We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2–12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (ΔHcal) and the van't Hoff enthalpy (ΔHv) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

In this work we report the effects of continuous UV-light (276 nm, ∼2.20 W.m−2) excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin’s structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes in protein structure. Structural damage includes insulin dimerization via dityrosine cross-linking or disulphide bond disruption, which affects the hormone’s structure and bioactivity.

The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the “protein structure code” has not been fully identified. Our manuscript presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence distance, secondary structure, and sequence length. The number of pairs found in a particular environment is stored in a cell in an 8 dimensional data tensor. When plotting the cell population against the number of cells that have the same population size, a scale free organization is found. When analyzing which amino acid paired residues contributed to the cells with a population above 50, pairs of Ala, Ile, Leu and Val dominate the results. This result is statistically highly significant. We postulate that such pairs form “structural stability points” in the protein structure. Our data shows that they are in buried α-helices or β-strands, in a spatial distance of 3.8–4.3Å and in a sequence distance >4 residues. We speculate that the scale free organization of the amino acid pair interactions in the 8D protein structure combined with the clear dominance of pairs of Ala, Ile, Leu and Val is important for understanding the very nature of the protein structure formation. Our observations suggest that protein structures should be considered as having a higher dimensional organization.

Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis.

Objective

Spiral computed tomography (SCT) is being evaluated as a screening tool for lung cancer. Our objective was to describe the effect of participation in SCT screening on participants’ risk perceptions, worry, and expectations regarding the accuracy of the screening result.

Methods

We surveyed sixty individuals with lung cancer family history who were participating in a SCT study for the primary purpose of improving genetic linkage analysis at baseline, and then 1 and 6 months post SCT.

Results

Of the sixty participants, forty received normal results, nineteen received non-negative results requiring follow-up, and one was diagnosed with lung cancer. At baseline, participants reported high levels of perceived lung cancer risk (64%), were concerned about getting lung cancer (94%) and the majority (84%) were not OK with receiving a non-negative SCT result. At 1 month post SCT, those with a non-negative screen (n=19) had lowered their expectations of test accuracy regarding non-negative results (54%) and reported increased levels in worry/concern (100%) and perceived risk (75%), but these effects diminished over time and returned almost to baseline levels at 6 months.

Conclusions

Persons at very high empiric risk for lung cancer expect their SCT screening test to be accurate and present with high levels of lung cancer risk perception and worry/concern overall. Our findings suggest a need for risk counseling and discussion on the limitations of screening tests to accurately detect lung cancer.

Background

The available health resources limit the amount of therapy that may be offered to children with cerebral palsy and the amount of training in each session may be insufficient to drive the neuroplastic changes, which are necessary for functional improvements to take place. The aim of this pilot study was to provide proof of concept that individualized and supervised interactive home-based training delivered through the internet may provide an efficient way of maintaining intensive training of children with cerebral palsy over prolonged periods.

Methods

9 children (aged 9-13 years) with cerebral palsy were included in the study. Motor, perceptual and cognitive abilities were evaluated before and after 20 weeks of home-based training delivered through the internet.

Results

The children and their families reported great enthusiasm with the training system and all experienced subjective improvements in motor abilities and self-esteem. The children on average trained for 74 hours during a 20 week period equalling just over 30 minutes per day. Significant improvements in functional muscle strength measured as the frontal and lateral step-up and sit-to-stand tests were observed. Assessment of Motor and processing skills also showed significant increases. Endurance measured as the Bruce test showed a significant improvement, whereas there was no significant change in the 6 min walking test. Balance (Romberg) was unchanged. Visual perceptual abilities increased significantly.

Conclusions

We conclude that it is feasible to deliver interactive training of children with cerebral palsy at home through the internet and thereby ensure more intensive and longer lasting training than what is normally offered to this group.

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.

We investigate the potential for using latency-based measures of retrieval processing capacity to assess changes in perfomance specific to individuals with mild cognitive impairment (MCI), a reliable precursor state to Alzheimer's Disease. Use of these capacity measures is motivated in part by exploration of the effects of atrophy on a computational model of a basic hippocampal circuit. We use this model to suggest that capacity may be a more sensitive indicator of undelying atrophy than speed of processing, and test this hypothesis by adapting a standard behavioral measure of memory (the free and cued selective reminding test, FCSRT) to allow for the collection of cued recall latencies. Participants were drawn from five groups: college-aged, middle-aged, healthy elderly, those with a diagnosis of MCI, and a sample of MCI control participants. The measure of capacity is shown to offer increased classificatory sensitivity relative to the standard behavioral measures, and is also shown to be the behavioral measure that correlated most strongly with hippocampal volume.

