pro-thrombotic effect. Leptin levels are increased in normal preg-nancy, and leptin levels have been linked to various pregnancy com-plications, including gestational diabetes, intrauterine growth restric-tion and preeclampsia. We sought to characterize methylationpatterns in the leptin gene in normal early pregnancy and preeclamp-tic pregnancies.STUDY DESIGN: The methylation profiles of 2 CpG sites in the leptingene were characterized in DNA from maternal blood longitudinallyin 14 women at �16 gestational weeks, at delivery and postpartum;and in 14 preeclamptics at delivery. All women were non-smokers andthe groups were matched for age and BMI. Genomic DNA was derivedfrom buffy coat, purified, and bisulfite modified then run on the Illu-mina Methylation Assay. Mean methylation levels at each CpG sitewere compared using a t-test.RESULTS: Leptin methylation patterns at both CpG sites differed sig-nificantly between the early pregnancy and postpartum samples(P�0.01, P�10-5). Both leptin CpG sites were hypomethylated dur-ing early pregnancy as compared to postpartum. Methylation patternswere similar in normotensive and preeclamptic patients at the time ofdelivery (P�0.64, P�0.71).CONCLUSION: Our data demonstrates that early pregnancy results indecreased methylation at two CpG sites on the leptin gene. As meth-ylation levels tend to be inversely correlated with gene transcriptionand resultant serum protein levels, this result suggests that alteredmethylation may be an epigenetic mechanism to control increasedleptin production in normal early pregnancy. The presence of pre-eclampsia at the time of delivery did not appear to impact methylationof the leptin gene compared to normotensive patients. This suggeststhat there may be another mechanism controlling the increase in cir-culating serum leptin seen in pregnancies effected by preeclampsia.

575 Routine testing for copy number variants (CNVs) bychromosomal microarray analysis in patients with a normalkaryotype: a single center’s 20 month experienceEran Bornstein1, Sau Cheung2, Kristen Maliszewski2, ArthurBeaudet2, Flavia Theil1, Ankita Patel2, Amber Pursley2, VictoriaMinior1, Michael Divon1

1Lenox Hill Hospital NSLIJ, Obstetrics & Gynecology, New York, NY, 2BaylorCollege of Medicine, Department of Molecular and Human Genetics,Houston, TXOBJECTIVE: To evaluate the clinical value of prenatal chromosomalmicroarray analysis (CMA) in patients with a normal karyotype andto determine the risk factors most likely to yield informative CMAresults.STUDY DESIGN: We conducted a retrospective analysis of all amnio-centesis (n�1180) and CVS (n�424) samples obtained during a 20month period in a single center. All patients were offered CMA inaddition to standard testing. 533 patients (33%) elected to pursueCMA studies, which were performed in a single laboratory primarilyusing uncultured samples. 500 cases were tested with a BCM V7(105K) array containing oligos corresponding to over 270 knowngenomic disorders, as well as 41 unique subtelomeric regions and all43 unique pericentromeric regions. The other 34 cases were testedwith a V8 (180K) array with exon coverage for 1700 genes. CMAresults were stratified based on indications for testing, and the additivedetection rate of CMA was compared among these indications usingFisher’s exact test.RESULTS: 523 women who elected to have CMA performed had a nor-mal karyotype. Stratification of CMA results based on the indicationfor testing is presented in the table. In our cohort, abnormal CMAresults were detected in 1.9% of the patients regardless of the indica-tion for testing. CMA abnormalities were detected in 5.5% of patientswith abnormal 1st or 2nd trimester sonographic findings, whereasnone were detected among patients who were tested due to AMA orparental concern alone (p�0.001).CONCLUSION: Prenatal CMA detected clinically relevant genetic ab-normalities that were not detected by traditional karyotype analysis.

The additive value of CMA in the prenatal population varies signifi-cantly based on the patient’s risk factors, with abnormal ultrasoundfindings comprising the highest risk group.

576 Simultaneous fetal cell detection and genetic diagnosisby immunophenotyping and chromosomal fluorescence insitu hybridization (FISH)George Sitar1, Giuseppe Calabrese2, Leonard Kellner4, JonathanHerman3, Hassan Bennani4, Javad Khosravi4

