Deubiquitylases in developmental ubiquitin signaling and congenital diseases.

Metazoan development from a one-cell zygote to a fully formed organism requires complex cellular differentiation and communication pathways. To coordinate these processes, embryos frequently encode signaling information with the small protein modifier ubiquitin, which is typically attached to lysine residues within substrates. During ubiquitin signaling, a three-step enzymatic cascade modifies specific substrates with topologically unique ubiquitin modifications, which mediate changes in the substrate's stability, activity, localization, or interacting proteins. Ubiquitin signaling is critically regulated by deubiquitylases (DUBs), a class of ~100 human enzymes that oppose the conjugation of ubiquitin. DUBs control many essential cellular functions and various aspects of human physiology and development. Recent genetic studies have identified mutations in several DUBs that cause developmental disorders. Here we review principles controlling DUB activity and substrate recruitment that allow these enzymes to regulate ubiquitin signaling during development. We summarize key mechanisms of how DUBs control embryonic and postnatal differentiation processes, highlight developmental disorders that are caused by mutations in particular DUB members, and describe our current understanding of how these mutations disrupt development. Finally, we discuss how emerging tools from human disease genetics will enable the identification and study of novel congenital disease-causing DUBs.

The association between maternal methylenetetrahydrofolate reductase C677T and A1298C polymorphism and birth defects and adverse pregnancy outcomes.

Published studies indicate the MTHFR C677T and A1298C polymorphisms are associated with abnormal homocysteine levels, which may cause various pregnancy complications and birth defects. However, the results obtained from different studies have been inconsistent. Therefore, this meta-analysis explores the association between MTHFR polymorphisms and birth defects and adverse pregnancy outcomes. The PubMed, ScienceDirect, Embase, and China Biology Medicine literature databases and ClinicalTrials were searched. Analyses of public bias, meta-regression, subgroups, and sensitivity were used to ensure the robustness of our results. MTHFR C677T was significantly associated with recurrent pregnancy loss in developing countries (odds ratio [OR], 1.34; 95% confidence interval [CI], 1.20-1.50) but not in developed countries (OR, 0.87; 95% CI, 0.68-1.11). No significant relationship was found between MTHFR A1298C and recurrent pregnancy loss (OR, 1.04; 95% CI, 0.93-1.18). MTHFR C677T and A1298C were not associated with preeclampsia (OR, 1.06; 95% CI, 0.97-1.16 and OR, 1.16; 95% CI, 0.97-1.39, respectively), and C677T was not associated with placental abruption (OR, 1.03; 95% CI, 0.87-1.21), intrauterine growth retardation (OR, 1.02; 95% CI, 0.90-1.15), or congenital heart disease (OR, 1.05; 95% CI, 0.89-1.25). MTHFR C677T, but not A1298C, was associated with neural tube defects (OR, 1.24; 95% CI, 1.08-1.42) and Down syndrome (OR, 1.65; 95% CI, 1.39-1.95). CONCLUSION: Although MTHFR C677T and A1298C are significantly associated with some types of congenital defects and adverse pregnancy outcomes, the impact of these polymorphisms is moderate.

MTHFD1 regulates nuclear de novo thymidylate biosynthesis and genome stability.

Disruptions in folate-mediated one-carbon metabolism (FOCM) are associated with risk for several pathologies including developmental anomalies such as neural tube defects and congenital heart defects, diseases of aging including cognitive decline, neurodegeneration and epithelial cancers, and hematopoietic disorders including megaloblastic anemia. However, the causal pathways and mechanisms that underlie these pathologies remain unresolved. Because folate-dependent anabolic pathways are tightly interconnected and best described as a metabolic network, the identification of causal pathways and associated mechanisms of pathophysiology remains a major challenge in identifying the contribution of individual pathways to disease phenotypes. Investigations of genetic mouse models and human inborn errors of metabolism enable a more precise dissection of the pathways that constitute the FOCM network and enable elucidation of causal pathways associated with NTDs. In this overview, we summarize recent evidence that the enzyme MTHFD1 plays an essential role in FOCM in humans and in mice, and that it determines the partitioning of folate-activated one carbon units between the folate-dependent de novo thymidylate and homocysteine remethylation pathways through its regulated nuclear localization. We demonstrate that impairments in MTHFD1 activity compromise both homocysteine remethylation and de novo thymidylate biosynthesis, and provide evidence that MTHFD1-associated disruptions in de novo thymidylate biosynthesis lead to genome instability that may underlie folate-associated immunodeficiency and birth defects.