Background

Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. MAP kinase 4 (MPK4) functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins.

Methodology/Principal Findings

We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that mks1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/mks1 double mutants reverted the mpk4 dwarf phenotype. Transformation of mks1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function mks1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses.

Conclusions

Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.

β-turns are the most common type of non-repetitive structures, and constitute on average 25% of the amino acids in proteins. The formation of β-turns plays an important role in protein folding, protein stability and molecular recognition processes. In this work we present the neural network method NetTurnP, for prediction of two-class β-turns and prediction of the individual β-turn types, by use of evolutionary information and predicted protein sequence features. It has been evaluated against a commonly used dataset BT426, and achieves a Matthews correlation coefficient of 0.50, which is the highest reported performance on a two-class prediction of β-turn and not-β-turn. Furthermore NetTurnP shows improved performance on some of the specific β-turn types. In the present work, neural network methods have been trained to predict β-turn or not and individual β-turn types from the primary amino acid sequence. The individual β-turn types I, I', II, II', VIII, VIa1, VIa2, VIba and IV have been predicted based on classifications by PROMOTIF, and the two-class prediction of β-turn or not is a superset comprised of all β-turn types. The performance is evaluated using a golden set of non-homologous sequences known as BT426. Our two-class prediction method achieves a performance of: MCC  = 0.50, Qtotal = 82.1%, sensitivity  = 75.6%, PPV  = 68.8% and AUC  = 0.864. We have compared our performance to eleven other prediction methods that obtain Matthews correlation coefficients in the range of 0.17 – 0.47. For the type specific β-turn predictions, only type I and II can be predicted with reasonable Matthews correlation coefficients, where we obtain performance values of 0.36 and 0.31, respectively.

Conclusion

The NetTurnP method has been implemented as a webserver, which is freely available at http://www.cbs.dtu.dk/services/NetTurnP/. NetTurnP is the only available webserver that allows submission of multiple sequences.

Objectives

Delayed diagnosis of colorectal cancer (CRC) is among the most common reasons for ambulatory diagnostic malpractice claims in the United States. Our objective was to describe missed opportunities to diagnose CRC before endoscopic referral, in terms of patient characteristics, nature of clinical clues, and types of diagnostic-process breakdowns involved.

Methods

We conducted a retrospective cohort study of consecutive, newly diagnosed cases of CRC between February 1999 and June 2007 at a tertiary health-care system in Texas. Two reviewers independently evaluated the electronic record of each patient using a standardized pretested data collection instrument. Missed opportunities were defined as care episodes in which endoscopic evaluation was not initiated despite the presence of one or more clues that warrant a diagnostic workup for CRC. Predictors of missed opportunities were evaluated in logistic regression. The types of breakdowns involved in the diagnostic process were also determined and described.

Results

Of the 513 patients with CRC who met the inclusion criteria, both reviewers agreed on the presence of at least one missed opportunity in 161 patients. Among these patients there was a mean of 4.2 missed opportunities and 5.3 clues. The most common clues were suspected or confirmed iron deficiency anemia, positive fecal occult blood test, and hematochezia. The odds of a missed opportunity were increased in patients older than 75 years (odds ratio (OR) = 2.3; 95% confidence interval (CI) 1.3–4.1) or with iron deficiency anemia (OR = 2.2; 95% CI 1.3–3.6), whereas the odds of a missed opportunity were lower in patients with abnormal flexible sigmoidoscopy (OR = 0.06; 95% CI 0.01–0.51), or imaging suspicious for CRC (OR = 0.3; 95% CI 0.1–0.9). Anemia was the clue associated with the longest time to endoscopic referral (median = 393 days). Most process breakdowns occurred in the provider–patient clinical encounter and in the follow-up of patients or abnormal diagnostic test results.

Conclusions

Missed opportunities to initiate workup for CRC are common despite the presence of many clues suggestive of CRC diagnosis. Future interventions are needed to reduce the process breakdowns identified.

Background

Although several types of computerized provider order entry (CPOE)-related errors may occur, errors related to inconsistent information within the same prescription (ie, mismatch between the structured template and the associated free-text field) have not been described, to our knowledge. We determined the nature and frequency of such errors and identified their potential predictive variables.

