Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome.

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. METHODS: We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. RESULTS: We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. CONCLUSION: This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success.

Total elbow replacement for the management of the ankylosed or fused elbow.

This study reports our experience with total elbow replacement for fused elbows. Between 1982 and 2004, 13 patients with spontaneously ankylosed elbows were treated with a linked semi-constrained non-custom total elbow implant. The mean age at operation was 54 years (24 to 80). The stiffness was a result of trauma in ten elbows, juvenile rheumatoid arthritis in one, and rheumatoid arthritis in two. The patients were followed for a mean of 12 years (2 to 26) and were evaluated clinically using the Mayo Elbow Performance Score, as well as radiologically. A mean arc from 37 degrees of extension to 118 degrees of flexion was achieved. Outcomes were good or excellent for seven elbows at final review. Ten patients felt better or much better after total elbow replacement. However, there was a high complication rate and re-operation was required in over half of patients. Two developed peri-operative soft-tissue breakdown requiring debridement. A muscle flap with skin grafting was used for soft-tissue cover in one. Revision was undertaken in one elbow following fracture of the ulnar component. Three patients developed a deep infection. Three elbows were manipulated under anaesthesia for post-operative stiffness. Prophylactic measures for heterotopic ossification were unsuccessful. Total elbow replacement for the ankylosed elbow should be performed with caution. However, the outcome can be reliable in the long term and have a markedly positive impact on patient function and satisfaction. The high potential for complications must be considered. We consider total elbow replacement to be an acceptable procedure in selected patients with reasonable expectations.

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

Sequence, structure and pathology of the fully annotated terminal 2 Mb of the short arm of human chromosome 16.

We have sequenced 1949 kb from the terminal Giemsa light band of human chromosome 16p, enabling us to fully annotate the region extending from the telomeric repeats to the previously published tuberous sclerosis disease 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes. This region can be subdivided into two GC-rich, Alu-rich domains and one GC-rich, Alu-poor domain. The entire region is extremely gene rich, containing 100 confirmed genes and 20 predicted genes. Many of the genes encode widely expressed proteins orchestrating basic cellular processes (e.g. DNA recombination, repair, transcription, RNA processing, signal transduction, intracellular signalling and mRNA translation). Others, such as the alpha globin genes (HBA1 and HBA2), PDIP and BAIAP3, are specialized tissue-restricted genes. Some of the genes have been previously implicated in the pathophysiology of important human genetic diseases (e.g. asthma, cataracts and the ATR-16 syndrome). Others are known disease genes for alpha thalassaemia, adult polycystic kidney disease and tuberous sclerosis. There is also linkage evidence for bipolar affective disorder, epilepsy and autism in this region. Sixty-three chromosomal deletions reported here and elsewhere allow us to interpret the results of removing progressively larger numbers of genes from this well defined human telomeric region.

Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster.

We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

Instructional technology in medical education: lessons learnt.

New instructional technologies, especially Web-based applications, may play an increasing role in medical education, particularly for distance and distributed learning. As medical educators turn to this medium, numerous benefits and opportunities, but also challenges and pitfalls, will arise. The successful development and implementation of instructional technologies in medicine require an appreciation of the medium's heterogeneous nature, its strengths, weaknesses and limitations. These in turn rely on partnerships with various experts and the early adoption of evaluation. We have summarized the lessons learnt from developing Web-based courses on palliative care in a framework for adopting instructional technologies. This framework incorporates development, implementation and evaluation.

Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58.

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Molecular cloning and phylogenetic analysis of the small cytoplasmic RNA from Listeria monocytogenes.

A molecular cloning strategy has been designed to isolate the gene that encodes the small cytoplasmic RNA (scRNA) component of bacterial signal recognition particles. Using this strategy a putative Listeria monocytogenes scRNA lambda gt11 recombinant clone was isolated. A previously described complementation assay developed to genetically select functional homologues of 4.5S RNA and scRNA of bacteria confirmed that the lambda gt11 recombinant clone isolated encoded for the scRNA from L. monocytogenes. A secondary structure for this scRNA is proposed and a phylogenetic comparison of the 276 base L. monocytogenes scRNA with previously characterised Gram-positive bacterial scRNAs is also presented.

Simple sequence repeats in the Helicobacter pylori genome.

