W. Allen Hogge

Altered Global Gene Expression in First Trimester Placentas of Women Destined to Develop Preeclampsia

BACKGROUND Preeclampsia is a pregnancy-specific disorder that remains a leading cause of maternal, fetal and neonatal morbidity and mortality, and is associated with risk for future cardiovascular disease. There are no reliable predictors, specific preventative measures or treatments other than delivery. A widely held view is that the antecedents of preeclampsia lie with impaired placentation in early pregnancy. Accordingly, we hypothesized dysregulation of global gene expression in first trimester placentas of women who later manifested preeclampsia. METHODS Surplus chorionic villus sampling (CVS) tissues were collected at 10-12 weeks gestation in 160 patients with singleton fetuses. Four patients developed preeclampsia, and their banked CVS specimens were matched to 8 control samples from patients with unaffected pregnancies. Affymetrix HG-U133 Plus 2.0 GeneChips were utilized for microarray analysis. Naïve Bayes prediction modeling and pathway analysis were conducted. qRT-PCR examined three of the dysregulated genes. RESULTS Thirty-six differentially expressed genes were identified in the preeclampsia placentas. qRT-PCR verified the microarray analysis. Thirty-one genes were down-regulated. Many were related to inflammation/immunoregulation and cell motility. Decidual gene dysregulation was prominent. No evidence was found for alterations in hypoxia and oxidative stress regulated genes. CONCLUSIONS To our knowledge, this is the first study to show dysregulation of gene expression in the early placentas of women approximately 6 months before developing preeclampsia, thereby reinforcing a placental origin of the disorder. We hypothesize that placentation in preeclampsia is compromised in the first trimester by maternal and fetal immune dysregulation, abnormal decidualization, or both, thereby impairing trophoblast invasion. Several of the genes provide potential targets for the development of clinical biomarkers in maternal blood during the first trimester. Supplementary materials are available for this article via the publishers online edition.

Noninvasive prenatal diagnosis of a fetal microdeletion syndrome.

This proof-of-principle study shows that it is possible to detect a genetic microdeletion carried by a fetus through analysis of DNA in circulating maternal blood.

Sequential pathways of testing after first-trimester screening for trisomy 21

OBJECTIVE: To evaluate the performance and use of second-trimester multiple-marker maternal serum screening for trisomy 21 by women who had previously undergone first-trimester combined screening (nuchal translucency, pregnancy-associated plasma protein A, and free β-hCG), with disclosure of risk estimates. METHODS: In a multicenter, first-trimester screening study sponsored by the National Institute of Child Health and Human Development, multiple-marker maternal serum screening with alpha-fetoprotein, unconjugated estriol, and total hCG was performed in 4,145 (7 with trisomy 21) of 7,392 (9 with trisomy 21) women who were first-trimester screen-negative and 180 (7 with trisomy 21) of 813 (52 with trisomy 21) who were first-trimester screen-positive. Second-trimester risks were calculated using multiples of the median and a standardized risk algorithm with a cutoff risk of 1:270. RESULTS: Among the first-trimester screen-negative cohort, 6 of 7 (86%) trisomy 21 cases were detected by second-trimester multiple-marker maternal serum screening with a false-positive rate of 8.9%. Among the first-trimester screen-positive cohort, all 7 trisomy 21 cases were also detected in the second trimester, albeit with a 38.7% false-positive rate. CONCLUSION: Our data demonstrate that a sequential screening program that provides patients with first-trimester results and offers the option for early invasive testing or additional serum screening in the second trimester can detect 98% of trisomy 21–affected pregnancies. However, such an approach will result in 17% of patients being considered at risk and, hence, potentially having an invasive test. LEVEL OF EVIDENCE: II-2

Insights into the pathogenesis and natural history of fetuses with multicystic dysplastic kidney disease

To better delineate the natural history of multicystic displastic kidney disease (MCDKD) and provide insights into the pathogenesis of this condition, we report our experience in 102 prenatally detected cases. MCDKD is most commonly an incidental finding on prenatal ultrasound examination. The abnormality may be unilateral (76 per cent) or bilateral (24 per cent). In unilateral cases, abnormality of the contralateral kidney is common (33 per cent). Associated non‐renal abnormalities occur frequently with both unilateral (26 per cent) and bilateral (67 per cent) MCDKD, and increase the risk for an abnormal chromosome study. Males are more likely to be affected than females with a ratio of 2.4:1, but females are twice as likely to have bilateral MCDKD and associated non‐renal abnormalities, and four times more likely to have an abnormal chromosome study. We suggest that the option of chromosomal analysis should be discussed with all patients diagnosed with MCDKD in their fetus, if there is bilateral renal involvement or if an associated non‐renal abnormality is present. Unilateral MCDKD without associated renal or non‐renal abnormalities was not associated with an abnormal chromosome study, and resulted in favourable outcomes. While unilateral MCDKD, lack of associated anomalies, normal chromosome study and adequate amniotic fluid are all reassuring findings, a complete neonatal urologic work‐up should be performed in all newborns. We believe the evaluation should include voiding cystourethrography to rule out vesicoureteral reflux. Our findings allow more precise counselling of patients regarding prognosis, and subsequent management of the fetus found to have MCDKD. Copyright

A microarray‐based approach for the identification of epigenetic biomarkers for the noninvasive diagnosis of fetal disease

We describe a novel microarray‐based approach for the high‐throughput discovery of epigenetic biomarkers for use in the noninvasive detection of fetal genetic disease.