1University of Pavia, Internal Medicine, Pavia, Italy, 2Chieti-University,Medical Genetics, Chieti, Italy, 3Long Island Jewish Medical Center, Ob/Gyn,New Hyde Park, NY, 4Kellbenx Incorporated, Genetics, Great River, NYOBJECTIVE: To confirm application of a novel monoclonal anti-fetalerythroblast antibody (4B9 mAb) for specific detection of fetal eryth-roblasts in nucleated cell fraction of maternal blood and simultaneousanalysis of chromosomal status of the identified cells by single or dual-probe FISH.STUDY DESIGN: 4B9 mAb specificity and method optimization wasinitially investigated using fetal liver, cord blood and cells obtained bychorionic villus sampling. Maternal bloods samples tested were frompregnancies at 10-11 weeks gestations. Where indicated, nucleatedcell fractions were isolated by previously described Ficoll cell densitycentrifugations. Immunocytochemical (ICC) staining performed bydirect or indirect detection of 4B9 mAb coupled to various detectionprobes including Rhodamine, FITCI, AMCA, or Alkaline phospha-tase. FISH was performed according to standard protocols.RESULTS: Approximately 30-40% of cells in nucleated cell fractions ofcord blood or in CVS wash fractions stained positively by 4B9 mAb. Inpreliminary analysis of 10 maternal blood samples, 1.5-2% of cells inthe isolated cell fractions also positively stained by indirect 4B9 mAbICC staining. Among various detection moieties, indirect staining byRhodamine-based anti-4B9 mAb detection generated best signalswhen combined with FISH analysis. As expected, 4B9-positive cells inthe nucleated cell fractions of pregnancies carrying female or malefetuses stained positively for XX or XY chromosomes, respectively. Inone case with confirmed trisomy 21, the majority of 4B9 positive cellshad trisomy 21 as revealed by dual-probe FISH approach.CONCLUSION: This study confirms high specificity of 4B9 mAb for cellmembrane epitopes expressed on fetal erythroid cells and demon-strates the feasibility of its use for simultaneous fetal cell identificationand genetic diagnosis by FISH. Availability of this novel mAb shouldenable development of non-invasive tests for comprehensive deter-mination of fetal genetic status.

577 PLAC4/COL6A2 single-nucleotide polymorphism allelicratio is predictive marker for the noninvasive detection oftrisomy 21Jun Zhang1, Hongying Hou1, Peiqiong Li2, Yuzhu Yin1, BenqiTeng1, Yanchou Ye1, Junwei Lin1

1The Third Affiliated Hospital of Sun Yat-sen University, Department ofObstetrics and Gynecology, Guangzhou, China, 2Guangzhou MedicalCollege, Department of Medical Genetics and Cell Biology, Guangzhou,ChinaOBJECTIVE: Chromosomal abnormalities can be developed as fast,cost-effective screening/diagnosis strategies. PLAC4 and COL6A2 aretranscribed from chromosome 21 in placental cells. Therefore, wehypothesized that maternal plasma mRNA encoded by alternative

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S246 American Journal of Obstetrics & Gynecology Supplement to JANUARY 2013

PLAC4 and COL6A2 are potential predictive markers for the nonin-vasive assessment of chromosome 21 dosage in the fetus.STUDY DESIGN: 100 pregnant women were recruited to study. Cell freeplasma RNA was extracted from maternal peripheral blood. Due tohigh heterozygosities, five SNP markers (rs8130833, rs4818219,rs59066201, rs7717, rs1044598) encoding the regions of PLAC4 andCOL6A2 were studied. Fluoresceins labeled specific primers wereused to amplify the markers. Digestion with restriction endonucleaseswas applied specifically to cut one allele while dumb to the other. PCRproducts were analyzed with capillary electrophoresis. Allelic ratioswere calculated from the areas of the fluorescent peaks from eachspecific fragmental size.RESULTS: At least one of the five SNP markers exhibited heterozygousin 100% T21 cases. 95% cases showed multiple heterozygous alleles.Allelic ratios of the heterozygous SNP markers are 0.7-1.3 in non T21fetus’s group vs 1.8-2.1 in T21 group.CONCLUSION: Trisomy 21 can be screening by noninvasive method us-ing maternal blood. Five SNP markers encoding the regions of PLAC4and COL6A2 can be accurately detected by fluorescent PCR and en-donuclease digestion followed by capillary electrophoresis. The ap-proach may be specifically useful for very fine mapping of the regionsof chromosome 21 that are critical for Down syndrome.