We describe an integrated system for the analysis of DNA sequence motifs within complete bacterial genome sequences. This system is based around ACeDB, a genome database with an integrated graphical user interface; we identify and display motifs in the context of genetic, sequence and bibliographic data. Tomb et aL (1997) previously reported the identification of contingency genes in Helicobacter pylori through their association with homopolymeric tracts and dinucleotide repeats. With this as a starting point, we validated the system by a search for this type of repeat and used the contextual information to assess the likelihood that they mediate phase variation in the associated open reading frames (ORFs). We found all of the repeats previously described, and identified 27 putative phase-variable genes (including 17 previously described). These could be divided into three groups: lipopolysaccharide (LPS) biosynthesis, cell-surface-associated proteins and DNA restriction/modification systems. Five of the putative genes did not have obvious homologues in any of the public domain sequence databases. The reading frame of some ORFs was disrupted by the presence of the repeats, including the alpha(1-2) fucosyltransferase gene, necessary for the synthesis of the Lewis Y epitope. An additional benefit of this approach is that the results of each search can be analysed further and compared with those from other genomes. This revealed that H. pylori has an unusually high frequency of homopurine:homopyrimidine repeats suggesting mechanistic biases that favour their presence and instability.

Teaching dietary counseling skills to residents: patient and physician outcomes. The CADRE Study Group.

Our objective was to determine whether an educational intervention and prompting intervention for physicians improved dietary counseling of patients with high blood cholesterol and resulted in beneficial changes in patients' diets and cholesterol levels. We instituted a factorial design, multicenter, randomized, placebo-controlled trial to test two interventions. We tested the trial at continuity care clinics of internal medicine residents at seven community and university medical centers in the northern and eastern United States. Our participants were 130 internal medicine residents and 254 adult outpatients with blood cholesterol levels of 240-300 mg/dL. Interventions included an educational program for resident physicians designed to improve their skills and confidence in dietary counseling (two one-hour sessions with specially prepared printed materials for use in counseling) and a prompting intervention, which was a fingerstick blood cholesterol determination prior to the patient's clinic visit. Resident physicians' knowledge, attitudes, and self-reported behaviors were assessed prior to the intervention and 10 months later using chart audits and questionnaires. Residents' behaviors were also assessed by exit interviews with patients. Patients' knowledge, attitudes, behaviors, and fingerstick blood cholesterol levels were measured at baseline and 10 months later. The educational program increased the percentage of physicians who were confident in providing effective dietary counseling (baseline of 26% to 67%-78%; P < .01). The prompting intervention approximately doubled the frequency of physician counseling (P = .0005) and increased the likelihood that patients would try to change their diets. When both interventions were combined, most outcomes were better, although not statistically significant. Cholesterol levels, however, decreased only marginally and were no different among groups at 10-month follow-up. Despite success in changing physicians' attitudes and behaviors and increasing patients' willingness to change their diets, there was no significant change in patients' cholesterol levels. Medical Subject Headings (MeSH): randomized controlled trial; cholesterol; patient education; behavior therapy; education, medical; diet.

Upstream stimulators for recoding.

Recent progress in elucidation of 5' stimulatory elements for translational recoding is reviewed. A 5' Shine-Dalgarno sequence increases both +1 and -1 frameshift efficiency in several genes; examples cited include the E. coli prfB gene encoding release factor 2 and the dnaX gene encoding the gamma and tau subunits of DNA polymerase III holoenzyme. The spacing between the Shine-Dalgarno sequence and the shift site is critical in both the +1 and -1 frameshift cassettes; however, the optimal spacing is quite different in the two cases. A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5' sequences have been shown to be vital for this frameshift event. Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase. The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation. A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur.

DNA sequence evolution: the sounds of silence.