Comprehensive analysis of HLA-G: implications for recurrent spontaneous abortion.

Miscarriage is one of the most common pregnancy complications. Recurrent spontaneous abortion is defined as 2 or more pregnancy losses and may be associated with genetic variation. Human leukocyte antigen-G (HLA-G) is a ligand for natural killer (NK) cell receptors and has the ability to block NK cell activity, which if not blocked can potentially harm a fetus. Consequently a deletion or mutation of the HLA-G gene could lead to miscarriage. Our cases (n = 238) include Caucasian women experiencing 2 or more spontaneous abortions, and controls (n = 233) include women with at least 1 live birth and no history of SA. We sequenced approximately 1400 base pairs (bp) of the HLA-G promoter region, genotyped the 14 bp exon 8 insertion/deletion and single nucleotide polymorphism (SNP) in the coding region of HLA-G. Promoter haplotypes were constructed from sequence information. Twenty-three SNPs were observed in the promoter region with minor allele frequency >0.02. Twelve SNPs differed significantly in frequency between cases and controls. Two haplotypes incorporating these 12 SNPs accounted for 90% of haplotypes and differed significantly in frequency between the 2 populations. Cases were more likely to carry haplotype 2 (P = .0078) and controls to have haplotype 6 (P = .0004). Cases also had a higher frequency of individuals homozygous for the 14 bp insertion. Among the 12 alleles carried on haplotype 2, 5 are predicted to disrupt transcription factor binding sites. The HLA-G promoter is highly associated with the risk of spontaneous abortion, but high linkage disequilibrium in the promoter prevents assignment of the causal variant.

THE USE OF INTERPHASE FISH FOR PRENATAL DIAGNOSIS OF PALLISTER–KILLIAN SYNDROME

Pallister–Killian syndrome (tetrasomy 12p) is a relatively rare aneuploidy syndrome characterized by the presence of mosaicism for an isochromosome 12p [i(12p)]. We report two new cases diagnosed following chorionic villus sampling and an abnormal ultrasound, respectively. Fluorescent in situ hybridization (FISH) was used to enumerate the number of interphase cells containing the isochromosome. The results of these studies illustrate the importance of the use of interphase FISH to detect the presence of the i(12p) in uncultured, non‐dividing cells. A review of the literature identified 23 additional cases of Pallister–Killian syndrome diagnosed prenatally. Approximately 50 per cent of these cases were associated with the presence of a congenital diaphragmatic hernia. We suggest that a perinatal‐lethal form of Pallister–Killian syndrome is underdiagnosed and recommend that all cases of prenatally detected diaphragmatic hernia be tested for Pallister–Killian syndrome using interphase FISH on uncultured amniocytes.

Choroid plexus cysts and trisomy 18: Risk modification based on maternal age and multiple-marker screening

Choroid plexus cysts are more common in fetuses with chromosomal aneuploidies, particularly trisomy 18. Although it is accepted that the risk of karyotypic abnormality justifies amniocentesis when associated abnormalities are present, disagreement continues as to the risk of trisomy 18 in a fetus with an isolated choroid plexus cyst. We propose consideration of maternal age and multiple-marker screening for chromosomal aneuploidy in the assessment of risk. Bayesian statistical modeling was used to calculate the risk of trisomy 18 from age-related risk figures for trisomy 18 and the incidence of isolated choroid plexus cysts in fetuses with trisomy 18. The risk was further modified on the basis of the ability of multiple-marker screening to detect fetuses with trisomy 18. From risk estimates calculated across maternal ages 20 to 45 years, the risk of trisomy 18 does not approach that of amniocentesis until a maternal age of > or = 37 years. Therefore in the presence of an isolated choroid plexus cyst and normal multiple-marker screen results amniocentesis is justified only in the patient with advanced maternal age.

