Apigenin as a Candidate Prenatal Treatment for Trisomy 21: Effects in Human Amniocytes and the Ts1Cje Mouse Model.

Human fetuses with trisomy 21 (T21) have atypical brain development that is apparent sonographically in the second trimester. We hypothesize that by analyzing and integrating dysregulated gene expression and pathways common to humans with Down syndrome (DS) and mouse models we can discover novel targets for prenatal therapy. Here, we tested the safety and efficacy of apigenin, identified with this approach, in both human amniocytes from fetuses with T21 and in the Ts1Cje mouse model. In vitro, T21 cells cultured with apigenin had significantly reduced oxidative stress and improved antioxidant defense response. In vivo, apigenin treatment mixed with chow was administered prenatally to the dams and fed to the pups over their lifetimes. There was no significant increase in birth defects or pup deaths resulting from prenatal apigenin treatment. Apigenin significantly improved several developmental milestones and spatial olfactory memory in Ts1Cje neonates. In addition, we noted sex-specific effects on exploratory behavior and long-term hippocampal memory in adult mice, and males showed significantly more improvement than females. We demonstrated that the therapeutic effects of apigenin are pleiotropic, resulting in decreased oxidative stress, activation of pro-proliferative and pro-neurogenic genes (KI67, Nestin, Sox2, and PAX6), reduction of the pro-inflammatory cytokines INFG, IL1A, and IL12P70 through the inhibition of NFκB signaling, increase of the anti-inflammatory cytokines IL10 and IL12P40, and increased expression of the angiogenic and neurotrophic factors VEGFA and IL7. These studies provide proof of principle that apigenin has multiple therapeutic targets in preclinical models of DS.

Human epidermal growth factor receptor 2 (Her2) testing for uterine serous carcinoma: Report of scenarios of unusual overexpression.

The human epidermal growth factor receptor 2 (Her2) is tested in many human cancers, including breast, bladder, pancreatic, ovarian and stomach. The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) have issued Clinical Practice Guidelines for reporting Her2 results for breast carcinomas (Wolf et al., 2018). For the last 1-2 years Her2/neu is tested in endometrial serous carcinoma, especially in recurrent tumors or non-responsive tumors as an option for additional treatment. College of American Pathologists (CAP) offers a template for prognostic marker reporting results for specimens with endometrial carcinomas (Fitzgibbons et al., 2019). Her2/neu testing by immunohistochemistry (IHC) mandates rigorous fixation time control, e.g., fixation time should fall within 6-72 h (Recommendations for Her2 Testing in Breast Cancer, 2013). For that reason, in breast cancers, Her2/neu testing is done on initial core biopsy specimens. The test is however, repeated on excision specimen in high grade tumors where Her2/neu expression was initially negative on core biopsies. For endometrial serous carcinoma no guidelines have been set or proposed as of yet. The Gynecologic Oncologists request this test because of proven benefit of adding Trastuzumab (Fader et al., 2018) and that is why it is important to documenting the findings in this report in the literature so that an informed request can be made by the treating oncologist when multiple tissue samples from the same patient are available for testing. Similarly pathologists also can decide which would be the best sample to test when no instruction is received. We report here three separate scenarios of uterine serous carcinomas in which the Her2/neu expressions were unique enough to justify documentation and therefore have implications for determining which specimen is ideal for the Her2 overexpression testing and likely to have highest possibility in identifying the Her2/neu overexpressed clone in the tumor which would expand the therapeutic options for the patients.

Ovotesticular Disorder of Sex Development (Ovotestis) in Simpson-Golabi-Behmel Syndrome: Expansion of the Clinical Spectrum.

Simpson-Golabi-Behmel syndrome type I (SGBS, OMIM312870), caused by defects of the GPC3 and GPC4 genes on chromosome Xq26, is an X-linked recessive macrosomia/multiple congenital anomaly disorder characterized by somatic overgrowth, coarse facial features, variable congenital anomalies, increased tumor risk, and mild-to-moderate neurodevelopmental anomalies. We report the postmortem findings in 3 second-trimester male siblings with SGBS who displayed ambiguous genitalia (in all 3) and gonadal dysgenesis (ovotestis) (in 1), thus expanding the SGBS spectrum to include these disorders of sex development.