Consequences of POR mutations and polymorphisms.

P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism.

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

Structure-function analyses of single nucleotide polymorphisms in human N-acetyltransferase 1.

Human N-acetyltransferase 1 (NAT1) alleles are characterized by one or more single nucleotide polymorphisms (SNPs) associated with rapid and slow acetylation phenotypes. NAT1 both activates and deactivates arylamine drugs and carcinogens, and NAT1 polymorphisms are associated with increased frequencies of many cancers and birth defects. The recently resolved human NAT1 crystal structure was used to evaluate SNPs resulting in the protein substitutions R64W, V149I, R187Q, M205V, S214A, D251V, E261K, and I263V. The analysis enhances knowledge of NAT1 structure-function relationships, important for understanding associations of NAT1 SNPs with genetic predisposition to cancer, birth defects, and other diseases.

N-Acetylcysteine and alpha-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-delta and PKC-zeta and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro.

Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of alpha-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-delta and protein kinase C-zeta in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-delta and protein kinase C-zeta. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-delta and protein kinase C-zeta activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-delta and protein kinase C-zeta were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-delta and protein kinase C-zeta.

Interstitial cells of Cajal and electrical activity in ganglionic and aganglionic colons of mice.

An antibody directed against Kit protein was used to investigate the distribution of interstitial cells of Cajal (ICC) within the murine colon. The ICC density was greatest in the proximal colon and decreased along its length. The distribution of the different classes of ICC in the aganglionic colons of lethal spotted (ls/ls) mice was found to be similar in age-matched wild-type controls. There were marked differences in the electrical activities of the colons from ls/ls mutants compared with wild-type controls. In ls/ls aganglionic colons, the circular muscle was electrically quiescent compared with the spontaneous spiking electrical activity of wild-type tissues. In ls/ls aganglionic colons, postjunctional neural responses were greatly affected. Inhibitory junction potentials were absent or excitatory junction potentials inhibited by atropine were observed. In conclusion, the distribution of ICC in the ganglionic and aganglionic regions of the colons from ls/ls mutants appeared similar to that of wild-type controls. The electrical activity and neural responses of the circular layer are significantly different in aganglionic segments of ls/ls mutants.

Abnormal expression of cdk5 in focal cortical dysplasia in humans.

Focal cortical dysplasia (FCD) is an important cause of refractory epilepsy in humans. The origin of its pathognomonic abnormal cell types and the links between abnormal cell morphology and epileptogenicity remain unknown. The developmentally-regulated kinase cdk5 and its neuronal activator p35 are known to be central to a number of key components in neuronal development, cellular morphology, cytoskeletal function, synaptic plasticity and neurodegeneration. Here we examine eight cases of human FCD for expression of cdk5. We show abnormal cdk5 immunoreactivity and aggregation of protein suggesting alterations in cdk5 may also be involved in this important epileptogenic human pathology.

Distinguishing the effects of maternal and offspring genes through studies of "case-parent triads".

A gene variant that increases disease risk will be overrepresented among diseased persons, even compared with their own biologic parents. This insight has led to tests based solely on the asymmetric distribution of a variant allele among cases and their parents (e.g., the transmission/disequilibrium test). Existing methods focus on effects of alleles that operate through the offspring genotype. Alleles can also operate through the mother's genotype, particularly for conditions such as birth defects that have their origins in fetal life. An allele working through the mother would have higher frequency in case-mothers than in case-fathers. The authors develop a log-linear method for estimating relative risks for alleles in the context of case-parent triads. This method is able to detect the effects of genes working through the offspring, the mother, or both. The authors assume Mendelian inheritance, but Hardy-Weinberg equilibrium is unnecessary. Their approach uses standard software, and simulations demonstrate satisfactory power and confidence interval coverage. This method is valid with a self-selected or hospital-based series of cases and helps to protect against misleading inference that can result when cases and controls are randomly sampled from a population not in Hardy-Weinberg equilibrium.

