Soluble syndecan-1 and glycosaminoglycans in preeclamptic and normotensive pregnancies.

Preeclampsia, an important cause of maternal and fetal morbidity and mortality, is associated with increased sFLT1 levels and with structural and functional damage to the glycocalyx contributing to endothelial dysfunction. We investigated glycocalyx components in relation to preeclampsia in human samples. While soluble syndecan-1 and heparan sulphate were similar in plasma of preeclamptic and normotensive pregnant women, dermatan sulphate was increased and keratan sulphate decreased in preeclamptic women. Dermatan sulphate was correlated with soluble syndecan-1, and inversely correlated with blood pressure and activated partial thromboplastin time. To determine if syndecan-1 was a prerequisite for the sFlt1 induced increase in blood pressure in mice we studied the effect of sFlt1 on blood pressure and vascular contractile responses in syndecan-1 deficient and wild type male mice. The classical sFlt1 induced rise in blood pressure was absent in syndecan-1 deficient mice indicating that syndecan-1 is a prerequisite for sFlt1 induced increase in blood pressure central to preeclampsia. The results show that an interplay between syndecan-1 and dermatan sulphate contributes to sFlt1 induced blood pressure elevation in pre-eclampsia.

Ketonuria is not associated with hyperemesis gravidarum disease severity.

OBJECTIVE: To assess the association between ketonuria and hyperemesis gravidarum (HG) disease severity. STUDY DESIGN: We included pregnant women hospitalised for HG who participated in the Maternal and Offspring outcomes after Treatment of HyperEmesis by Refeeding (MOTHER) trial and women who were eligible, chose not to be randomised and agreed to participate in the observational cohort. Between October 2013 and March 2016, in 19 hospitals in the Netherlands, women hospitalised for HG were approached for study participation. The presence of ketonuria was not required for study entry. Ketonuria was measured at hospital admission with a dipstick, which distinguishes 5 categories: negative and 1+ through 4 + . The outcome measures were multiple measures of HG disease severity at different time points: 1) At hospital admission (study entry): severity of nausea and vomiting, quality of life and weight change compared to pre-pregnancy weight, 2) One week after hospital admission: severity of nausea and vomiting, quality of life and weight change compared to admission, 3) Duration of index hospital admission and readmission for HG at any time point RESULTS: 215 women where included. Ketonuria was not associated with severity of nausea and vomiting, quality of life or weight loss at hospital admission, nor was the degree of ketonuria at admission associated with any of the outcomes 1 week after hospital admission. The degree of ketonuria was also not associated with the number of readmissions. However, women with a higher degree of ketonuria had a statistically significant longer duration of hospital stay (per 1+ ketonuria, difference: 0.27 days, 95 % CI: 0.05 to 0.48). CONCLUSIONS: There was no association between the degree of ketonuria at admission and severity of symptoms, quality of life, maternal weight loss, or number of readmissions, suggesting that ketonuria provides no information about disease severity or disease course. Despite this, women with a higher degree of ketonuria at admission were hospitalised for longer. This could suggest that health care professionals base length of hospital stay on the degree of ketonuria. Based on the lack of association between ketonuria and disease severity, we suggest it has no additional value in the clinical management of HG.

Higher decidual EBI3 and HLA-G mRNA expression in preeclampsia: Cause or consequence of preeclampsia.

The maternal immune system must adapt to tolerate the invasion of the allogeneic feto-placental unit. It is generally accepted that improper adaptation causes pregnancy complications like preeclampsia. The Epstein-Barr virus-induced gene 3 (EBI3) protein is a subunit of immune-modulatory cytokines interleukin 27 (IL-27) and IL-35. EBI3 has been reported to associate with HLA-G. In this small pilot study we find higher decidual EBI3 (p<0.05) and HLA-G (p<0.01) mRNA expression in preeclampsia (n=7) compared to normotensive (n=8) pregnancies. Whether the higher EBI3 and HLA-G mRNA expression is a consequence or cause of preeclampsia remains to be answered. Further research to determine the effects on IL-27 and IL-35 is needed.

