From next-generation resequencing reads to a high-quality variant data set.

Sequencing has revolutionized biology by permitting the analysis of genomic variation at an unprecedented resolution. High-throughput sequencing is fast and inexpensive, making it accessible for a wide range of research topics. However, the produced data contain subtle but complex types of errors, biases and uncertainties that impose several statistical and computational challenges to the reliable detection of variants. To tap the full potential of high-throughput sequencing, a thorough understanding of the data produced as well as the available methodologies is required. Here, I review several commonly used methods for generating and processing next-generation resequencing data, discuss the influence of errors and biases together with their resulting implications for downstream analyses and provide general guidelines and recommendations for producing high-quality single-nucleotide polymorphism data sets from raw reads by highlighting several sophisticated reference-based methods representing the current state of the art.

Analysis of computer-aided techniques for virtual planning in nasoalveolar moulding.

We compared two methods of planning virtual alveolar moulding as the first step in nasoalveolar moulding to provide the basis for an automated process to fabricate nasoalveolar moulding appliances by using computer-assisted design and computer-aided manufacturing (CAD/CAM). First, the initial intraoral casts taken from seven newborn babies with complete unilateral cleft lip and palate were digitised. This was repeated for the target models after conventional nasoalveolar moulding had been completed. The initial digital model for each patient was then virtually modified by two different modelling techniques to achieve the corresponding target model: parametric and freeform modelling with the software Geomagic(®). The digitally-remodelled casts were quantitatively compared with the actual target model for each patient, and the comparison between the two modified models and the target model showed that freeform modelling of the initial cast was successful (mean (SD) deviation n=7, +0.723 (0.148) to -0.694 (0.157)mm) but needed continuous orientation and was difficult to automate. The results from the parametric modelling (mean (SD) deviation, n=7, +1.168 (0.185) to -1.067 (0.221)mm) were not as good as those from freeform modelling. During parametric modelling, we found some irregularities on the surface, and transverse growth of the maxilla was not accounted for. However, this method seems to be the right one as far as automation is concerned. In addition, an external algorithm must be implemented because the function of the commercial software is limited.

[Liver abscess in a dog. A case report]. / Leberabszess bei einem Hund. Ein Fallbericht.

The presented case report describes diagnostic and therapy of a liver abscess in a male Golden Retriever dog. The dog was adversely affected by fever, apathy, and vomitus. Diagnostic imaging including radiography, sonography and computed tomography, revealed an abscess-forming lesion of 10 × 5 cm in the left middle liver lobe with detectable multiple gas accumulation within the lesion. The surgical therapy included lobectomy with adjacent omentopexy. Four days after the operation the dog was discharged in a good general condition and with physiological body temperature. Detecting multiple gas accumulation in circular, inhomogeneous lesions by sonography allowed confirmation of the diagnosis. Surgical removal of the affected liver lobe led to recovery of the patient. In human medicine, less invasive methods are preferred, e.g. percutaneous drainage and alcoholization of the lesion. Exclusive medicamentous therapy yields the worst outcome in humans.

Impedance measurement for real time detection of neuronal cell death.

Detection of neuronal cell death is a standard requirement in cell culture models of neurodegenerative diseases. Although plenty of viability assays are available for in vitro applications, most of these are endpoint measurements providing only little information on the kinetics of cell death. Here, we validated the xCELLigence system based on impedance measurement for real-time detection of cell death in a neuronal cell line of immortalized hippocampal neurons (HT-22 cells), neuronal progenitor cells (NPC) and differentiated primary cortical neurons. We found a good correlation between impedance measurements and endpoint viability assays in HT-22 cells and NPC, for detecting proliferation, cell death kinetics and also neuroprotective effects of pharmacological inhibitors of apoptosis. In primary neurons we could not detect dendritic outgrowth during differentiation of the cells. Cell death in primary neurons was detectable by the xCELLigence system, however, the changes in the cell index on the basis of impedance measurements depended to a great extent on the severity of the insult. Cell death induced by ionomycin, e.g. shows as a fast paced process involving a strong cellular disintegration, which allows for impedance-based detection. Cell death accompanied by less pronounced morphological changes like glutamate induced cell death, however, is not well accessible by this approach. In conclusion, our data show that impedance measurement is a convenient and reliable method for the detection of proliferation and kinetics of cell death in neuronal cell lines, whereas this method is less suitable for the assessment of neuronal differentiation and viability of primary neurons.

Prenatal ultrasound and the risk of childhood brain tumour and its subtypes.

We carried out a nationwide case-control study of childhood brain tumours in Sweden (n=512) by histological subtype in relation to prenatal ultrasound, extracting data from antenatal records and the Medical Birth Register. We found no increased risk for brain tumour after ultrasound exposure, either for all tumours or for any subgroup.

Prenatal X-ray exposure and childhood brain tumours: a population-based case-control study on tumour subtypes.