Methods

In this prospective study, we enrolled pharmacists to report prescriptions containing inconsistent communication over a 4-month period at a tertiary care facility. We also electronically retrieved all prescriptions written during the study period containing any comments in the free-text field and then randomly selected 500 for manual review to determine inconsistencies between free-text and structured fields. Of these, prescriptions without inconsistencies were categorized as controls. Data on potentially predictive variables from reported and unreported errors and controls were collected. For all inconsistencies, we determined their nature (eg, drug dosage or administration schedule) and potential harm and used multivariate logistic regression models to identify factors associated with errors and harm.

Results

Of 55 992 new prescriptions, 532 (0.95%) were reported to contain inconsistent communication, a rate comparable to that obtained from the unreported group. Drug dosage was the most common inconsistent element among both groups. Certain medications were more likely associated with errors, as was the inpatient setting (odds ratio, 3.30; 95% confidence interval, 2.18–5.00) and surgical subspecialty (odds ratio, 2.45; 95% confidence interval, 1.57–3.82). About 20% of errors could have resulted in moderate to severe harm, for which significant independent predictors were found.

Conclusions

Despite standardization of data entry, inconsistent communication in CPOE poses a significant risk to safety. Improving the usability of the CPOE interface and integrating it with workflow may reduce this risk.

The Arabidopsis acd11 mutant exhibits runaway, programmed cell death (PCD) due to loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian glycolipid transfer protein (GLTP). We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11 null mutant, resulting in delayed PCD development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death, and transgenic plants showed normal induction of hypersensitive cell death upon infection with avirulent strains of Pseudomonas syringae. The significance of these findings with respect to the function(s) of ACD11 in sphingolipid transport and cell death regulation is discussed.

Background

Estimation of the reliability of specific real value predictions is nontrivial and the efficacy of this is often questionable. It is important to know if you can trust a given prediction and therefore the best methods associate a prediction with a reliability score or index. For discrete qualitative predictions, the reliability is conventionally estimated as the difference between output scores of selected classes. Such an approach is not feasible for methods that predict a biological feature as a single real value rather than a classification. As a solution to this challenge, we have implemented a method that predicts the relative surface accessibility of an amino acid and simultaneously predicts the reliability for each prediction, in the form of a Z-score.

Results

An ensemble of artificial neural networks has been trained on a set of experimentally solved protein structures to predict the relative exposure of the amino acids. The method assigns a reliability score to each surface accessibility prediction as an inherent part of the training process. This is in contrast to the most commonly used procedures where reliabilities are obtained by post-processing the output.

Conclusion

The performance of the neural networks was evaluated on a commonly used set of sequences known as the CB513 set. An overall Pearson's correlation coefficient of 0.72 was obtained, which is comparable to the performance of the currently best public available method, Real-SPINE. Both methods associate a reliability score with the individual predictions. However, our implementation of reliability scores in the form of a Z-score is shown to be the more informative measure for discriminating good predictions from bad ones in the entire range from completely buried to fully exposed amino acids. This is evident when comparing the Pearson's correlation coefficient for the upper 20% of predictions sorted according to reliability. For this subset, values of 0.79 and 0.74 are obtained using our and the compared method, respectively. This tendency is true for any selected subset.

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. In a recent publication we show that MPK4 and its substrate MKS1 interact with WRKY33 in vivo, and that WRKY33 is released from complexes with MPK4 upon infection. Transcriptome analysis of a wrky33 loss-of-function mutant identified a subset of defense-related genes as putative targets of WRKY33. These genes include PAD3 and CYP71A13, which encode cytochrome P450 monoxygenases required for synthesis of the antimicrobial phytoalexin camalexin. Chromatin immunoprecipitation confirmed that WRKY33 bound the promoter of PAD3 when plants were inoculated with pathogens. Here we further discuss the involvement of two other targets of WRKY33, NUDT6 and ROF2 in defense responses against invading pathogens.

Background

Sepsis and complications to sepsis are major causes of mortality in critically ill patients. Rapid treatment of sepsis is of crucial importance for survival of patients. The infectious status of the critically ill patient is often difficult to assess because symptoms cannot be expressed and signs may present atypically. The established biological markers of inflammation (leucocytes, C-reactive protein) may often be influenced by other parameters than infection, and may be unacceptably slowly released after progression of an infection. At the same time, lack of a relevant antimicrobial therapy in an early course of infection may be fatal for the patient. Specific and rapid markers of bacterial infection have been sought for use in these patients.