Placental expression of heparan sulfate 3-O-sulfotransferase-3A1 in normotensive and pre-eclamptic pregnancies.

INTRODUCTION: The endothelial glycocalyx, consisting of membrane-bound proteoglycans and attached glycosaminoglycans plays an important role in vascular homeostasis. We aimed to assess whether glycocalyx mRNA transcripts are differentially expressed in placental tissue of pre-eclamptic and normotensive women. METHODS: We evaluated the expression of transcripts encoding for proteins involved in glycocalyx synthesis and degradation using a microarray analysis of placental mRNA obtained from pre-eclamptic and normotensive women. Participants were recruited from the department of obstetrics at a university hospital in Amsterdam, The Netherlands. The most prominent differentially expressed transcript was validated by qPCR on 112 additional placenta samples. RESULTS: Of 78 preselected genes involved in glycocalyx synthesis and degradation, only HS3ST3A1 mRNA was differentially expressed in placental tissue obtained from pre-eclamptic women (N = 12) compared to normotensive women (N = 12, fold change = 0.61, p = 0.02). Validation with qPCR in additional placental samples of 64 normotensive and 48 pre-eclamptic women confirmed that normalized mRNA expression of HS3ST3A1 was decreased by 27% (95% CI 14%-41%) in placental tissue obtained from pre-eclamptic compared to normotensive women (p < 0.001). HS3ST3A1 expression was positively correlated with neonatal birth weight in normotensive women (r = 0.35, p < 0.01) and inversely correlated with mean arterial pressure of women with pre-eclampsia (r = 0.32, p = 0.02). CONCLUSIONS: The mRNA expression of HS3ST3A1, which encodes for a 3-O sulfating enzyme of heparan sulfate (3-OST-3A1), is decreased in pre-eclamptic placental tissue. Expression of this glycocalyx synthesis transcript is correlated with maternal blood pressure and neonatal birth weight, suggesting a possible role in pre-eclampsia-associated placental dysfunction.

Increased glucocerebrosidase expression and activity in preeclamptic placenta.

INTRODUCTION: Lysosomal glucosidase beta acid (GBA) deficiency is inherent to Gaucher disease, Parkinsonism and Lewy-body dementia. Increased GBA expression has never been associated with human disease. We describe increased GBA expression and activity in placenta from preeclamptic pregnancies. METHODS: 112 placenta biopsies were available for qPCR, analysis of GBA gene expression and activity. Microanalysis was performed on 20 placenta samples. Alternatively spliced placental GBA transcripts were cloned, expressed in HEK293 cells and analyzed by Western blot and activity assay. RESULTS: GBA is expressed in the syncytiotrophoblast layer of human placenta already at 5 weeks of gestation. We identified five novel GBA transcripts in placenta that enzymatically inactive when expressed in HEK293 cells. Both GBA RNA expression and enzymatic activity are upregulated in preeclamptic placenta. Microarray analysis of 20 placenta tissues identified 158 genes co-regulating with GBA expression and gene enrichment analysis highlights lysosomal function. In our micro-array data GBA expression does not correlate with FLT1 expression, currently the most powerful marker for preeclampsia. There are 89 transcripts that are negatively correlated with GBA expression of which BMP4 and TFEB are interesting as they are essential to early placenta function. DISCUSSION: Although very speculative, we hypothesize that increased GBA expression might relate to placentation through decreased BMP4 signaling or vascularization through downregulation of TFEB. Ceramide, the product of hydrolysis of glucosylceramide by GBA and involved in the regulation of cell differentiation, survival and apoptosis, is another putative candidate linking increased GBA activity to preeclampsia. Both pathways merit further investigation.

Pathophysiological aspects of thyroid hormone disorders/thyroid peroxidase autoantibodies and reproduction.

