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.

Cancer predisposition caused by elevated mitotic recombination in Bloom mice.

Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.

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.

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.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

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.

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.

Cloning, mapping and characterization of the human RAB27A gene.

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.

Pyrimidine dimer-DNA glycosylases: studies on bacteriophage T4-infected and on uninfected Escherichia coli.

Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M. luteus and phage T4 UV endonucleases. In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ([H5] dT):poly (dA), after the photo-reversal of thymine-thymine dimers. The activity has also been demonstrated in vivo following infection of UV-irradiated E. coli uvr- cells with phage T4. Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from [3H] labeled E. coli DNA. In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase. However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one. In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E. coli infected with this mutant show no detectable phage-coded AP endonuclease at 28 degrees C. Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear. The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E. coli with phage T4 denV+ yields a calculated average repair patch size of approximately 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is approximately 70 nucleotides. Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD. When uninfected E. coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E. coli. The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation.

Robertsonian (15q;15q) translocation in a child with Angelman syndrome: evidence of uniparental disomy.

A balanced Robertsonian translocation 45,XY,t(15q15q) was detected in a patient with mental retardation, microcephaly, and hypertonia. Deletion of the 15q11q13 region was unlikely based on fluorescence in situ hybridization studies that revealed hybridization of appropriate DNA probes to both arms of the Robertsonian chromosome. Inheritance of alleles from 13 highly polymorphic DNA markers on chromosome 15 showed paternal uniparental isodisomy. The clinical, cytogenetic, and molecular results are consistent with a diagnosis of Angelman syndrome.

Identification and molecular confirmation of a small chromosome 10q duplication [dir dup(10)(q24.2-->q24.3)] inherited from a mother mosaic for the abnormality.

We describe a family in which two siblings exhibited developmental delay, reduced muscle tone and mild muscle weakness. Cytogenetic evaluation demonstrated that both children had a tandem duplication of a small portion of the long arm of chromosome 10 [46,XX or XY,dir dup(10)(q24.2-->q24.3)], inherited from their clinically normal mother, who was found to be mosaic for the duplicated chromosome 10. Fluorescence in situ hybridization approaches, including total chromosome painting and the use of regional specific cosmid probes, were used to confirm the chromosome 10q origin of the duplicated material. This is the smallest confirmed duplication of this portion of chromosome 10 reported to date.

Novel assay for Roberts syndrome assigns variable phenotypes to one complementation group.

Roberts syndrome (RS) is a rare autosomal recessive disorder characterized by heterogeneous clinical features, the most notable being tetraphocomelia, cleft lip, and cleft palate. Cells derived from most RS patients exhibit abnormal cytogenetic and cellular phenotypes that include the premature separation of para- and pericentromeric heterochromatin visible on C-banded metaphase chromosomes, a phenomenon referred to as heterochromatic splaying. Previously, it was shown that these abnormal phenotypes can be complemented following somatic cell hybridization between RS cells and control cells. In the current study, a permanent cell line was established from a new RS patient with a more severe phenotype than represented by previously established cells in culture. With a newly developed assay designed to facilitate rapid evaluation of in vitro complementation, we assigned this new patient to the same genetic complementation group defined by other, less severely affected patients. The results demonstrate that a single complementation group defines RS patients with heterochromatic splaying regardless of clinical severity.

Elevation of sister chromatid exchange frequency in transformed human fibroblasts following exposure to widely used aminoglycosides.

Aminoglycosides are a class of antibiotics that interfere with protein translation. Geneticin and hygromycin are two such agents, which have been shown to exhibit highly toxic effects in mammalian cells. Cloned bacterial genes, which inactivate these antibiotics, have facilitated the establishment of dominant selection systems, which are widely used in eukaryotic molecular genetics. We have examined the effect of aminoglycosides on the sister chromatid exchange (SCE) frequency in transformed human fibroblast cell lines. Geneticin and hygromycin were both found to increase SCE frequency in all cell lines examined, including a cell line derived from a patient with Bloom syndrome, a disorder exhibiting an elevated spontaneous SCE frequency. Induction was seen to occur in a dose-responsive manner and was also observed in cells expressing the resistance genes that inactivate the cellular toxicity of these antibiotics. The implications of these findings for somatic cell genetics and for human gene therapy protocols are discussed.

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.

Mechanoreceptors in human cruciate ligaments. A histological study.

We obtained human cruciate ligaments at the time of total knee replacement and from autopsy and amputation specimens, and examined histological sections of the ligaments for the presence of mechanoreceptors using the Bodian, Bielschowsky, and Ranvier gold-chloride stains for axons and nerve-endings. The cruciate ligaments obtained at the time of total knee replacement were too distorted by disease processes to be of use. The autopsy and amputation specimens, however, contained fusiform mechanoreceptor structures measuring 200 by seventy-five micrometers, with a single axon exiting from the capsule of the receptor. One to three receptors were found at the surface of each ligament beneath the synovial membrane, and were absent from the joint capsules and menisci. Morphologically the receptors resembled Golgi tendon organs, and it seems likely that they provide proprioceptive information and contribute to reflexes inhibiting injurious movements of the knee. This is the first histological demonstration of mechanoreceptors in human cruciate ligaments.

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum.

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.