Gene Expression in First Trimester Preeclampsia Placenta

Background. The goal of this study was to further validate eight candidate genes identified in a microarray analysis of first trimester placentas in preeclampsia. Material and method. Surplus chorionic villus sampling (CVS) specimens of 4 women subsequently diagnosed with preeclampsia (PE) and 8 control women (C) without preeclampsia analyzed previously by microarray and 24 independent additional control samples (AS) were submitted for confirmatory studies by quantitative real-time polymerase chain reaction (qRT-PCR). Results. Downregulation was significant in FSTL3 in PE as compared to C and AS (p = .04). PAEP was downregulated, but the difference was only significant between C and AS (p = .002) rather than between PE and either of the control groups. Expression levels for CFH, EPAS1, IGFBP1, MMP12, and SEMA3C were not statistically different among groups, but trends were consistent with microarray results; there was no anti-correlation. S100A8 was not measurable in all samples, probably because different probes and primers were needed. Conclusions. This study corroborates reduced FSTL3 expression in the first trimester of preeclampsia. Nonsignificant trends in the other genes may require follow-up in studies powered for medium or medium/large effect sizes. qRT-PCR verification of the prior microarray of CVS may support the placental origins of preeclampsia hypothesis. Replication is needed for the candidate genes as potential biomarkers of susceptibility, early detection, and/or individualized care of maternal—infant preeclampsia.

The X Chromosome and Recurrent Spontaneous Abortion: The Significance of Transmanifesting Carriers

If, as we have argued, a subset of female carriers of X-linked recessive lethal traits are genetically predisposed to spontaneous abortion, and if such carriers can reliably be ascertained via their skewed pattern X chromosome inactivation, it becomes possible to test the hypothesis that X-linked recessive lethal traits are a significant cause of RSA in the general population. Intrinsic to this argument are the assumptions that the trait is cell autonomous—that is, that it causes death or growth disadvantage to the cells with the mutant X active and that hemizygous males survive at least until the pregnancy is clinically observable through a positive bHCG test.To test this hypothesis, we have initiated a case-control study wherein we compare the frequency of highly skewed X chromosome inactivation in women with two or more unexplained spontaneous abortions to the frequency in female controls (Lanasa et al. 1998xSee all References1998). The women characterized with idiopathic RSA have undergone a complete evaluation to rule out any of the known causes of RSA described above (Stephenson 1996xFrequency of factors associated with habitual abortion in 197 couples. Stephenson, MD. Fertil Steril. 1996; 66: 24–29Abstract | Full Text PDF | PubMedSee all References1996). The controls are women from the same demographic region, with no known history of spontaneous abortion; furthermore, the cases and controls are age-distribution matched, so that the distribution of ages between the two groups is the same. Defining skewed X inactivation as preferential use of one X chromosome in ≥90% of peripheral leukocytes, we have found 7 (14.6%) of 48 to have skewed X inactivation. In contrast, only 1 (1.5%) of 68 control females exhibit this extent of nonrandom X inactivation. This finding is statistically significant, with P < .01 (Fishers exact test, one-tailed).The frequency of nonrandom X inactivation is somewhat lower (1.5%) in our control group than has been reported previously. Other groups have estimated that the frequency of skewed inactivation (at the level of ≥90% silencing of one copy of the chromosome) as 3.2% (Gale et al. 1997xAcquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age. Gale, RE, Fielding, AK, Harrison, CN, and Linch, DC. Br J Haematol. 1997; 98: 512–519Crossref | PubMedSee all References1997) or 3.5% (Plenge et al. 1997xA promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Plenge, RM, Hendrich, BD, Schwartz, C, Arena, JF, Naumova, A, Sapienza, C, Winter, RM et al. Nat Genet. 1997; 17: 353–356Crossref | PubMed | Scopus (189)See all References1997) in women of the same age in the population at large. Although case-control comparisons across studies may be perilous, it is interesting to note that, even when these higher estimates of skewing frequency are used for the control group, the frequency we observe in our group of RSA-affected women remains significant at the level of P < .05 (Fishers exact test, one-tailed).Although the results presented here are clearly preliminary, it is interesting to speculate about the frequency of X-linked recessive lethal traits in the general population. Given a frequency of idiopathic RSA of 1 in 250 in the general population, and an affection rate of ∼1 in 7 in our case population, the population prevalence of X-linked lethals leading to RSA could be as high as 1 in 1,750. In fact, our ascertainment methodology will miss a large number of carriers, since, on average, a carrier would have to become pregnant five times to show two spontaneous abortions. Furthermore, there is great selective pressure against such traits. As X-linked recessive lethal traits can be passed on only to daughters, the carrier frequency should be halved in each generation. If 1 in 1,750, in fact, approximates the carrier frequency, then the new mutation rate must be 1 in 3,500. Since the highest-known single-gene–mutation rate is that of dystrophin at 1 in 10,000, a mutation rate of 1 in 3,500 indicates extensive genetic heterogeneity. This is consistent with the hypothesis that there are a significant number of vital genes on the X chromosome.RSA is a major womens health concern. As the application of molecular genetics to RSA advances, it will be possible to begin characterizing those genes that cause spontaneous abortion in the recessive state. The X chromosome inactivation assay affords a methodology by which female carriers of X-linked recessive lethal defects can be identified. Over time, then, by assembling familial pedigrees, the individual causative genes can be identified. The X-inactivation assay should become an important diagnostic tool in the clinical evaluation of women with RSA, as secondary skewed X inactivation will be the common denominator by which carriers of X-linked recessive lethal traits can be identified.