BACKGROUND: Thyroid hormone disorders and thyroid peroxidase autoantibodies (TPO-Ab) in women are associated with subfertility and early pregnancy loss. Here, we aim to provide a comprehensive overview of the literature on the pathophysiology of these associations. METHODS: A review of the literature in the English language was carried out. Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register from 1975 until March 2014. RESULTS: From a total of 6108 primary selected articles from the literature search, 105 articles were selected for critical appraisal. Observational data indicate that altered thyroid hormone levels are associated with disturbed folliculogenesis, spermatogenesis, lower fertilization rates and lower embryo quality. Triiodothyronine (T3) in combination with FSH enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway. T3 is considered a biological amplifier of the stimulatory action of gonadotrophins on granulosa cell function. T3 increases the expression of matrix metalloproteinases (MMP), MMP-2, MMP-3, fetal fibronectin and integrin α5ß1T3 in early placental extravillous trophoblasts. Thyroid hormone transporters and receptors are expressed in the ovary, early embryo, endometrium, uterus and placenta. No other data explaining the associations could be retrieved from the literature. The presence of TPO-Ab is negatively associated with spermatogenesis, fertilization and embryo quality, but no data are available on the potential pathophysiological mechanisms. CONCLUSIONS: Thyroid hormone disorders and TPO-Ab are associated with disturbed folliculogenesis, spermatogenesis, fertilization and embryogenesis. The pathophysiology of these associations remains largely unknown, as evidence is limited and includes studies using small sample sizes, and often restricted to animal models. There are no studies on the pathophysiology underlying the association between TPO-Ab and reproduction. The available evidence, although limited, supports a role of thyroid hormone in fertility and early pregnancy. This justifies clinical intervention studies on the effects of thyroid hormone supplementation in women with subclinical hypothyroidism and in women prone to develop hypothyroidism due to the presence of TPO-Ab. In addition, more research is needed to identify the underlying mechanisms. This would be of particular interest in women undergoing IVF to pinpoint the effects of thyroid hormone on different parameters of reproduction.

Maternal cardiac function, uteroplacental Doppler flow parameters and pregnancy outcome: a systematic review.

OBJECTIVE: To investigate the existing evidence for a link between maternal cardiac function, abnormal uteroplacental flow and poor perinatal outcome in women with and without known cardiac disease. METHODS: PubMed and EMBASE databases were searched systematically for studies relating cardiac functional parameters and uteroplacental Doppler flow with pregnancy outcome in women with pre-existing congenital cardiac disease and women without known cardiac disease. Only studies based on echocardiography were included. RESULTS: From 1732 citations, 10 articles were included. In women with known congenital heart disease, a relationship was found between abnormal uteroplacental Doppler flow patterns and cardiac function before and during pregnancy. Conversely, women without a history of congenital heart disease, but with abnormal uterine artery resistance and pregnancy complications, more often showed global left ventricular diastolic dysfunction (33%; P = 0.0001), impaired myocardial relaxation (72%; P < 0.0001) and left ventricular systolic dysfunction (17%; P = 0.006), even up to 1 year postpartum. CONCLUSION: There is increasing evidence for an association between pre-existing subclinical cardiac dysfunction, poor placentation (reflected by uteroplacental Doppler flow abnormalities) and poor pregnancy outcome. It may be postulated that pre-existing suboptimal cardiac performance, as a result of either congenital heart disease or a subclinical latent condition, is one of the common denominators of poor placentation, leading to poor pregnancy outcome.

Active caspase-3 is removed from cells by release of caspase-3-enriched vesicles.

