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.

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.

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.

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.

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.

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.

Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis.

The analysis of a human thyroid serial analysis of gene expression (SAGE) library shows the presence of an abundant SAGE tag corresponding to the mRNA of thyroglobulin (TG). Additional, less abundant tags are present that can not be linked to any other known gene, but show considerable homology to the wild-type TG tag. To determine whether these tags represent TG mRNA molecules with alternative cleavage, 3'-RACE clones were sequenced. The results show that the three putative TG SAGE tags can be attributed to TG transcripts and reflect the use of alternative polyadenylation cleavage sites downstream of a single polyadenylation signal in vivo. By screening more than 300 000 sequences corresponding to human, mouse and rat transcripts for this phenomenon we show that a considerable percentage of mRNA transcripts (44% human, 22% mouse and 22% rat) show cleavage site heterogeneity. When analyzing SAGE-generated expression data, this phenomenon should be considered, since, according to our calculations, 2.8% of human transcripts show two or more different SAGE tags corresponding to a single gene because of alternative cleavage site selection. Both experimental and in silico data show that the selection of the specific cleavage site for poly(A) addition using a given polyadenylation signal is more variable than was previously thought.

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.

Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics.

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

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.

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.

Serial analysis of gene expression as a tool to assess the human thyroid expression profile and to identify novel thyroidal genes.

The assessment of the expression profile of normal human thyroid tissue using serial analysis of gene expression (SAGE) generated a collection of 10,994 sequence transcripts (tags). Each tag represented a messenger RNA transcript, and, in total, 6099 different tags could be distinguished. The presence and abundance of thyroid-specific transcripts showed the overall expression profile to be from a normal thyroid cell. The expression level of several transcripts was confirmed on Northern blot. Seventy percent of tags could not be attributed to a known human gene and, therefore, possibly correspond to novel genes putatively involved in thyroid function. The tag sequence generated by the SAGE technique can be used to further characterize these novel genes. In this way, application of the SAGE technique to thyroid tissue gives insight in the expression profile of a normal thyroid gland and provides the information to characterize novel genes involved in thyroid pathology, such as congenital hypothyroidism and thyroid neoplasia.

Type II and type III deiodinase activity in human placenta as a function of gestational age.

Thyroid hormones are essential for fetal development. T4 can be activated by type I (ID-I) and type II (ID-II) iodothyronine deiodinase or inactivated by type III deiodinase (ID-III). The influence of placental ID-II and ID-III on the regulation of fetal thyroid hormone levels was investigated. Using [125I]T4 and [125I]T3, respectively, ID-II and ID-III activities were measured in homogenates of normal human placentas from 6-43 weeks gestational age and in placentas from five term neonates with a total thyroid hormone synthesis defect. ID-II and ID-III activities related to protein or DNA concentration decreased and total placental ID-III activity increased significantly during pregnancy, whereas the increase in total placental ID-II activity was not significant. Absolute placental ID-II activity was approximately 200 times lower than ID-III activity at all gestational ages. Therefore, fluctuations in ID-II activity were not likely to have a significant influence on fetal thyroid hormone concentrations, but may play a role in the regulation of intraplacental T3 generation. The high ID-III activity most likely influences the thyroid hormone economy of the fetus. Severely hypothyroid newborns showed strongly decreased serum T4 levels, but serum T3 and placental ID-III activities were similar to those in euthyroid newborns. These results suggest that placental ID-III activity is regulated by serum T3, but not by serum T4.

A premature stopcodon in thyroglobulin messenger RNA results in familial goiter and moderate hypothyroidism.

Impaired thyroglobulin (Tg) synthesis is one of the putative causes for dyshormonogenesis of the thyroid gland. This type of hypothyroidism is characterized by intact iodide trapping, normal organification of iodide, and usually low serum Tg levels in relation to high TSH, and when untreated the patients develop goiter. In thyroid tissue from a 13-yr-old patient suspected of a thyroglobulin synthesis defect, the Tg mRNA was studied. The complete coding region of 8307 bp was directly sequenced and revealed a homozygous point mutation: a C886T transition in exon 7. Upon translation this mutation would result in a stopcodon at amino acid position 277, replacing the arginine residue. A Tg cDNA construct containing the mutation was expressed in rabbit reticulocyte lysate resulting in a truncated protein of 30 kDa. Expression in the presence of microsomal membranes resulted in a gel shift of this Tg molecule, indicating glycosylation ability. Two other siblings had a clinical presentation like the index patient, while their parents were unaffected. Additional restriction fragment length polymorphism analysis of the pedigree verified that the homozygous nonsense mutation cosegregated with the clinical phenotype. Clinically, hypothyroidism was not severe in the affected siblings because the truncated Tg glycoprotein was still capable of thyroid hormonogenesis.

The screening for mutations in the thyroglobulin cDNA from six patients with congenital hypothyroidism.

The six patients described in this study were clinically diagnosed with congenital hypothyroidism. Based on clinical and pathophysiological parameters, the cause of the thyroid dyshormonogenesis was suspected to be a defect in the synthesis of thyroglobulin, the matrix protein for thyroid hormone synthesis in the thyroid gland. After RNA isolation from six goitrous tissues and control thyroid tissues, RT-PCR was used to amplify 20 overlapping thyroglobulin (TG) cDNA fragments. Two alternative splice transcripts were identified: a transcript with a deletion of nucleotides 177-274 and a transcript with a deletion of nucleotides 3430-3736 that result in frame shifts and the introduction of premature stop codons. Two alternatively spliced transcripts not changing the reading frame were also identified: a transcript containing a deletion of nucleotides 4529-4699 and a transcript with a deletion of nucleotides 7301-7561. All these transcripts were expressed in thyroid tissue of both patients and controls. Nucleotide sequence analysis and comparison to the revised TG sequence (1997) revealed one revision and eight polymorphisms that did not result in amino acid changes and four polymorphisms that did change amino acid codons. In three patients a homozygous mutation was present at nucleotide position 229, causing a glycine to serine amino acid substitution. The clinical description 'thyroglobulin synthesis defect' in this population cannot be explained by major mutations in the coding region of the TG gene. Furthermore, the presence and level of expression of the alternatively spliced transcripts do not co-segregate with thyroid dyshormonogenesis, since in normal thyroid tissue the same alternatively spliced transcripts are present.

Structural organization of the human androgen receptor gene.

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.