Silent sites (positions that can undergo synonymous substitutions) in protein-coding genes can illuminate two evolutionary processes. First, despite being silent, they may be subject to natural selection. Among eukaryotes this is exemplified by yeast, where synonymous codon usage patterns are shaped by selection for particular codons that are more efficiently and/or accurately translated by the most abundant tRNAs; codon usage across the genome, and the abundance of different tRNA species, are highly co-adapted. Second, in the absence of selection, silent sites reveal underlying mutational patterns. Codon usage varies enormously among human genes, and yet silent sites do not appear to be influenced by natural selection, suggesting that mutation patterns vary among regions of the genome. At first, the yeast and human genomes were thought to reflect a dichotomy between unicellular and multicellular organisms. However, it now appears that natural selection shapes codon usage in some multicellular species (e.g. Drosophila and Caenorhabditis), and that regional variations in mutation biases occur in yeast. Silent sites (in serine codons) also provide evidence for mutational events changing adjacent nucleotides simultaneously.

Combination antidepressant therapy in primary care.

Increasing clinical experience with the selective serotonin reuptake inhibitors and tricyclic antidepressants make combination antidepressant therapy at times a reasonable alternative to single-agent therapy in primary care patients with depression. This article describes three cases that illustrate possible rationales for combination antidepressant therapy: reduced side effects, synergistic treatment effects, reduced treatment response time, prescriber familiarity, and clinical experience. The combination of selective serotonin reuptake inhibitors and tricyclic antidepressants may be useful in treating patients who experience intolerable side effects or who are resistant to therapy with a single antidepressant. Further research should be done to define the role of combination antidepressant therapy in the treatment of primary care patients with depression.

Patient-carried card prompts vs computer-generated prompts to remind private practice physicians to perform health maintenance measures.

BACKGROUND: We compared the efficacy of a computer-generated prompt sheet placed on the front of patients' charts with a patient-carried prompt card to remind physicians to perform selected health maintenance items. METHODS: A randomized prospective single-blind study was performed in private practice offices in rural eastern North Carolina, with 28 family practitioners and nine general internists participating. Twenty-two physicians were randomized to the computer prompt group and 22 to the card prompt group. Four physicians in the computer group did not complete the study because of computer software problems, and three physicians did not complete the study because their limited staff was unable to enter patient data into the computer. Before the intervention, 20 patient charts (10 males and 10 females) from each physician were audited for the performance of influenza vaccinations, stool for occult blood, pap smears, breast examinations performed by the physicians, and mammograms. One year after the intervention was instituted, chart audits for the above five items were done again on 20 different patient charts for each physician. RESULTS: There was a 7% increase in the performance of influenza vaccinations in the card group (17% vs 24%) compared with a 6% increase in the computer group (20% vs 26%). There was a 5% decrease in the performance of stool for occult blood in the card group (28% vs 23%) compared with a 1% increase in the computer group (30% vs 31%). There was an 11% decrease in the performance of pap smears in the card group (26% vs 15%) compared with a 3% increase in the computer group (23% vs 26%). There was a 2% decrease in the performance of breast examinations by the physician in the card group (35% vs 33%) compared with a 3% increase in the computer group (30% vs 33%). Finally, there was a 3% increase in the performance of mammograms in the card group (22% vs 25%) compared with an 11% increase in the computer group (15% vs 26%). CONCLUSIONS: Our data show a greater increase in performance of health maintenance items in the computer-prompted group. The performance of stool for occult blood, pap smears, breast examinations performed by the physician, and mammograms were increased more in the computer-prompted group than in the card group. However, there was not a statistically significant difference after intervention for any of the audited health maintenance items for either the computer group or the card group. Overall, health maintenance measures were performed in only a minority of appropriate patients.

Cloning and sequencing of the Atlantic salmon (Salmo salar) cytochrome c oxidase subunit III gene (coxIII) and analysis of coxIII expression during parr-smolt transformation.

Smoltification is the process whereby salmon alter their metabolism in preparation for movement from freshwater to seawater. Differential screening of a cDNA library prepared from post-smolt salmon liver mRNA led to the selection of a smoltification-induced sequence. Analysis of this cDNA revealed that it partially encoded subunit III of the enzyme cytochrome c oxidase. The complete coxIII sequence was amplified from salmon genomic DNA using consensus oligonucleotides based on ATPase 6 and tRNA(GLY) sequences from Pacific salmonid species. Cytochrome c oxidase subunit III liver mRNA levels were found to be significantly increased in salmon smolts. Northern blot analysis revealed a coxIII transcript of approximately 750 bp in all salmon tissues tested except blood. The DNA sequence of coxIII employs the mammalian mitochondrial genetic code and is strongly conserved when compared with that of other species.