Persistent eNOS in lung hypoplasia caused by left pulmonary artery ligation in the ovine fetus.

Because increased flow and shear stress upregulate endothelial (e) nitric oxide synthase (NOS) in adult endothelial cells in vivo and in vitro, we hypothesized that decreased pulmonary blood flow would decrease eNOS content in the late-gestation ovine fetus. To investigate the effects of decreased blood flow and the potential role of altered eNOS content in lung hypoplasia, we studied an animal model of lung hypoplasia after left pulmonary artery (LPA) ligation in nine fetal lambs (114-124 days gestation; term = 147 days). After at least 14 days, animals were killed, and lungs were harvested for histology, immunostaining, Western blot analysis for eNOS protein content, and biochemical assays of NOS activity. LPA ligation markedly reduced left lung size. Histology demonstrated loose connective tissue and airway immaturity in the left lungs. eNOS immunostaining demonstrated equal staining in the left pulmonary vessels compared with the right. Solitary endothelial cells staining for eNOS and factor VIII-related antigen were observed throughout the mesenchyme of left, but not right, lungs. eNOS protein content and activity were similar in left and right lungs. We conclude that, despite the absence of pulmonary blood flow and marked lung hypoplasia, eNOS content and NOS activity were not reduced after LPA ligation in the late fetal lung. We speculate that low pulmonary blood flow does not downregulate fetal pulmonary vascular eNOS expression and that other factors, such as paracrine or autocrine stimuli, may account for the persistence of eNOS in the developing lung circulation.

[Increased AFP in maternal serum as an indication for invasive diagnosis]. / APF-Erhöhung im mütterlichen Serum als Indikation zur invasiven Diagnostik.

The association of increased maternal serum alpha-fetoprotein levels (MS-AFP) with certain morphologic anomalies of the fetus is fully established. These anomalies are abdominal wall defects (e.g. omphalocele, gastroschisis, complete eventration), neural tube defects (anencephalus, spina bifida, encepalocele) and other malformations (e.g. coccygeal teratoma). The present study compares MS-AFP levels with amniotic fluid alpha-fetoprotein (AF-AFP) and acetylcholinesterase activity, the results of ultrasound, genetic and morphologic examination. Between April 1992 and November 1995 60 patients were referred to our clinic for further diagnosis after detection of elevated MS-AFP. After an ultrasound examination was carried out amniocentesis (AC) was performed in 54 cases, AC + placentesis (PC) in 3 patients and PC only in 3 cases with anhydramnion. Mean maternal age was 29 years (range 20-37 years). Punction was performed at a median of 19 + 1 weeks of gestation (15 + 2-23 + 2 weeks of gestation). MS-AFP, AF-AFP and acetylcholinesterase activity was measured. MS-AFP and AF-AFP were given as multiples of the median (MoM). Values < or = 1-2.0 MoM were considered normal. Elevated levels were defined to be greater than 2 MoM. In all cases the chromosomal finding was normal or norm variant. Sonographical anomalies were detected in 7 fetuses (3 cases with spina bifida, 1 case with omphalocele and 3 cases with abdominal wall defects in connection with other malformations). All sonographic diagnoses were confirmed post partum. The MS-AFP varied between 0.9-6.0 MoM. In 7 cases MS-AFP previously (referring center) evaluated as increased was found to be within normal range in our department. This cases included normal values were found in 30 cases and 25 findings were pathological. 2 out of 7 structurally abnormal fetuses had normal values, 5 of them had elevated MS-AFP. In the group of normal fetuses the highest MS-AFP we found was 6.6 MoM. In the AC-group the AF-AFP ranged between 0.7-8.7 MoM, it was within normal range in 50 and pathological in 8 cases. In the group of fetuses without structural anomalies the highest AF-AFP was 7.5 MoM. In the group of fetuses with structural anomalies the AF-AFP values were 1x normal and 3x pathological (> 3.5 MoM). Acetylcholinesterase activity in the amniotic fluid was negative in 51 cases, positive in 4 cases and faint positive in 2 cases. In all fetuses with structural anomalies in which an AC could be performed, acetylcholinesterase activity was positive. On the other hand we found only 1 of the structurally normal fetuses with positive acetylcholinesterase activity. Elevated MS-AFP and AF-AFP may be an indicator for morphological anomalies. Yet there were many positive results without any sonographical findings. The determination of a positive acetylcholinesterase activity in the amniotic fluid is more specific. All malformations have been detected by ultrasound examination before the results of the AF-AFP were available.