Methods

Multi-centre randomized controlled interventional trial. Powered for superiority and non-inferiority on all measured end points. Complies with, "Good Clinical Practice" (ICH-GCP Guideline (CPMP/ICH/135/95, Directive 2001/20/EC)). Inclusion: 1) Age ≥ 18 years of age, 2) Admitted to the participating intensive care units, 3) Signed written informed consent.

Exclusion: 1) Known hyper-bilirubinaemia. or hypertriglyceridaemia, 2) Likely that safety is compromised by blood sampling, 3) Pregnant or breast feeding.

Computerized Randomisation: Two arms (1:1), n = 500 per arm: Arm 1: standard of care. Arm 2: standard of care and Procalcitonin guided diagnostics and treatment of infection.

Primary Trial Objective: To address whether daily Procalcitonin measurements and immediate diagnostic and therapeutic response on day-to-day changes in procalcitonin can reduce the mortality of critically ill patients.

Discussion

For the first time ever, a mortality-endpoint, large scale randomized controlled trial with a biomarker-guided strategy compared to the best standard of care, is conducted in an Intensive care setting. Results will, with a high statistical power answer the question: Can the survival of critically ill patients be improved by actively using biomarker procalcitonin in the treatment of infections? 700 critically ill patients are currently included of 1000 planned (June 2008). Two interim analyses have been passed without any safety or futility issues, and the third interim analysis is soon to take place. Trial registration number at clinicaltrials.gov: Id. nr.: NCT00271752).

Objectives:

To study if genes with common single nucleotide polymorphisms (SNPs) associated with obesity-related phenotypes influence weight loss (WL) in obese individuals treated by a hypo-energetic low-fat or high-fat diet.

Design:

Randomised, parallel, two-arm, open-label multi-centre trial.

Setting:

Eight clinical centres in seven European countries.

Participants:

771 obese adult individuals.

Interventions:

10-wk dietary intervention to hypo-energetic (−600 kcal/d) diets with a targeted fat energy of 20%–25% or 40%–45%, completed in 648 participants.

Outcome Measures:

WL during the 10 wk in relation to genotypes of 42 SNPs in 26 candidate genes, probably associated with hypothalamic regulation of appetite, efficiency of energy expenditure, regulation of adipocyte differentiation and function, lipid and glucose metabolism, or production of adipocytokines, determined in 642 participants.

Results:

Compared with the noncarriers of each of the SNPs, and after adjusting for gender, age, baseline weight and centre, heterozygotes showed WL differences that ranged from −0.6 to 0.8 kg, and homozygotes, from −0.7 to 3.1 kg. Genotype-dependent additional WL on low-fat diet ranged from 1.9 to −1.6 kg in heterozygotes, and from 3.8 kg to −2.1 kg in homozygotes relative to the noncarriers. Considering the multiple testing conducted, none of the associations was statistically significant.

Conclusions:

Polymorphisms in a panel of obesity-related candidate genes play a minor role, if any, in modulating weight changes induced by a moderate hypo-energetic low-fat or high-fat diet.

Editorial Commentary

Background: Obesity is an important cause of death and disease, particularly in the developed world. It is understood that both environmental and genetic factors contribute towards obesity. Numerous studies have associated particular gene variants with a tendency towards obesity, but it is not known whether such gene variants affect the degree to which obese individuals will lose weight when dieting.

What this trial shows: As part of a randomised trial, 771 participants were assigned to one of two different low-energy diets for 10 weeks: one low in fat or one high in fat. The researchers then did a genetic analysis of 642 participants completing the intervention, to find out whether any of 42 distinct genetic variations in 26 genes were associated with weight loss in the trial. The genetic variants were chosen for study as they were known or already thought to be associated with appetite regulation or various aspects of metabolism and fat tissue development and function. The investigators found that none of the genetic variants studied had a significant association with weight loss in the trial. It was also seen that the majority of genetic variants were not associated with efficacy of one dietary intervention over another.

Strengths and limitations: Although a large number of participants was recruited into the trial, the genetic analysis involved multiple comparisons—168 tests of 42 genetic variants. This increases the likelihood that any significant associations found could have resulted from chance alone. Significant associations from this study will require additional confirmation in larger studies.

Contribution to the evidence: This study adds data indicating that variation in the genes studied did not have an important influence on weight loss.