An Integrated Human/Murine Transcriptome and Pathway Approach To Identify Prenatal Treatments For Down Syndrome.

Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls, and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn, and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice.

Updated 2013 College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) guideline recommendations for human epidermal growth factor receptor 2 (HER2) fluorescent in situ hybridization (FISH) testing increase HER2 positive and HER2 equivocal breast cancer cases; retrospective study of HER2 FISH results of 836 invasive breast cancers.

For dual probe HER2 FISH assay, the 2013 CAP/ASCO guideline recommendations lowered the HER2/CEP17 ratio cut off for HER2 amplification to ≥2.0 and introduced an average HER2 copy number criterion for HER2 amplification (≥6.0/cell) and HER2 equivocal categories (≥4 and <6/cell). The HER2/CEP17 equivocal category is eliminated. The aim of this study is to assess the impact of 2013 HER2 FISH testing guideline recommendations update on the assignment of HER2 status with dual probe HER2 FISH assay. Dual probe HER2 FISH assay results on breast cancers from 09/2009 to 07/2015 that underwent reflex HER2 FISH testing after equivocal HER2 (2+) immunohistochemistry (IHC) were reviewed. HER2 copy number, CEP17 signals, and HER2/CEP ratios were noted. HER2 status was assigned as HER2 negative (HER2-), HER2 equivocal (HER2e), and HER2 amplified (HER2+) by applying both 2007 and 2013 CAP/ASCO HER2 FISH guideline recommendations and results were compared. New guidelines reclassified HER2 FISH status in a significant proportion of cases (8.3 %, 69/836; p = .021). There were 22 (2.6 %) more HER2+, 17 (2.1 %) more HER2e, and 39 (4.1 %) fewer HER2- tumors. Change of HER2 status correlated significantly with ≥3 CEP17 signals (38 vs. 2 %; p < .001). The 2013 CAP/ASCO guideline recommendations for HER2 FISH testing by dual probe assay increased the HER2 amplified and HER2 equivocal tumors. Increase in HER2 equivocal tumors would potentially increase the frequency of repeat HER2 testing. Tumors with ≥3 CEP17 signals, so-called chromosome 17 polysomy, are more likely to be impacted and classified as HER2 equivocal.

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.

Genetic and phenotypic diversity of NHE6 mutations in Christianson syndrome.

OBJECTIVE: Recently, Christianson syndrome (CS) has been determined to be caused by mutations in the X-linked Na(+) /H(+) exchanger 6 (NHE6). We aimed to determine the diagnostic criteria and mutational spectrum for CS. METHODS: Twelve independent pedigrees (14 boys, age = 4-19 years) with mutations in NHE6 were administered standardized research assessments, and mutations were characterized. RESULTS: The mutational spectrum was composed of 9 single nucleotide variants, 2 indels, and 1 copy number variation deletion. All mutations were protein-truncating or splicing mutations. We identified 2 recurrent mutations (c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were unique. In our study, 7 of 12 mutations (58%) were de novo, in contrast to prior literature wherein mutations were largely inherited. We also report prominent neurological, medical, and behavioral symptoms. All CS participants were nonverbal and had intellectual disability, epilepsy, and ataxia. Many had prior diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included eye movement abnormalities (79%), postnatal microcephaly (92%), and magnetic resonance imaging evidence of cerebellar atrophy (33%). Regression was noted in 50%, with recurrent presentations involving loss of words and/or the ability to walk. Medical symptoms, particularly gastrointestinal symptoms, were common. Height and body mass index measures were below normal ranges in most participants. Behavioral symptoms included hyperkinetic behavior (100%), and a majority exhibited high pain threshold. INTERPRETATION: This is the largest cohort of independent CS pedigrees reported. We propose diagnostic criteria for CS. CS represents a novel neurogenetic disorder with general relevance to autism, intellectual disability, Angelman syndrome, epilepsy, and regression.

Lethal hypoplasia and developmental anomalies of the lungs in a newborn with intrauterine adrenal hemorrhage and cerebral infarcts: a proposed pulmonary disruption sequence.

