Diagnostic utility of microarray testing in pregnancy loss.

OBJECTIVES: To determine the frequency of clinically significant chromosomal abnormalities identified by chromosomal microarray in pregnancy losses at any gestational age and to compare microarray performance with that of traditional cytogenetic analysis when testing pregnancy losses. METHODS: Among 535 fetal demise specimens of any gestational age, clinical microarray-based comparative genomic hybridization (aCGH) was performed successfully on 515, and a subset of 107 specimens underwent additional single nucleotide polymorphism (SNP) analysis. RESULTS: Overall, clinically significant abnormalities were identified in 12.8% (64/499) of specimens referred with normal or unknown karyotypes. Detection rates were significantly higher with earlier gestational age. In the subset with normal karyotype, clinically significant abnormalities were identified in 6.9% (20/288). This detection rate did not vary significantly with gestational age, suggesting that, unlike aneuploidy, the contribution of submicroscopic chromosomal abnormalities to fetal demise does not vary with gestational age. In the 107 specimens that underwent aCGH and SNP analysis, seven cases (6.5%) had abnormalities of potential clinical significance detected by the SNP component, including female triploidy. aCGH failed to yield fetal results in 8.3%, which is an improvement over traditional cytogenetic analysis of fetal demise specimens. CONCLUSIONS: Both the provision of results in cases in which karyotype fails and the detection of abnormalities in the presence of a normal karyotype demonstrate the increased diagnostic utility of microarray in pregnancy loss. Thus, chromosomal microarray testing is a preferable, robust method of analyzing cases of pregnancy loss to better delineate possible genetic etiologies, regardless of gestational age.

Further Evidence of Contrasting Phenotypes Caused by Reciprocal Deletions and Duplications: Duplication of NSD1 Causes Growth Retardation and Microcephaly.

Microduplications of the Sotos syndrome region containing NSD1 on 5q35 have recently been proposed to cause a syndrome of microcephaly, short stature and developmental delay. To further characterize this emerging syndrome, we report the clinical details of 12 individuals from 8 families found to have interstitial duplications involving NSD1, ranging in size from 370 kb to 3.7 Mb. All individuals are microcephalic, and height and childhood weight range from below average to severely restricted. Mild-to-moderate learning disabilities and/or developmental delay are present in all individuals, including carrier family members of probands; dysmorphic features and digital anomalies are present in a majority. Craniosynostosis is present in the individual with the largest duplication, though the duplication does not include MSX2, mutations of which can cause craniosynostosis, on 5q35.2. A comparison of the smallest duplication in our cohort that includes the entire NSD1 gene to the individual with the largest duplication that only partially overlaps NSD1 suggests that whole-gene duplication of NSD1 in and of itself may be sufficient to cause the abnormal growth parameters seen in these patients. NSD1 duplications may therefore be added to a growing list of copy number variations for which deletion and duplication of specific genes have contrasting effects on body development.

Investigation of TBR1 Hemizygosity: Four Individuals with 2q24 Microdeletions.

TBR1 encodes a transcription factor with critical roles in corticogenesis, including cortical neuron migration and axon pathfinding, establishment of regional and laminar identity of cortical neurons, and control of glutamatergic neuronal cell fate. Based upon TBR1's role in cortical development, we sought to investigate TBR1 hemizygosity in individuals referred for genetic evaluation of intellectual disability and developmental delay. We describe 4 patients with microdeletions identified by molecular cytogenetic techniques, encompassing TBR1 and spanning 2q24.1q31.1, ranging in size from 2.17 to 12.34 Mb. Only the patient with the largest deletion had a possible cortical malformation. Mild ventriculomegaly is the only common brain anomaly, present in all patients; a Chiari I malformation is seen in 2 patients, and mega cisterna magna is seen in a third. Our findings are consistent with Tbr1 mouse models showing that hemizygosity of the gene requires additional genetic factors for the manifestation of severe structural brain malformations. Other syndromic features are present in these patients, including autism spectrum disorders, ocular colobomas, and craniosynostosis, features that are likely affected by the deletion of genes other than TBR1.

Variability of yellow tulp (Moraea pallida Bak.) toxicity.

Yellow tulp (Moraea pallida Bak.), collected predominantly during the flowering stage from a number of sites in South Africa, showed large variation in digoxin equivalent values, indicating variability in yellow tulp toxicity. Very low values were recorded for tulp collected from certain sites in the Northern Cape.