Cleavage of Rho associated Coiled Coil kinase I (ROCK I) by caspase-3 contributes to membrane blebbing. Whether caspase-3 and ROCK I also play a role in the release of membrane vesicles is unknown. Therefore, we transfected a human breast cancer cell line (MCF-7) that is caspase-3 deficient, lacks membrane blebbing, and does not release membrane vesicles, with caspase-3. Cells expressing caspase-3 demonstrate both ROCK I-mediated membrane blebbing, and release of small (400-600nm) membrane vesicles in a ROCK I-independent manner. These membrane vesicles contain caspase-3, and are enriched in caspase-3 activity compared to the releasing cells. Caspase-3-containing vesicles are taken up by untransfected cells but the cells do not show any sign of apoptosis. In conclusion, we show that the release of caspase-3-enriched membrane vesicles and membrane blebbing are two differentially regulated processes. Furthermore, we hypothesize that packaging of caspase-3 into membrane vesicles contributes to cellular homeostasis by the removal of caspase-3, and concurrently, protects the cells' environment from direct exposure to caspase-3 activity.

Maternal circulating PlGF concentrations and placenta-related pregnancy complications: First results from the CoLab AngF Study.

INTRODUCTION: Circulating angiogenic factors are potential markers for preeclampsia, but heterogeneous studies have failed to identify precise predictive/diagnostic properties. The Global CoLaboratory is investigating how to merge published data of angiogenic factors for meta-analysis on an individual sample basis. OBJECTIVE: To amalgamate pregnancy angiogenic factor studies, investigate diagnostic and predictive properties of these markers in preeclampsia and placenta-related pregnancy complications, and to test if measures from disparate platforms can be standardised. This is the first report using PlGF measures to diagnose preeclampsia. METHODS: Data were derived from 15 cohorts, within and outside the CoLaboratory network. Women were classified as either case (confirmed diagnosis of preeclampsia at sampling) or non-case (no preeclampsia at sampling). Individual PlGF measurements from four different analytical platforms were used, along with transformations of the data (e.g. log-transformations, transformations to a baseline platform). Transformed measurements were standardised both for specific platforms and globally, stratifying on gestational age. Different statistical techniques were compared. RESULTS: The database currently contains 1442 cases and 11,512 non-cases, which were used to define an algorithm to merge PlGF measurements from different platforms. Non-case distributions were used to standardise case results. Diagnostic PlGF measurements in relation to preeclampsia will be presented and confirm feasibility. CONCLUSIONS: Future studies can extend this approach to other angiogenic factors, prediction as well as diagnosis and to other placenta-related disorders.

Accuracy of circulating placental growth factor, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1 and soluble endoglin in the prediction of pre-eclampsia: a systematic review and meta-analysis.

BACKGROUND: Biomarkers have been proposed for identification of women at increased risk of developing pre-eclampsia. OBJECTIVES: To investigate the capacity of circulating placental growth factor (PlGF), vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG) to predict pre-eclampsia. SEARCH STRATEGY: Medline and Embase through October 2010 and reference lists of reviews, without constraints. SELECTION CRITERIA: We included original publications on testing of PlGF, VEGF, sFLT1 and sENG in serum or plasma of pregnant women at <30 weeks of gestation and before clinical onset of pre-eclampsia. DATA COLLECTION AND ANALYSIS: Two reviewers independently identified eligible studies, extracted descriptive and test accuracy data and assessed methodological quality. Summary estimates of discriminatory performance were obtained. MAIN RESULTS: We included 34 studies. Concentrations of PlGF (27 studies) and VEGF (three studies) were lower in women who developed pre-eclampsia: standardised mean differences (SMD) -0.56 (95% CI -0.77 to -0.35) and -1.25 (95% CI -2.73 to 0.23). Concentrations of sFLT1 (19 studies) and sENG (ten studies) were higher: SMD 0.48 (95% CI 0.21-0.75) and SMD 0.54 (95% CI 0.24-0.84). The summary diagnostic odds ratios were: PlGF 9.0 (95% CI 5.6-14.5), sFLT1 6.6 (95% CI 3.1-13.7), sENG 4.2 (95% CI 2.4-7.2), which correspond to sensitivities of 32%, 26% and 18%, respectively, for a 5% false-positive rate. AUTHOR'S CONCLUSIONS: PlGF, sFLT1 and sENG showed modest but significantly different concentrations before 30 weeks of gestation in women who developed pre-eclampsia. Test accuracies of all four markers, however, are too poor for accurate prediction of pre-eclampsia in clinical practice.