We report a case of a 31-week-gestation male newborn who died soon after birth from intractable respiratory failure and persistent pulmonary hypertension. The pregnancy had been complicated by intermittent bleeding between 13 and 20 weeks' gestation, attributed to peripheral placental separation, as well as bilateral fetal adrenal hemorrhage, first detected at 17 weeks' gestation. Postmortem examination revealed small, calcified adrenal glands as well as several remote cerebral and cerebellar infarcts. The lungs were hypoplastic (lung weight/body weight ratio: 1.64%; 10th percentile for 28-36 weeks' gestation: 2.27%) and distorted by exaggerated lobulation. Microscopically, the lungs exhibited several developmental anomalies, including focal acinar dysgenesis suggestive of arrested development in the pseudoglandular stage of development (8-16 weeks' gestation) (mainly in the upper lobes), and features of bronchial obstruction, including focal lobular hyperplasia and microcystic maldevelopment (mainly in the lower lobes). The adrenal and cerebral findings were consistent with a severe early-gestation hypoxic-ischemic insult, likely related to peripheral placental separation and chronic abruption. The co-occurrence and timing of these well-recognized hypoxic lesions provide further evidence that certain developmental lung anomalies, such as focal acinar dysplasia, focal lobular hyperplasia, and microcystic maldevelopment, may, at least in some cases, have a hypoxic/ischemic etiology.

Monochorionic twins discordant for mosaic trisomy 14.

Monochorionic-diamniotic twins are usually monozygotic twins and known to be associated with adverse obstetric and perinatal outcomes. Cases of discordant karyotype of monozygotic twins are rare and most involves sex chromosomes. We present the first case of monochorionic twins with discordant karyotype manifested as mosaic trisomy 14 in one twin (B) and a normal karyotype in the other (A). We describe the postmortem pathological and imaging findings of the trisomic twin and for the first time neuropathological findings of this entity. Metaphase chromosome analysis of twin B using fetal tissue showed a 47,XX, +14 karyotype. Chromosomal microarray analysis (CMA) using fetal tissue revealed 38% mosaicism. CMA with placental tissue from both sides demonstrated normal karyotype and confirmed monozygosity, highlighting the value of array based testing on diagnosing mosaicism and zygosity.

An unbalanced translocation involving loss of 10q26.2 and gain of 11q25 in a pedigree with autism spectrum disorder and cerebellar juvenile pilocytic astrocytoma.

We report on a pedigree with a pair of brothers each with minor anomalies, developmental delay, and autistic-symptoms who share an unbalanced translocation (not detectable by karyotype). The unbalanced translocation involves a 7.1 Mb loss of the terminal portion of 10q, and a 4.2 Mb gain of 11q. One of the brothers also developed a cerebellar juvenile pilocytic astrocytoma. The father was found to be a balanced carrier and the couple had a previous miscarriage. We demonstrate that the breakpoint for the triplicated region from chromosome 11 is adjacent to two IgLON genes, namely Neurotrimin (NTM) and Opioid Binding Protein/Cell Adhesion Molecule-like (OPCML). These genes are highly similar neural cell adhesion molecules that have been implicated in synaptogenesis and oncogenesis, respectively. The children also have a 10q deletion and are compared to other children with the 10q deletion syndrome which generally does not involve autism spectrum disorders (ASDs) or cancer. Together these data support a role for NTM and OPCML in developmental delay and potentially in cancer susceptibility.

Proof of concept study to assess fetal gene expression in amniotic fluid by nanoarray PCR.

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care.

Transcriptomic analysis of cell-free fetal RNA suggests a specific molecular phenotype in trisomy 18.