Altered levels of scavenging enzymes in embryos subjected to a diabetic environment.

Maternal diabetes during pregnancy is associated with an increased rate of congenital malformations in the offspring. The exact molecular etiology of the disturbed embryogenesis is unknown, but an involvement of radical oxygen species in the teratological process has been suggested. Oxidative damage presupposes an imbalance between the activity of the free oxygen radicals and the antioxidant defence mechanisms on the cellular level. The aim of the present study was to investigate if maternal diabetes in vivo, or high glucose in vitro alters the expression of the free oxygen radical scavenging enzymes superoxide dismutase (CuZnSOD and MnSOD), catalase and glutathione peroxidase in rat embryos during late organogenesis. We studied offspring of normal and diabetic rats on gestational days 11 and 12, and also evaluated day-11 embryos after a 48 hour culture period in 10 mM or 50 mM glucose concentration. Both maternal diabetes and high glucose culture caused growth retardation and increased rate of congenital malformations in the embryos. The CuZnSOD and MnSOD enzymes were expressed on gestational day 11 and both CuZnSOD, MnSOD and catalase were expressed on day 12 with increased concentrations of MnSOD transcripts when challenged by a diabetic milieu. There was a good correlation between mRNA, protein, and activity levels, suggesting that the regulation of these enzymes occurs primarily at the pretranslational level. Maternal diabetes in vivo and high glucose concentration in vitro induced increased MnSOD expression, concomitant with increased total SOD activity, and a tentative decrease in catalase expression and activity in the embryos. These findings support the notion of enhanced oxidative stress in the embryo as an etiologic agent in diabetic teratogenesis.

Prevention of diabetes-associated embryopathy by overexpression of the free radical scavenger copper zinc superoxide dismutase in transgenic mouse embryos.

OBJECTIVES: It has recently been suggested that oxygen free radicals are involved in the high incidence of fetal dysmorphogenesis that is associated with diabetic pregnancies. The purpose of the current investigation was to study the effect of copper zinc superoxide dismutase, a free radical scavenging enzyme, on the prevention of diabetes-associated embryopathy in mice. STUDY DESIGN: Mice used in this study were either transgenic, bearing the human copper zinc superoxide dismutase gene, or nontransgenic controls. Diabetes was generated by streptozotocin administration on days 6 and 7 of gestation. Hyperglycemia developed on day 8 and was maintained through day 10 (critical period of organogenesis). On day 10 fetuses were examined for external anomalies, and their crown-rump lengths and deoxyribonucleic acid content were determined. RESULTS: Induction of maternal diabetes produced a significant reduction in mean crown-rump length of control embryos (4.48 +/- 0.7 mm vs 3.65 +/- 0.6 mm, p = 0.0001), whereas transgenic embryos were not affected (4.72 +/- 0.6 mm vs 4.45 +/- 0.8 mm, p > 0.05). After induction of diabetes fetal loss and malformation rates were significantly higher in control embryos (6.0% vs 23.8% and 8.4% vs 16.5%, respectively). Transgenic embryos were practically unaffected by diabetes and showed fetal loss and malformation rates of 5.9% and 4.4%, respectively, after induction of diabetes. CONCLUSIONS: Elevated levels of copper zinc superoxide dismutase, a key enzyme in the metabolism of free oxygen radicals, elicit a protective effect against diabetes-associated embryopathy.

Scanning gel densitometry of amniotic fluid acetylcholinesterase and butyrylcholinesterase: quantification of 'faint-positive' bands in fetal malformations.

The quantification of 'faint-positive' cholinesterases in amniotic fluid was performed in 40 patients with positive acetylcholinesterase (AchE) tests. Fetuses with anencephaly and open spina bifida presented an AchE/butyrylcholinesterase (A/B) ratio of more than 0.3 with clearly dense bands. Fetuses with gastroschisis showed a 'faint-positive' AchE band (A/B < 0.3). In 5 fetuses (1 teratoma, 3 open spina bifida, 1 unaffected) we found faint AchE and butyrylcholinesterase bands.