Hydrogen sulfide producing enzymes in pregnancy and preeclampsia.

Preeclampsia, a human pregnancy specific disorder is characterized by an anti-angiogenic state. As hydrogen sulfide (H(2)S) has pro-angiogenic and anti-oxidative characteristics, we hypothesized that H(2)S levels could play a role in the pathogenesis of preeclampsia and studied the placental expression of the H(2)S-producing enzymes cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS). CBS and CSE protein are expressed in the fetal-placental endothelium and CBS only in Hofbauer cells. CBS mRNA expression is decreased (p = 0.002) in early-onset preeclampsia, while CSE mRNA is unchanged. Thus, down regulation of CBS during early-onset preeclampsia may result in less H(2)S-production and may aid in the anti-angiogenic state.

OS065. Placental growth factor as a diagnostic and prognostic test forplacental complications of pregnancy.

INTRODUCTION: Placental growth factor (PlGF) levels in maternal circulation are altered in pre-eclampsia and intrauterine growth restriction (IUGR) (Benton et al.) and may have utility in identifying cases associated with placental dysfunction and stratifying pregnancy survival. OBJECTIVES: We sought to determine if a positive PlGF test measured on the Triage PlGF rapid assay (Alere, San Diego) agrees with the clinical diagnosis and predicts preterm delivery. METHODS: EDTA-blood was collected from women admitted to the AMC Obstetrics Department after 20 weeks of gestation with informed consent and according to the protocol approved by the AMC Medical Ethical Board (10/127). Plasma free PlGF levels from women diagnosed with early-onset pre-eclampsia (n=28), normotensive IUGR (N=6) and pregnancy complications excluding pre-eclampsia and IUGR (n=18) were quantified using the Triage PlGF immunoassay. Samples were collected before GA 34+6 and analyzed in batch assay. Results were interpreted against GA-dependent cutoffs set at the 5th centile for gestational age in normal pregnancy (Knudsen et al). PlGF levels below the cutoffs were assigned "positive" according to the product insert. The proportion of subjects with a positive PlGF test result was calculated for each group, together with the proportion of subjects requiring preterm delivery. RESULTS: Twenty-eight women developed early-onset pre-eclampsia and, of these, 27/28 (96.4%) had a positive PlGF test. The woman with negative PlGF test presented to clinic at GA 34+3 with hypertension and suspected pre-eclampsia, but delivered at GA 39+4. Six women developed normotensive IUGR, of which 4 had a positive PlGF test, and in each the PlGF level was below the limit of detection of the test. The 2 women with a negative PlGF test had twin pregnancies. Eighteen women developed pregnancy complications excluding pre-eclampsia and IUGR. Six had at least one serial sample with a positive PlGF test. Of these, one woman had partial placental abruption, 3 had PPROM, one had spontaneous labour at GA 33+3, and one had bleeding after elective embryo reduction and neonatal death during delivery at 23+3. The proportion of women with a positive PlGF test, where the date of delivery is known, requiring preterm delivery was 34/37 (91.9%). CONCLUSION: These preliminary data suggest that a positive PlGF test by Triage may identify placentally-mediated pregnancy complications and that a very low level of PlGF identifies women at increased risk for preterm delivery.

Unique skin changes in a case of Albright hereditary osteodystrophy caused by a rare GNAS1 mutation.