578 Gene expression analysis of VEZF1, a lymphatictranscription factor, in fetal cystic hygromas and increasednuchal translucencyInna Landres1, Elizabeth Looke-Stewart2, Samprit Banerjee3,Rebecca Baergen4, Venkat Pulijaal4, Stephen Chasen1, HeidiStuhlmann2

1Weill Cornell Medical College, Obstetrics and Gynecology, New York, NY,2Weill Cornell Medical College, Cell and Developmental Biology, New York,NY, 3Weill Cornell Medical College, Public Health, New York, NY, 4WeillCornell Medical College, Pathology and Laboratory Medicine, New York, NYOBJECTIVE: Abnormal proliferation of lymphatic vessels has been pro-posed as a mechanism for increased nuchal translucency (NT) andfetal cystic hygromas. Vascular endothelial zinc finger 1 (VEZF1) is atranscription regulator of blood and lymphatic vessel formation and,in a mouse model, VEZF1 mutant embryos have a phenotype remi-niscent of human cystic hygromas. Our objective was to investigatethe involvement of VEZF1 in human fetuses with abnormal NT.STUDY DESIGN: This was a prospective case control study performed ata single academic center between September 2010 and June 2012.Inclusion criteria were presence of abnormal NT � 3mm or cystichygroma, or control subjects with normal NT (�2.5mm) undergoingCVS or amniocentesis. Cells were obtained from CVS, amniocentesisor products of conception and from available nuchal tissues. Quanti-tative Real Time Reverse Transcription PCR (qPCR) was performedto determine expression levels for VEZF1.RESULTS: We analyzed 34 cases with NT � 3mm and 32 controls withnormal NT. Of the 34 cases, 18 were euploid and 16 were aneuploid.There was no difference in mean gestational age, maternal age, BMI,parity and ethnicity between cases and controls. Using a linear mixedmodel, a statistically significant decrease in expression of VEZF1 wasnoted in cases vs. controls (fold change decrease of 0.8108 in cases vs.controls, p � 0.023), after controlling for cell origin (CVS or amnio-centesis), RIN number (RNA integrity value) and gestational age. Thedecrease in expression of VEZF1 was significant for euploid (foldchange decrease of 0.7818 in euploid cases vs. controls, p�0.014) butnot aneuploid cases (fold change decrease of 0.8731 in aneuploid casesvs. controls, p�0.307).CONCLUSION: A significant decrease in expression of VEZF1 by qPCRwas noted in euploid fetuses with abnormal NT. Future studies willinclude protein analysis, DNA sequencing and immunohistochemis-try studies of nuchal tissue samples.

579 Genetic risk factors for preeclampsiaJanet Burlingame1, Hyeong Jun Ahn3, John Chen3,Johann Urschitz2

1University of Hawaii, John A. Burns School of Mediicne, Obstetrics,Gynecology and Womens Health, Honolulu, HI, 2University of Hawaii,Institute for Biogenesis Research, Honolulu, HI, 3University of Hawaii, JohnA. Burns School of Medicine, Biostatistics Core, Honolulu, HIOBJECTIVE: To investigate associations between preeclampsia and sin-gle nucleotide polymorphisms (SNP) previously associated with met-abolic and cardiovascular disease in our local pregnant population.STUDY DESIGN: OpenArray technology (Applied Biosystems) was usedto screen maternal DNA from the Hawaii Biospecimen Repository for32 single nucleotide polymorphisms (SNPs). Chi-squared test statis-tics and odds ratios (OR) were calculated to determine SNP associa-tions with preeclampsia.RESULTS: Figure 1 details the findings of our study. In the Filipinapopulation, EXT2 gene SNP rs1113132 (p � 0.032) demonstrated adominant inheritance pattern for preeclampsia. In the Pacific Islanderpopulation, HHEX gene SNPs rs1111875 (p � 0.0032) and rs7923837(p � 0.0063) demonstrated dominant inheritance patterns, andKCNQ1 gene SNP rs2237892 (p � 0.025) and G6PC2 gene SNPrs16856187 (p � 0.048) demonstrated recessive inheritance patternsfor preeclampsia.CONCLUSION: We have demonstrated that in the local Filipina and Pa-cific Islander populations genetic polymorphisms previously associ-ated with metabolic but not cardiovascular disease are also associatedwith preeclampsia. The risk of developing preeclampsia is increased inwomen with diabetes. Preeclampsia can also be a harbinger for thedevelopment of both cardiovascular, microvascular and metabolicdiseases. In this study the strongest association was with preeclampsiawas with a SNP in the HHEX gene (rs111875 and rs7923837 SNPS).HHEX or hematopoietically expressed homeobox is coded for onchromosome 11 and has previously been associated with pancreasdevelopment and diabetes. It is a gene that encodes a transcriptionfactor that is also involved in the Wnt signaling pathway. As a tran-scription factor it could play many roles in the pathogenesis of pre-eclampsia as well as type 2 diabetes. These SNPs may serve as a linkbetween the development of preeclampsia and metabolic diseasessuch as diabetes. *The research described was supported in part by theNIH grants NCRR U54MD007584 and NIMHD G12MD007601.

Genetic associations between single nucleotide polymorphisms (SNPs) andpreeclampsia in Filipina and Pacific Islander populations using differentgenetic models (COD: co-dominant, DOM: dominant, REC: recessive, ODO:over dominant, ADD: log additive)

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