We investigated childhood brain tumours by histological subtype in relation to prenatal X-ray among all children, less than 15 years of age, born in Sweden between 1975 and 1984. For each case, one control was randomly selected from the Medical Birth Register, and exposure data on prenatal X-ray were extracted blindly from antenatal medical records. Additional information on maternal reproductive history was obtained from the Medical Birth Register. We found no overall increased risk for childhood brain tumour after prenatal abdominal X-ray exposure (adjusted odds ratio (OR): 1.02, 95% confidence interval (CI): 0.64-1.62); primitive neuroectodermal tumours had the highest risk estimate (OR: 1.88, 95% CI: 0.92-3.83).

Treatment of chronic sarcoidosis with an azathioprine/prednisolone regimen.

In a few patients with chronic sarcoidosis, prolonged, unacceptably high doses of corticosteroids are required to achieve symptomatic relief. In these cases, a corticosteroid-sparing drug might be administered to allow long-term treatment without the adverse effects of corticosteroids. This study examines azathioprine as a prednisolone-sparing treatment. In an open study, the course of 11 patients with chronic sarcoidosis was analysed. In an induction phase, 2 mg azathioprine x kg body weight (BW)(-1) x day(-1) in combination with 0.6-0.8 mg prednisolone x kg BW(-1) x day(-1) were administered with prednisolone being reduced to 0.1 mg x kg BW(-1) x day(-1) within 2-3 months. This was followed by a 21-22-month maintenance phase with 2 mg azathioprine x kg BW(-1) x day(-1) and 0.1 mg prednisolone x kg BW(-1) day(-1). Clinical parameters and immunological findings of bronchoalveolar lavage (BAL) were analysed. All patients had significant symptomatic relief and improvements or resolutions of physiological, serological and radiographic findings without suffering from serious adverse effects. Nine of 11 patients completed therapy after 19-26 months, and 2/11 patients terminated therapy after 8 and 12 months, respectively. Eight patients had remissions lasting 4-73 months. Three relapses occurred after 8, 18, and 22 months. During the induction phase, BAL cell composition changed and their activity in terms of cytokine release was suppressed. This preliminary study suggests that azathioprine may be effective as a corticosteroid-sparing agent in long-term therapy of sarcoidosis, but a much larger study is necessary to give the definitive answer.

Demonic attributions in nondelusional disorders.

OBJECTIVE: Belief in demonic influence has repeatedly been described as a delusion in schizophrenic patients. The goal of this explorative study was to examine the frequency, as well as the psychodynamic and social functions of such beliefs in a sample of nondelusional patients. METHOD: The sample consisted of 343 psychiatric outpatients who described themselves as religious. In semistructured interviews they were asked to give their view of demonic causality of their illness. RESULTS: A high prevalence of such beliefs was not only found in schizophrenic patients (56%) but also in the following groups of nondelusional patients: affective disorders (29%), anxiety disorders (48%), personality disorders (37%) and adjustment disorders (23%). Belief in demonic oppression tended to be associated with lower educational level and rural origin, and was significantly influenced by church affiliation. CONCLUSIONS: Beliefs in possession or demonic influence are not confined to delusional disorders and should not be qualified as a mere delusion. Rather they have to be interpreted against the cultural and religious background which is shaping causal models of mental distress in the individual.

Clearance of acanthosis nigricans associated with the HAIR-AN syndrome after partial pancreatectomy: an 11-year follow-up.

We describe a woman with the syndrome characterised by hyperandrogenism, insulin resistance and acanthosis nigricans (the HAIR-AN syndrome), and an associated insulinoma (islet B-cell tumour), whose signs and symptoms cleared after partial pancreatectomy.

Mosaic allelic insulin-like growth factor 2 expression patterns reveal a link between Wilms' tumorigenesis and epigenetic heterogeneity.

Numerous observations link the loss of imprinting of insulin-like growth factor 2 (IGF2) and an overdosage of this growth factor gene with cancer, in general, and with Wilms' tumorigenesis, in particular. It is not known, however, if loss of imprinting correlates with specific stages of neoplasia or if allelic expression patterns vary within the tumor. By applying an allele-specific in situ hybridization technique to formalin-fixed thin sections, we show that the parental IGF2 alleles can be differentially expressed, not only in Wilms' tumors, but also in nephrogenic rests (which represent premalignant lesions) of Wilms' tumor patients. Moreover, a subpopulation of mesenchymal cells, which surrounds tumor nodules, expresses IGF2 biallelically irrespective of the imprinted state of IGF2 within the tumor. These data show that Wilms' tumorigenesis involves epigenetic heterogeneity as visualized by variable allelic IGF2 expression patterns.

Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells.