Albright hereditary osteodystrophy (AHO) is a syndrome of short stature, obesity, brachydactyly and subcutaneous calcifications with pseudohypoparathyroidism (PHP; leading to hypocalcaemia, hyperphosphataemia and elevated levels of parathyroid hormone, PTH). It was first described over 60 years ago. Since then, much has been learned about the aetiology of AHO which has been shown to be caused by heterozygous loss-of-function mutations within the GNAS1 gene. GNAS1 is subject to imprinting leading to phenotypic heterogeneity within kindreds with one mutation. Patients with AHO often present with symptoms of hypocalcaemia and/or with subcutaneous calcifications. The latter is thought to be the typical skin abnormality in AHO. We describe a family with AHO and hormone resistance (PHP type Ia) resulting from a rare mutation in GNAS1. The proband presented with small subcutaneous calcifications in the helix of the right ear and concentrated in a sharply demarcated zone of subcutaneous and dermal hypoplasia. This abnormality has so far not been described in patients with AHO. We speculate on the mechanism of dermal hypoplasia and resistance to PTH and suggest that subcutanous or dermal hypoplasia might be another feature which can be present in patients with AHO.

Seven placental transcripts characterize HELLP-syndrome.

The human placenta is prerequisite for the development of gestational hypertensive diseases like early-onset preeclampsia (PE) and Hemolysis, Elevated Liver enzymes and Low platelets (HELLP) syndrome. Both syndromes are associated with extensive maternal and perinatal mortality, and morbidity with life long consequences. We aimed to investigate differences in gene expression between placental tissue obtained from normotensive pregnant women and women with PE and HELLP syndrome. Firstly, comparison of Serial Analysis of Gene Expression profiles of 28 weeks' control placenta (available after idiopathic premature delivery) to a HELLP/PE placenta matched for gestational age identified 404 differentially expressed transcripts. Secondly, using sqPCR, the expression levels of 37 of these transcripts were analyzed in placentas of 36 pregnant women, 22 with preeclampsia and HELLP syndrome. Thirdly, nearest centroid classification determined the HELLP specific molecular signature consisting of the upregulated expression of genes encoding the vascular endothelial growth factor receptor (FLT1), leptin (LEP), pappalysin 2 (PAPPA2), and WW domain containing transcription regulator 1 (WWTR1) combined with down regulated expression of the genes encoding cadherin-associated protein (CTNNAL), glutathione S-transferase pi (GSTP1) and calgranulin A (S100A8). This set discriminates HELLP placenta from control and PE placenta with a 24% misclassification rate (95% CI 8.3-41.9%), independent from known risk factors like parity and ethnicity. The transcripts involved correspond to diverse molecular pathways, exemplifying the multigenic molecular basis of the disorder. This distinct placental molecular signature suggests that HELLP is not a PE variant but a separate disease entity. Our data may prove fundamental for the further molecular analysis of PE and HELLP syndrome.

Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism.

BACKGROUND: Several genetic defects are associated with permanent congenital hypothyroidism. Immunologic, environmental, and iatrogenic (but not genetic) factors are known to induce transient congenital hypothyroidism, which spontaneously resolves within the first months of life. We hypothesized that molecular defects in the thyroid oxidase system, which is composed of at least two proteins, might be involved in the pathogenesis of permanent or transient congenital hypothyroidism in babies with defects in iodide organification, for which the oxidase system is required. METHODS: Nine patients were recruited who had idiopathic congenital hypothyroidism (one with permanent and eight with transient hypothyroidism) and an iodide-organification defect and who had been identified by the screening program for congenital hypothyroidism. The DNA of the patients and their relatives was analyzed for mutations in the genes for thyroid oxidase 1 (THOX1 ) and 2 (THOX2 ). RESULTS: The one patient with permanent and severe thyroid hormone deficiency and a complete iodide-organification defect had a homozygous nonsense mutation in the THOX2 gene that eliminates all functional domains of the protein. Three of the eight patients with mild transient congenital hypothyroidism and a partial iodide-organification defect had heterozygous mutations in the THOX2 gene that prematurely truncate the protein, thus abolishing its functional domains. CONCLUSIONS: Biallelic inactivating mutations in the THOX2 gene result in complete disruption of thyroid-hormone synthesis and are associated with severe and permanent congenital hypothyroidism. Monoallelic mutations are associated with milder, transient hypothyroidism caused by insufficient thyroidal production of hydrogen peroxide, which prevents the synthesis of sufficient quantities of thyroid hormones to meet the large requirement for thyroid hormones at the beginning of life.