A fluorescent investigation of subcellular damage in H9c2 cells caused by pavetamine, a novel polyamine.

Gousiekte, which can be translated literally as "quick disease", is one of the six most important plant toxicoses that affect livestock in South Africa. It is a plant-induced cardiomyopathy of domestic ruminants characterised by the sudden death of animals within a period of 4-8weeks after the initial ingestion of the toxic plant. The main ultrastructural change in sheep hearts is degradation of myofibres. In this study, fluorescent probes were used to investigate subcellular changes induced by pavetamine, the toxic compound that causes gousiekte, in H9c2 cells. The sarcoplasmic reticula (SR) and mitochondria showed abnormalities that were not present in the control cells. The lysosomes of treated cells were more abundant and enlarged than those of the control cells. There was increased activity of cytosolic hexosaminidase and acid phosphatase, indicating increased lysosomal membrane permeability. Lysosomes play an important role in both necrosis and apoptosis. The degradation of the myofibres may be a consequence of the increased lysosomal membrane permeability. Pavetamine was also found to cause alterations in the organisation of F-actin. F-actin in the nucleus is a transcription regulator and can therefore influence protein synthesis. Actin filament organisation also regulates the cardiac L-type Ca(2+) channels. Fluorescent staining demonstrated that pavetamine may damage a number of organelles, all of which can influence the proper functioning of the heart.

Damage to some contractile and cytoskeleton proteins of the sarcomere in rat neonatal cardiomyocytes after exposure to pavetamine.

Pavetamine, a cationic polyamine, is a cardiotoxin that affects ruminants. The animals die of heart failure after a period of four to eight weeks following ingestion of the plants that contain pavetamine. This immunofluorescent study was undertaken in rat neonatal cardiomyocytes (RNCM) to label some of the contractile and cytoskeleton proteins after exposure to pavetamine for 48 h. Myosin and titin were degraded in the RNCM treated with pavetamine and the morphology of alpha-actin was altered, when compared to the untreated cells, while those of beta-tubulin seemed to be unaffected. F-actin was degraded, or even absent, in some of the treated cells. On an ultrastructural level, the sarcomeres were disorganized or disengaged from the Z-lines. Thus, all three contractile proteins of the rat heart were affected by pavetamine treatment, as well as the F-actin of the cytoskeleton. It is possible that these proteins are being degraded by proteases like the calpains and/or cathepsins. The consequence of pavetamine exposure is literally a "broken heart".

Cytotoxicity and ultrastructural changes in H9c2(2-1) cells treated with pavetamine, a novel polyamine.

Intake of pavetamine, a novel polyamine, synthesized by certain rubiaceous plants, is the cause of gousiekte ("Quick disease") in ruminants. The disease is characterized by a latent period of 4-8 weeks, followed by heart failure. The aim of this study was to firstly investigate the cytotoxicity in H9c2(2-1) cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and LDH (lactate dehydrogenase) release assays. Maximum cell death occurred after pavetamine exposure of cells for 72h at a concentration of 200muM (55%+/-9.84), as measured by the MTT assay. LDH release was only observed after 72h exposure to pavetamine. Secondly, the ultrastructural changes induced by pavetamine in H9c2(2-1) cells were investigated. Changes in the mitochondria and sarcoplasmic reticula were observed. The nucleus was not affected during the first 48h exposure of cells to pavetamine and no chromatin condensation occurred. However, after 72h exposure to pavetamine, the nucleus became fragmented and membrane blebbing occurred. It was concluded that the ultimate cell death of H9c2(2-1) cells treated with pavetamine, was through necrosis and not apoptosis. Thirdly, the effect of pavetamine on the mitochondrial membrane potential (DeltaPsi) was evaluated by using the JC-1 (5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) and TMRM (tetramethylrhodamine methyl ester perchlorate) probes. Pavetamine treatment led to significant hyperpolarization of the mitochondrial membrane potential. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly, indicating that the MPTP (mitochondrial permeability transition pore) plays no role in the cytotoxicity of pavetamine.

Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents.

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Evaluation of activated charcoal as treatment for Yellow tulp (Moraea pallida) poisoning in cattle.

