampsia in the Netherlands. Eur J Hum Genet 2001;9:758-64. 33. Laasanen J, Hiltunen M, Romppanen EL, Punnonen K, Mannermaa A, Heinonen S. Microsatellite marker association at chromosome region 2p13 in Finnish patients with preeclampsia and obstetric cholestasis suggests a common risk locus. Eur J Hum Genet 2003;11:232-6. 34. Lachmeijer AM, Dekker GA, Pals G, Aarnoudse JG, ten Kate LP, Arngrimsson R. Searching for preeclampsia genes: the current position. Eur J Obstet Gynecol Reprod Biol 2002;105:94-113. 35. Roberts JM, Cooper DW. Pathogenesis and genetics of preeclampsia. Lancet 2001;357:53-6. 36. Woodage T, Venter JC, Broder S. Application of the human genome to obstetrics and gynecology. Clin Obstet Gynecol 2002;45:711-32. 245 37. Moses EK, Lade JA, Guo G, Wilton AN, Grehan M, Freed M, et al. A genome scan in families from Australia and New Zealand confirms the presence of a maternal susceptibility locus for preeclampsia, on chromosome 2. Am J Hum Genet 2000;67:1581-5. 38. Chesley LC, Annitto JE, Cosgrove RA. The familial factor in toxemia of pregnancy. Obstet Gynecol 1968;32:303-11. 39. Ward K, Hata A, Jeunemaitre X, Helin C, Nelson L, Namikawa C, et al. A molecular variant of angiotensinogen associated with preeclampsia. Nat Genet 1993;4:59-61. 40. Arngrimsson R, Purandare S, Connor M, Walker JJ, Bjornsson S, Soubrier F, et al. Angiotensinogen: A candidate gene involved in preeclampsia? Nat Genet 1993;4:114-5. 41. Bunting R, Leitch J. Buphthalmos in trisomy 13. Eye 2005;19: 487-8. 42. Phadke SR, Patil SJ. Partial trisomy 13 with features similar to c syndrome. Indian J Pediatr 2004;41:614-7.