Mapping, cloning and genetic characterization of the region containing the Wilson disease gene.

Wilson disease (WD) is an autosomal recessive disorder of copper transport which map to chromosome 13q14.3. In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region. Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval. A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene. Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.

Acceptance of physical activity monitoring in cancer patients during radiotherapy, the GIROfit phase 2 pilot trial.

Background: In radiotherapy the timely identification of patients needing intervention and supportive care due to side effects is an important task especially in the outpatient setting. Activity trackers as an increasingly used lifestyle device may enable physicians to monitor patient's physical activity (PA) and to intervene early during the course of radiotherapy. Objective: The primary aim of this trial was to assess patient acceptance of PA monitoring in an outpatient setting and to correlate changes in PA with toxicity and changes in quality of life. Methods: Patients undergoing radio(chemo-)therapy with a curative intent were eligible to participate in this prospective pilot phase II trial. Patients were instructed to wear a commercially available activity tracker during the course of radiotherapy and four weeks afterwards. Quality of life (QoL) and fatigue was scored using the Functional assessment of Chronic Illness Therapy questionnaire. A linear regression was performed to determine baseline activity and changes in step counts during radiotherapy. Results: We included 23 patients in this trial. Two withdrew consent before the start of treatment, two patients were excluded after prophylactic feeding tube placement and prolonged recovery. Compliance in the remaining 19 patients was high, with availability of step-counts on 92% of the days. Baseline step counts were 6274 for breast cancer patients and 3621 for patients with other entities. Decreasing activity during radiotherapy coincided with the development of side effects and declines in quality of life. Conclusions: Activity trackers as tool to monitor PA during and after radiotherapy were accepted by a majority of the patients included in the current trial. Observed changes in PA correlated with patient reported side effects and QoL in some of the patients.

Auger-spectroscopy in quantum Hall edge channels and the missing energy problem.

Quantum Hall edge channels offer an efficient and controllable platform to study quantum transport in one dimension. Such channels are a prospective tool for the efficient transfer of quantum information at the nanoscale, and play a vital role in exposing intriguing physics. Electric current along the edge carries energy and heat leading to inelastic scattering, which may impede coherent transport. Several experiments attempting to probe the concomitant energy redistribution along the edge reported energy loss via unknown mechanisms of inelastic scattering. Here we employ quantum dots to inject and extract electrons at specific energies, to spectrally analyse inelastic scattering inside quantum Hall edge channels. We show that the missing energy puzzle could be untangled by incorporating non-local Auger-like processes, in which energy is redistributed between spatially separate parts of the sample. Our theoretical analysis, accounting for the experimental results, challenges common-wisdom analyses which ignore such non-local decay channels.

Radiation Pneumonitis after Intensity-Modulated Radiotherapy for Esophageal Cancer: Institutional Data and a Systematic Review.

Radiation pneumonitis (RP) is a serious complication that can occur after thoracic radiotherapy. The goal of this study is to investigate the incidence of RP after radiochemotherapy with intensity modulated radiotherapy (IMRT) in patients with esophageal cancer and correlate this with dose volume histogram (DVH) related parameters. For this purpose, the clinical course of 73 patients was evaluated and irradiation doses to the lungs were extracted from radiotherapy treatment plans. Furthermore, a systematic review on this topic was conducted across PubMed. In our institutional cohort, Common Terminology Criteria for Adverse Events (CTCAE) grade II or higher RP occurred in four patients (5.5%). The systematic review identified 493 titles of which 19 studies reporting 874 patients qualified for the final analysis. No grade IV or V RP after radiochemotherapy with IMRT for esophageal cancer was reported in the screened literature. Grade II or higher RP is reported in 6.6% of the patients. A higher incidence can be seen with increasing values for lung V20. In conclusion, our institutional data and the literature consistently show a low incidence of symptomatic RP after radiochemotherapy in patients with esophageal cancer treated with IMRT. However, efforts should be made to keep the lung V20 below 23% and specific caution is warranted in patients with pre-existing lung conditions.

Tumor response, toxicity, and survival after neoadjuvant organ-preserving chemotherapy for advanced laryngeal carcinoma. The Department of Veterans Affairs Cooperative Laryngeal Cancer Study Group.

PURPOSE: In 1984, the Department of Veterans Affairs Cooperative Studies Program began a trial in which patients with resectable squamous cell carcinoma of the larynx were randomized to receive standard surgery followed by radiation therapy or to receive neoadjuvant therapy with cisplatin and fluorouracil (5-FU) followed by radiation therapy for those achieving a greater than 50% tumor response to chemotherapy. This analysis reviews the tumor responses, toxicity, compliance, and long-term survival for those patients randomized to the chemotherapy arm. PATIENTS AND METHODS: One hundred sixty-six patients were randomized to the chemotherapy arm. Standard tumor response data, chemotherapy toxicity, and survival have been examined using standard statistical methods. RESULTS: The high response rates and acceptable toxicity to cisplatin and 5-FU of previously untreated patients were confirmed. Long-term disease-free survival was more likely to occur in patients who achieved a complete response to chemotherapy, particularly in those who had a confirmed histologic response to chemotherapy. Pretreatment histologic growth patterns were highly predictive of responses to chemotherapy. CONCLUSION: Neoadjuvant chemotherapy was well tolerated and did not negatively affect the definitive treatment that followed. The survival of nonresponding patients who underwent prompt salvage surgery was also not impaired. The role of organ preservation should be explored in other head and neck sites.