The efficacy of activated charcoal as a treatment for cattle (n = 57) poisoned by Yellow tulp (Moraea pallida) was investigated. Treatment with activated charcoal resulted in full recovery, irrespective of the degree of posterior paresis, provided that this clinical sign did not develop within the first 12 hours after initial exposure to Yellow tulp-infested grazing. For instance, despite treatment, 1 of 7 cattle succumbed after manifesting mild posterior paresis 6 to 8 h after initial exposure and 3 of 3 treated cattle died after developing severe posterior paresis within 6 to 12 h.

Cardiotoxic effects of pavetamine extracted from Pavetta harborii in the rat.

Previous studies have shown that crude extracts from Pavetta harborii as well as dried plant material have cardiotoxic effects on rats and sheep that can lead to heart failure. The active component has since been isolated and identified. This substance has been named pavetamine. The aim of this study was to determine whether pavetamine has cardiotoxic effects similar to those seen in previous reports, when administered to rats intraperitoneally. Sprague Dawley rats received two doses, initially 4 mg/kg and then 3 mg/kg pavetamine respectively and were monitored for 35 days before cardiodynamic parameters were measured by inserting a fluid-filled catheter into the left ventricle via the right carotid artery. These values were compared to those of control rats that had received only saline. Pavetamine significantly reduced systolic function and body mass in the treated rats, which indicates that it has the potential to induce heart failure in this animal model.

Changes in sheep oesophageal diameter and function during Geigeria ornativa (vermeerbos) poisoning and subsequent recovery.

Changes in the oesophageal diameter and function together with changes in body weight, feed intake and the cardiac pulmonary flow index were investigated during experimentally induced poisoning with Geigeria ornativa and subsequent recovery. This was performed under varying conditions for individual sheep. Results showed an increase in the oesophageal diameter index (ODI) during vermeersiekte, accompanied with a decrease in oesophageal function (OF). Cessation of G. ornativa intake resulted in a considerable although incomplete recovery of the ODI. Recovery of the OF for the different sheep, however, varied between 0 and 100%. Detrimental changes in the oesophageal diameter and function were also measured in sheep receiving only subclinical doses of G. ornativa. Decreases in body weight and feed intake commenced 1 to 3 weeks before the onset of vermeersiekte, while indications of a decline in these 2 parameters were also noticed with ingestion of subclinical amounts of G. ornativa. An increase in the cardiac pulmonary flow index (CPFI) to a value indicating the onset of heart failure was found in 1 of the sheep showing clinical signs of vermeersiekte. The CPFI returned to normal after termination of G. ornativa intake.

The role of fluorescence polarization immuno-assay in the diagnosis of plant-induced cardiac glycoside poisoning livestock in South Africa.

Poisoning with cardiac glycoside-containing plants is collectively the most important plant-associated poisoning of livestock in southern Africa. As a diagnosis of this significant poisoning is currently based on circumstantial evidence, a practical chemical procedure indicating the presence of cardiac glycosides in plants and animal specimens would be of considerable benefit. The fluorescence polarization immunoassay (FPIA) method, used to determine digoxin plasma levels in humans and dogs, was adapted to estimate cardiac glycoside levels in known cardiac-glycoside-containing plants as well as in the rumen and organs of dosed sheep. Positive FPIA values were obtained with bufadienolide-containing plants, while negative results were obtained with plants not known to contain cardiac glycosides. The FPIA has aided in the diagnosis of cardiac glycoside poisoning in livestock and game in 30 outbreaks examined at the Division of Toxicology, Onderstepoort Veterinary Institute. Each outbreak is briefly described. As a result of this assay, a better understanding of cardiac glycoside poisoning has been reached.

A study of the pathology and pathogenesis of the myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants.

Myocardial lesions were studied in sheep in which gousiekte was induced by experimental dosage of Pachystigma pygmaeum, Fadogia homblei or Pavetta harborii. The single most consistent diagnostic histological feature in 33 animals was hypertrophy of myocardial fibres in the subendocardial region. Fibrosis in the subendocardial region of the apex or left ventricular wall was often scarce or absent in animals with a short latent period, and was not always prominent even in sheep with an intermediate or long latent period. The presence or absence of fibrosis cannot therefore be used to confirm or exclude gousiekte, particularly in cases with shorter latent periods. Light microscopical and ultrastructural lesions in sheep with gousiekte correspond to a large extent to changes reported in humans with dilated cardiomyopathy of unknown cause. It appears that the myocardial lesions in gousiekte represent a final common pathway of cellular damage rather than a manifestation of a specific type of heart disease. The predilection for hypertrophy of myofibres in the subendocardial region is probably related to diminished perfusion that potentiates the primary myocardial dysfunction.