[Value of an assay of enzymatic activity in amniotic fluid for the detection of fetal anomalies]. / Intérêt du dosage d'activités enzymatiques dans le liquide amniotique pour la détection d'anomalies foetales.

Gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP) and its intestinal and placental isomers were measured at between 12 and 32 weeks after the LMP in the amniotic fluid of 492 pregnancies, including 400 used to establish normal values. Three cases of trisomy 21, 2 of trisomy 18 and one case of mucoviscidosis were tested. Amniotic GGT decreased during normal pregnancies while total ALP and the intestinal isoenzyme remained stable. These intestinal enzyme levels were lowered in the presence of chromosomal abnormalities (autosomal trisomy) or mucoviscidosis. Levels of these enzymes were determined in 5 multiple pregnancies which culminated in the birth of normal infants.

Fetal liver function tests: umbilical cord gamma-glutamyltransferase as a marker for fetal abnormality.

The objective was to assess the association of fetal liver function tests in various (noninfectious) abnormal fetal conditions. Liver function tests and complete blood counts were evaluated in 72 consecutive fetal blood specimens obtained by cordocentesis. The indications for cordocentesis included: fetal malformation (24), red blood cell alloimmunization (23), possible fetal infection (17), oligohydramnios (5), and immunologic thrombocytopenic purpura (3). Statistical analysis included analysis of variance and linear regression analysis. Liver function tests including total protein, albumin, total bilirubin, alanine and aspartate aminotransferase were all within the range of previously published normal values. However, fetal gamma-glutamyltransferase levels (mean +/- SEM) were 157.1 +/- 15.1 IU/l (norm: 24.4 +/- 1.2 IU/l; p < 0.001). There were no statistically significant differences in the gamma-glutamyltransferase levels between the various groups of fetal abnormalities. Mean fetal gamma-glutamyltransferase levels in 8 normal fetuses were 106.2 +/- 17.5 IU/l. In conclusion, fetal gamma-glutamyltransferase levels are significantly elevated in several abnormal fetal conditions.

Determination of amniotic fluid acetylcholinesterase activity in the antenatal diagnosis of foetal malformations: the first ten years.

The concentration of amniotic fluid acetylcholinesterase activity is elevated in cases of foetal open malformations, the levels being higher in cases of open neural tube defects than in cases of abdominal wall defects. Determination of amniotic fluid acetylcholinesterase activity is an established procedure for the antenatal diagnosis of foetal neural tube defects. Performance data, technical advantages and limitations for three procedures for the determination of acetylcholinesterase activity are reviewed in this paper: an immunoassay, a gel electrophoretic procedure and a spectrophotometric procedure. An immunoassay using the monoclonal antibody 4F19 and the gel electrophoretic procedure show nearly identical diagnostic performances, with detection rates for open spina bifida close to 100% and overall false positive rates of approximately 0.2%. The spectrophotometric procedure is not suitable for the antenatal diagnosis of foetal open neural tube defects and abdominal wall defects. It is possible to distinguish open neural tube defects from abdominal wall defects by determination of the ratio of acetylcholinesterase activity to butyrylcholinesterase activity, either by combining the 4F19 immunoassay with a butyrylcholinesterase immunoassay or by gel electrophoresis followed by densitometry, on samples that display elevated levels of acetylcholinesterase activity.

Amniotic fluid microvillar enzyme activity in fetal malformations.

Prenatal diagnosis of cystic fibrosis based on amniotic fluid microvillar enzyme activity assay has become routine practice in the past few years. Normal (median) values of these enzymes were determined in 177 normal healthy pregnancies between 15-20 gestational weeks and were related to enzyme values measured in 50 pregnancies complicated with congenital malformations, 6 monogenic inherited diseases and 4 chromosomal aberrations. It is concluded that increased trehalase activity has diagnostic importance in detecting fetal kidney diseases, and radial-renal syndrome (with elevated GGT activity), while low enzyme activities may indicate chromosomal aberrations (with no signs of intestinal obstruction). With the collection of further data, the analysis of these enzymes might provide an opportunity to set up diagnostic procedures for the detection of other, non-CF-related cases.