Up to date with human thyroglobulin.

The coding region of the human thyroglobulin (TG) mRNA has been resequenced, and comparison with the TG sequence originally published in 1987 showed many variations. All of the variations were validated in 20--40 other alleles, and this resulted in the revision of 41 nucleotide positions. This review presents the revised wild-type human TG sequence, including all known exon/exon boundaries and additional data on the TG mRNA population, concerning alternative splicing and variability of the polyadenylation cleavage site. The amino acid sequence derived shows one additional, 12 changed, and 10 polymorphic residues. Protein characteristics, such as acceptor and donor tyrosine residues, N-glycosylation sites, cysteine-rich repeats, the proposed receptor domain, and antigenic epitopes, are included, and their relationship to the revised sequence is discussed. Furthermore, all reported TG mutations causing dyshormonogenesis in humans and animals are designated in the nucleotide and amino acid sequences. This up-to-date profile of the human TG molecule presents the features of importance for its complex role in thyroid hormonogenesis, and is the basis for future studies on the structure--function relationship.

Cloning of tissue-specific genes using serial analysis of gene expression and a novel computational substraction approach.

A paradigm of molecular medicine is the identification of functionally specialized genes in the search of defects responsible for human disease. To identify novel genes relevant for thyroid physiology, we applied serial analysis of gene expression (SAGE) and identified 4260 tag sequences that did not match any known gene present in the GenBank database ("no-match" tags). These no-match tags represent still uncharacterized transcripts. Most of them are expected to correspond to housekeeping genes and only a few to genes with a tissue-restricted pattern of expression. To pinpoint the best candidates for tissue-specificity in a large series of tags, we used a computer-based approach. We compared the relative abundance of 80 no match tags in our thyroid SAGE library with the expression level in 14 other SAGE libraries derived from 9 different human tissues. Based on the expression data, we developed the "tissue preferential expression" (TPE) algorithm to discriminate tags expressed specifically in the thyroid. We then selected four tags as preferentially expressed in thyroid. Results were validated by RT-PCR and northern blot on multiple-tissue RNA samples. Finally, the screening of a thyroid cDNA library with expressed sequence tag (EST) sequences related to the selected tags allowed the isolation of four novel thyroid-specific cDNAs. We demonstrate that the computational substraction of SAGE tags by the proposed TPE algorithm is a rapid and reliable way to expedite the cloning of tissue-specific genes through the combined use of SAGE and EST databases.

Absence of activating mutations in ras and gsp oncogenes in a cohort of nine patients with sporadic pediatric thyroid tumors.

BACKGROUND: Characterization of the genetic background of pediatric thyroid carcinomas could aid in distinguishing between differently staged tumors with respect to treatment and prognosis. Two known genetic factors associated with thyroid carcinoma, the proto-oncogenes gsp and ras were investigated. PROCEDURE: DNA was extracted from paraffin sections from both tumor and normal thyroid tissue of nine patients (ages 9-16 years). Of these patients, eight were diagnosed with papillary carcinoma and one with follicular adenoma. The coding exons of gsp and the three known ras genes (H, K, and N-ras) were screened for mutations using SSCP-analysis. RESULTS: There were no mutations present in the ras and gsp proto-oncogenes hot spots, however, LOH of H-ras (chromosome location 11p15.5) was found in tumor tissue from one patient and a homozygous mutation in exon 12 of gsp causing a Pro-->Ser conversion was present in the thyroid tumor tissue from another patient. Two silent polymorphisms were detected, H-ras exon1, 86T-->C and gsp exon 5, 81T-->C. CONCLUSIONS: Our results indicate that the ras/gsp mutations found are probably late events in the tumorigenesis representing general oncogenic stress. In conclusion, it seems that ras/gsp activation is not a factor in the mechanism causing sporadic thyroid carcinoma in children.