In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine deoxyribonucleosides than the fruit fly multisubstrate deoxyribonucleoside kinase (EC 2.7.1.145). In addition, Ag-dNK could also phosphorylate some medically interesting nucleoside analogs, like stavudine (D4T), 2-chloro-deoxyadenosine (CdA) and 5-bromo-vinyl-deoxyuridine (BVDU). Although the biological significance of multisubstrate deoxyribonucleoside kinases and their diversity among insects remains unclear, the observed variation provides a whole range of applications, as species specific and highly selective targets for insecticides, they have a potential to be used in the enzymatic production of various (di-)(deoxy-)ribonucleoside monophosphates, and as suicide genes in gene therapy.

Comparative genomic hybridization (CGH) was applied to squamous cellcarcinomas (SCC) of the lung to define chromosomal imbalances that are associated with the metastatic phenotype. In total, 64 lung SCC from 50 patients were investigated, 25 each with or without evidence of metastasis formation. The chromosomal imbalances summarized by a CGH histogram of the 50 cases revealed deletions most frequently on chromosomes 1p21–p31, 2q34–q36, 3p, 4p, 4q, 5q, 6q14–q24, 8p, 9p, 10q, 11p12–p14, 13q13–qter, 18q12–qter and 21q21. DNA over-representations were most pronounced for chromosomes 1q11–q25, 1q32–q41, 3q, 5p, 8q22–qter, 11q13, 12p, 17q21–q22, 17q24–q25, 19, 20q and 22q. In ten cases, paired samples of primaries and at least one metastasis were analysed. The comparison revealed a considerable chromosomal instability and genetic heterogeneity; however, the CGH pattern indicated a clonal relationship in each case. The difference in histograms from the metastatic and non-metastatic tumour groups was most useful in pinpointing chromosomal imbalances associated with the metastatic phenotype, indicating that the deletions at 3p12–p14, 3p21, 4p15–p16, 6q24–qter, 8p22–p23, 10q21–qter and 21q22, as well as the over-representations at 1q21–q25, 8q, 9q34, 14q12 and 15q12–q15, occurred significantly more often in the metastatic tumour group. The comparison of the paired samples confirmed these findings in individual cases and suggested distinct genetic changes, in particular the extension of small interstitial deletions, during tumour progression. Importantly, metastasis-associated lesions were frequently detectable in the primary tumour providing a method of identifying patients at risk for tumour dissemination. Individual profiles and histograms are accessible at our web site http://amba.charite.de/cgh. © 2000 Cancer Research Campaign

We analysed 78 carcinomas of the lung for allelic losses on chromosome 10q. The tumours were of different stage and grade and comprised 22 small-cell lung carcinomas (SCLC), 40 squamous cell carcinomas (SCC), 11 adenocarcinomas, four large-cell carcinomas and one carcinoid. They were investigated by six polymorphic markers located between 10q21 and 10qter. We observed a high incidence of loss of heterozygosity (LOH) in SCLC (91%) and metastatic SCC (56%). Non-metastatic SCC showed deletions in three cases (14%) and no LOH was found in the other types of non-small-cell lung cancer. The statistical analysis indicated that the presence of LOH correlated significantly with advanced tumour stages in the entire collective and in particular within the SCLC and SCC subgroups. For SCC, a positive association was found between LOH and metastases formation, while in SCLC the number of non-metastatic tumours was too small for a final conclusion. Whereas SCLC was frequently characterized by multiple allelic losses, suggesting the deletion of the entire chromosomal arm, SCC showed interstitial imbalances. A high incidence of allelic loss was observed between the markers D10S677 and D10S1223. The analysis of five informative cases suggested the presence of two non-overlapping regions between the loci D10S677/D10S1237 and D10S1213/D10S1223. In SCLC, we did not find mutations in the putative tumour-suppressor gene MXI1. The data indicate that LOH on chromosome 10q is associated with tumour progression in SCC and SCLC. Thus it may become a useful genetic marker in the assessment of the malignant potential of these tumour types.

Images

A locus for autosomal dominant juvenile onset primary open angle glaucoma (POAG) was recently assigned to chromosome region 1q21-q31. In the present study, a large Greek family with autosomal dominant adult onset POAG was investigated using microsatellite markers. Exclusion of linkage of the adult onset POAG gene to the region D1S194-D1S191 was obtained in this pedigree. Therefore, the data provide evidence that juvenile and adult onset POAG are genetically distinct disease entities.

We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome.

Images