Trisomy 18 is a common human aneuploidy that is associated with significant perinatal mortality. Unlike the well-characterized "critical region" in trisomy 21 (21q22), there is no corresponding region on chromosome 18 associated with its pathogenesis. The high morbidity and mortality of affected individuals has limited extensive investigations. In order to better understand the molecular mechanisms underlying the congenital anomalies observed in this condition, we investigated the in utero gene expression profile of second trimester fetuses affected with trisomy 18. Total RNA was extracted from cell-free amniotic fluid supernatant from aneuploid fetuses and euploid controls matched for gestational age and hybridized to Affymetrix U133 Plus 2.0 arrays. Individual differentially expressed transcripts were obtained by two-tailed t tests. Over-represented functional pathways among these genes were identified with DAVID and Ingenuity(®) Pathways Analysis. Results show that three hundred and fifty-two probe sets representing 251 annotated genes were statistically significantly differentially expressed between trisomy 18 and controls. Only 7 genes (2.8% of the annotated total) were located on chromosome 18, including ROCK1, an up-regulated gene involved in valvuloseptal and endocardial cushion formation. Pathway analysis indicated disrupted function in ion transport, MHCII/T cell mediated immunity, DNA repair, G-protein mediated signaling, kinases, and glycosylation. Significant down-regulation of genes involved in adrenal development was identified, which may explain both the abnormal maternal serum estriols and the pre and postnatal growth restriction in trisomy 18. Comparison of this gene set to one previously generated for trisomy 21 fetuses revealed only six overlapping differentially regulated genes. This study contributes novel information regarding functional developmental gene expression differences in fetuses with trisomy 18.

Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu.

Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p<30), and onset of aneuploidy. At high passage (p>85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.

Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses.

To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS.

Utility of fetal karyotype in the evaluation of phenotypically abnormal stillbirths.

The objectives of this study are to test the hypothesis that stillbirths without aneuploidy-associated phenotypes have a low incidence of karyotypic abnormalities, similar among those with and without other anatomic defects. We employed a uniform postmortem protocol to examine fetuses and placentas in 962 consecutive stillbirths measuring > or =20 weeks in clinically determined gestational age submitted to the Women and Infants Hospital Division of Perinatal Pathology from 1990 through 2005. Classification of anatomic (macroscopic) abnormalities was based on a priori criteria. Anatomic fetal abnormalities were noted in 387 cases. Conventional karyotype analysis was successfully performed on 346 fetal tissue samples, 114 in anatomically normal and 232 in anatomically abnormal fetuses. The distribution of karyotypic abnormalities among cases with and without anatomic abnormalities was compared. Of the 962 stillbirths, 40% (387) had malformations. Tissue culture for karyotype analysis was attempted in 412 cases from both groups and failed in 66 cases (16%). At the 450 to 500-band resolution level, 60 of the remaining 346 karyotypes were abnormal. Of the 232 malformation cases with successful karyotyping, 59 had phenotypic attributes indicative of aneuploidy, all of which had later karyotype confirmation. Of the remaining 173 anomalous fetuses with karyotype analysis, only 1 demonstrated a karyotypic abnormality. All 114 karyotypes performed in stillbirths without anatomic abnormalities were normal. Among > or =20-week stillbirths, aneuploid karyotypes are uncommon except in fetuses with suspect phenotypes. The 95% probability estimates of karyotype abnormality in the phenotypically abnormal and normal stillbirths, 5.5% and 5.6%, respectively, do not differ. These data do not have sufficient power to detect a small difference in rates ofkaryotypic abnormalities between the 2 groups of > or =20-week stillbirths. However, this series indicates that this technology is uninformative among stillborn fetuses that lack aneuploidy phenotypes.

Effects of chemotherapy during pregnancy on the placenta.