Supernumerary tricentric derivative chromosome 15 in two boys with intractable epilepsy: another mechanism for partial hexasomy.

Rearrangements of chromosome 15q, including isodicentric 15 chromosomes and interstitial duplications and triplications, have been previously reported in association with autism spectrum disorders. We have identified two boys with exceptionally large der(15) chromosomes that are tricentric and contain four copies of the proximal long arm, including the Prader Willi/Angelman critical region, and leading to hexasomy of the involved segment. Biallelic inheritance of maternal alleles and methylation analysis indicate that the markers are maternally derived. Clinical assessment of the boys indicated severe cognitive impairment associated with marked delays in gross and fine motor skills. Social and language deficits were present in both, although the severity of the mental retardation precluded diagnosis of autism (both were considered to have pervasive developmental disorder-not otherwise specified). Neurologic manifestations included infantile spasms evolving into intractable early-onset myoclonic seizures, psychomotor regression, and profound diffuse hypotonia. These patients represent the most severe end of the spectrum of phenotypes associated with segmental aneuploidy for chromosome 15q11-q13.

Conditioned feed aversion as a means to prevent tulp (Homeria pallida) poisoning in cattle.

Conditioned feed aversion was investigated as a means to prevent tulp (Homeria pallida) poisoning in cattle on tulp-infested grazing. Aversion treatment with a combination of epoxyscillirosidin and lithium chloride together with a tulp-hexane extract, which served as identification factor for tulp, resulted in a significantly lower (P < 0.001) proportion of severe tulp poisoning. In a first trial where 21 averted and 21 non-averted control cattle were exposed to a tulp-infested grass pasture, only two of the averted cattle were severely poisoned compared to 13 of the non-averted control cattle. In a second trial, with cattle being exposed to a pure stand of tulp supplemented with maize residues, only two of 21 averted cattle were severely poisoned compared to 14 of 21 non-averted control cattle. Occurrence of mild tulp poisoning, however, did not differ much between averted and non-averted control cattle. The results show that conditioned feed aversion effectively restricted severe poisoning in cattle on tulp-infested grazing.

Continuous exposure to an aversive mixture as a means of maintaining aversion to vermeerbos (Geigeria ornativa O. Hoffm.) in the presence of non-averted sheep.

Continuous exposure to an aversive mixture was investigated as a means of maintaining aversion to vermeerbos in sheep subjected to the social influence of non-averted sheep. The use of an aversive mixture was based on a hypothesis that continuous exposure to an acceptable aversive mixture (containing both the aversive substance and the identification factors of vermeerbos mixed with maize meal) would tempt sheep to consume small quantities of the aversive mixture each day and that this would keep them averted to vermeerbos, despite the social influence of non-averted sheep. Persistent aversion to a vermeerbos-maize meal mixture (1:99 by mass) by sheep continuously exposed to such an aversive mixture, after an initial aversion conditioning with lithium chloride (LiCl, 160 mg/kg BM), was demonstrated. Aversion in adjacent controls not exposed to the aversive mixture only lasted for some time. A similar result was obtained when sheep were challenged for intake of a pure stand of established vermeerbos. Three sheep continuously exposed to an aversive mixture after an initial aversion conditioning totally refused grazing the vermeerbos during a 42-day trial, despite the social influence of three non-averted control sheep grazing vermeerbos on an adjacent site. These results were confirmed by a second replication the following year. Joint grazing for an hour a day by averted and non-averted sheep during the last seven days of this replication also resulted in total avoidance of vermeerbos by the averted animals, despite continued intake of vermeerbos by the control sheep

Analysis of candidate genes in the CLN6 critical region using in silico cloning.

CLN6, the gene for variant late infantile neuronal ceroid lipofuscinosis, was mapped to a 4 cM region on chromosome 15q22-23. Subsequently the critical region was narrowed to less than 1 cM between microsatellite markers D15S988 and D15S1000 by additional marker typing in an expanded family resource. A physical map was constructed across this region using YAC and PAC clones and sequence was generated from two PAC clones. This sequence was analysed together with overlapping sequence generated by the Human Genome Project to identify genes within the region using an in silico cloning approach. In all, 29 genes have been identified and 18 have been analysed for mutations by direct sequencing. This powerful new approach will lead to the identification of CLN6.

Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant.

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.