Alterations in the cellular redox potential by homocysteine promote endothelial cell (EC) dysfunction, an early event in the progression of atherothrombotic disease. In this study, we demonstrate that homocysteine causes endoplasmic reticulum (ER) stress and growth arrest in human umbilical vein endothelial cells (HUVEC). To determine if these effects reflect specific changes in gene expression, cDNA microarrays were screened using radiolabeled cDNA probes generated from mRNA derived from HUVEC, cultured in the absence or presence of homocysteine. Good correlation was observed between expression profiles determined by this method and by Northern blotting. Consistent with its adverse effects on the ER, homocysteine alters the expression of genes sensitive to ER stress (ie, GADD45, GADD153, ATF-4, YY1). Several other genes observed to be differentially expressed by homocysteine are known to mediate cell growth and differentiation (ie, GADD45, GADD153, Id-1, cyclin D1, FRA-2), a finding that supports the observation that homocysteine causes a dose-dependent decrease in DNA synthesis in HUVEC. Additional gene profiles also show that homocysteine decreases cellular antioxidant potential (glutathione peroxidase, NKEF-B PAG, superoxide dismutase, clusterin), which could potentially enhance the cytotoxic effects of agents or conditions known to cause oxidative damage. These results successfully demonstrate the use of cDNA microarrays in identifying homocysteine-respondent genes and indicate that homocysteine-induced ER stress and growth arrest reflect specific changes in gene expression in human vascular EC.

Reproductive technologies 1998: options available for the cancer patient.

Recent advances in the field of reproduction have potentially opened opportunities for the preservation of the reproductive potential of young cancer patients with good long-term prognosis for survival. In the postpubertal male, cryopreservation of ejaculated sperm is both feasible and potentially successful. Semen parameters at the time of procurement are of minor significance; intracytoplasmic sperm injection (ICSI) can bypass sperm concentration and motility problems and can lead to successful fertilization. For the prepubertal male there are no clinically applicable options insofar as extraction and cryopreservation of postmeiotic sperm cells (mature spermatozoa or round spermatids) is not feasible. To date, efforts for culture of testicular tissue and in vitro maturation of male germ cells have not been successful. In both pre- and postpubertal females, cryopreservation of ovarian cortical tissue or enzymatically extracted follicles and the in vitro maturation of preantral follicles are of potential clinical use, but, to date, these approaches have been successful only in laboratory animals. An additional option available to the postpubertal female is the stimulation of ovaries with exogenous gonadotropins and retrieval of mature oocytes for cryopreservation. The recent application of ICSI in previously cryopreserved human mature oocytes has improved fertilization rates and has resulted in live births. Unfortunately, a shortcoming of this approach is the limited number of oocytes that can be harvested after stimulation of the ovaries. Further, all these approaches potentially harbor the risk of the cryopreservation of malignant cells with their subsequent reintroduction in the patient at a later date. This is a more realistic concern for patients suffering from hematologic or gonadal tumors. Finally, even though cryopreservation of embryos has been successfully used for many years, this option is not available to the pediatric and adolescent patient. It should not be forgotten that, even if the patients' own gametes are not available in the future, donor sperm and eggs provide the option for offspring and can give the opportunity to the females to carry a pregnancy as long as their uterus has not been affected by the cancer treatment. Given the rapid progress we are witnessing in the field of reproductive medicine, it is probable that in the very near future most of the options described and newer ones will be clinically available.

Evaluation of adnexal masses in adolescents.

The detection of adnexal masses in adolescents is worrisome to patients, their families, and physicians. Reassurance can be given that the vast majority of these lesions are benign. Furthermore, a significant fraction of benign masses are functional ovarian cysts, most of which resolve spontaneously and never need surgery. Imaging is critical in determining the management of these patients. Sonography is the preferred first-line diagnostic tool. When surgery is necessary, physicians must recognize the importance of conserving the ovaries and uterus to avoid the loss of reproductive and endocrine function.

The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene.

Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent.

Random monoallelic expression of the imprinted IGF2 and H19 genes in the absence of discriminative parental marks.

The IGF2 and H19 genes are genomically imprinted and expressed preferentially from the paternal and maternal alleles, respectively, during human prenatal development. The exact role of the parental imprint(s), however, is not known. To explore this issue in some detail, we have examined human androgenetic cells which by definition should be incapable of allelic discrimination given the paternal origin of both genomes. Allele-specific in situ hybridisation analysis of dispermic complete hydatidiform moles shows that IGF2 and H19 can be found to be transcriptionally active in a variegated manner, which results in the generation of random monoallelic expression patterns. This data shows that imprinted genes can be expressed monoallelically in the absence of discriminating parental marks and raises the question whether or not mechanisms underlying monoallelic expression preceded the acquisition of parental imprints during evolution.

The H19 transcript is associated with polysomes and may regulate IGF2 expression in trans.

The imprinted H19 gene produces a fully processed transcript that does not exhibit any conserved open reading frame between mouse and man. Although transcriptional control elements associated with the mouse H19 locus have been shown to control the neighboring Igf2 gene in cis, the prevailing view is that the cytoplasmic H19 transcript does not display any function. In contrast to earlier reports, we show here that the H19 transcript is associated with polysomes in a variety of cell types, in both mouse and man. A possible trans-function of the H19 gene is suggested by a reciprocal correlation in trans between cytoplasmic H19 and IGF2 mRNA levels, as well as IGF2 mRNA translatability. We discuss these results in terms of their challenge to the prevailing dogma on the function of the enigmatic H19 gene, as well as with respect to the ontogeny of the Beckwith-Wiedemann syndrome, and propose that the human H19 gene is an antagonist of IGF2 expressivity in trans.