Whereas the effects of chemotherapy during pregnancy for mother and fetus are well described, its effects on the placenta remain largely undetermined. We performed a retrospective clinicopathologic analysis of the placenta following chemotherapy. Charts were reviewed for type of malignancy, type and timing of chemotherapy, and fetal and pregnancy outcome. Placentas were studied by standard pathologic analysis as well as computer-assisted morphometry and fluorescence in situ hybridization (FISH) analysis. Patients (n = 13) underwent chemotherapy during pregnancy for carcinoma of breast (3), ovary (2), cervix (2), salivary gland (1), lymphoma/leukemia (4), or rhabdomyosarcoma (1). Eleven patients were treated with DNA-active cytotoxic agents during the 2nd and/or 3rd trimesters; their placentas showed nonspecific findings, including villous hypermaturity, distal villous hypoplasia, villous edema, and excessive extravillous trophoblast, and 4/11 (36%) were small-for-age. In one case (rhabdomyosarcoma), the mother was exposed to cytotoxic agents throughout the entire pregnancy. In this case, associated with severe congenital anomalies, the placenta showed striking nuclear pleomorphism of the extravillous trophoblast of the chorion laeve, associated with FISH-demonstrated hyperpolyploidy. One patient was treated with the targeted tyrosine kinase inhibitor, imatinib, in 2 consecutive pregnancies; these placentas showed no specific anomalies. Our findings suggest that chemotherapy during the 1st trimester induces excessive polyploidization of the chorion laeve trophoblast, likely representing an adaptive response to intraamniotic toxins. Second and 3rd trimester exposure to cytotoxic agents may predispose to placental underdevelopment. However, without appropriate controls (untreated patients with equivalent malignancies), the specific effects of chemotherapy in this group are difficult to assess.

Fas-ligand-induced apoptosis of respiratory epithelial cells causes disruption of postcanalicular alveolar development.

Premature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia- associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)(7)-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA(+)/(TetOp)(7)-FasL(+) mice compared with single-transgenic CCSP-rtTA(+) littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. These data demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis.

Activin subunit and receptor expression in normal and cleft human fetal palate tissues.

Craniofacial malformations, such as cleft palate, present serious complications in the newborn and are often of unknown etiology. Activin BA subunit deletion leads to cleft palate in mice, but the expression of this protein in the human palate has not been explored. Our goal was to determine the spatial and temporal expression of inhibin/activin subunits; the binding protein, follistatin; and activin receptors in the human fetal palate. Residual human fetal palate tissues, with or without cleft, were collected during routine autopsy at Women and Infants Hospital. Inhibin/activin alpha and beta subunits, follistatin, and activin receptor protein and mRNA expression were studied by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) experiments, respectively. Dimeric activin A levels were compared in cleft and normal palate tissue homogenates by immunoassay. Activin BA, follistatin, and activin receptor type IIA proteins were observed in normal and cleft palate tissues throughout pregnancy (gestational weeks 11 to 40). Proteins were predominantly found in developing bone cells, with no significant group differences. Inhibin/activin BA subunit, follistatin, and activin receptor mRNAs were also detected in normal and cleft fetal palate tissues, but inhibin alpha and BB subunit were absent. Inhibin/activin BA subunit expression was consistent with the presence of dimeric activin A, but levels did not differ significantly between cleft and control tissues. Inhibin/activin BA subunit, follistatin, and activin receptor proteins and mRNAs are present in the human fetal palate. These data suggest that activin signalling has the potential to be associated with human palate development.

Array-CGH analysis of cell-free fetal DNA in 10 mL of amniotic fluid supernatant.

BACKGROUND: Previously, we showed that analysis of amniotic fluid (AF) supernatant cell-free fetal (cff) DNA using DNA microarrays (array-CGH) allows for detection of whole chromosome differences between test and reference DNA. Subsequent technical advances have increased both the yield and quality of extracted cffDNA. Here we determined whether array-CGH using smaller volumes of both fresh and frozen AF cffDNA could identify fetal aneuploidy. METHODS: CffDNA was extracted from 10 mL of residual AF supernatant. The test AF samples (n = 10) included one with a normal karyotype, and nine with the following fetal aneuploidies: trisomies 13 (n = 1), 18 (n = 3), 21 (n = 2), trisomy 9 mosaicism (47,XX,+ 9[18]/46,XX[2]), triploidy (69,XXY) and Turner syndrome (45,X). RESULTS: Array-CGH using AF cffDNA from aneuploid fetuses, compared to euploid reference AF cffDNA, detected whole chromosome aneuploidy in 8 of 9 cases tested, including the case of trisomy 9 mosaicism. The case of triploidy was not detected. CONCLUSIONS: CffDNA extracted from 10 mL AF supernatant can be analyzed using array-CGH to correctly identify human chromosome abnormalities. This technology allows for rapid screening of AF samples for whole chromosomal changes by using routinely discarded supernatant, and may augment standard prenatal karyotyping techniques by providing additional molecular information.