A single gene for human TRAF-3 at chromosome 14q32.3 encodes a variety of mRNA species by alternative polyadenylation, mRNA splicing and transcription initiation.

Human TRAF-3 is a signaling molecule that interacts with the cytoplasmic tails of CD40 and other TNF-receptor family members. TRAF-3 mRNA is expressed as two major classes of approximately 2 and 8 kb and a number of TRAF-3 encoding cDNA clones differ in discrete gene segments. Because this variety of mRNA species could result from mRNA processing events and/or multiple genes, the structure and localization of TRAF-3 encoding gene elements were determined. FISH and radiation hybrid mapping demonstrated that TRAF-3 is located at chromosome 14q32.3, approximately 1 Mb centromeric to the Ig heavy chain gene complex. Physical mapping of four overlapping genomic PAC clones established that TRAF-3 transcripts are encoded by a single gene, comprised of 13 exons and spanning 130 kb. Alternative polyadenylation in the mRNA segment encoded by exon 12 accounts for the difference between the 2 kb and the 8 kb classes of transcripts. Alternative mRNA splicing in the coding region (encoded by exons 3-12) generates transcripts which delete exons 8 (75 nt), 7+8 (156 nt) or 8+9 (168 nt) and that encode distinct protein isoforms (delta25, delta52 and delta56 aa, respectively). Alternative splicing of exon 2 (139 nt) and alternative transcriptional initiation result in mRNA species with distinct 5'UTRs. Together, these data indicate that a single TRAF-3 gene encodes a variety of mRNA species by a combination of alternative polyadenylation, alternative mRNA splicing and/or alternative initiation.

Bioinformatic analysis of autism positional candidate genes using biological databases and computational gene network prediction.

Common genetic disorders are believed to arise from the combined effects of multiple inherited genetic variants acting in concert with environmental factors, such that any given DNA sequence variant may have only a marginal effect on disease outcome. As a consequence, the correlation between disease status and any given DNA marker allele in a genomewide linkage study tends to be relatively weak and the implicated regions typically encompass hundreds of positional candidate genes. Therefore, new strategies are needed to parse relatively large sets of 'positional' candidate genes in search of actual disease-related gene variants. Here we use biological databases to identify 383 positional candidate genes predicted by genomewide genetic linkage analysis of a large set of families, each with two or more members diagnosed with autism, or autism spectrum disorder (ASD). Next, we seek to identify a subset of biologically meaningful, high priority candidates. The strategy is to select autism candidate genes based on prior genetic evidence from the allelic association literature to query the known transcripts within the 1-LOD (logarithm of the odds) support interval for each region. We use recently developed bioinformatic programs that automatically search the biological literature to predict pathways of interacting genes (PATHWAYASSIST and GENEWAYS). To identify gene regulatory networks, we search for coexpression between candidate genes and positional candidates. The studies are intended both to inform studies of autism, and to illustrate and explore the increasing potential of bioinformatic approaches as a compliment to linkage analysis.

Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization.

We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.

Cloning of the breakpoints of a de novo inversion of chromosome 8, inv (8)(p11.2q23.1) in a patient with Ambras syndrome.

Ambras syndrome (AMS) is a unique form of universal congenital hypertrichosis. In patients with this syndrome, the whole body is covered with fine long hair, except for areas where normally no hair grows. There is accompanying facial dysmorphism and teeth abnormalities, including retarded first and second dentition and absence of teeth. In 1993, Baumeister et al. reported an isolated case of Ambras syndrome in association with a pericentric inversion of chromosome 8. Subsequently, another patient with congenital hypertrichosis and rearrangement of chromosome 8 was reported by Balducci et al. (1998). Both of these patients have a breakpoint in 8q22 in common suggesting that this region of chromosome 8 contains a gene involved in regulation of hair growth. In order to precisely determine the nature of the rearrangement in the case of Ambras syndrome, we have used fluorescent in situ hybridization (FISH) analysis. We have cloned the inversion breakpoints in this patient and generated a detailed physical map of the inversion breakpoint interval. Analysis of the transcripts that map in the vicinity of the breakpoints revealed that the inversion does not disrupt a gene, and suggests that the phenotype is caused by a position effect.

A comparative trial of cefazolin and moxalactam as prophylaxis for preventing infection after abdominal hysterectomy.

In a randomized, double-blind clinical trial, 208 women who underwent abdominal hysterectomy received either cefazolin (N = 108) or moxalactam (N = 100) as perioperative antimicrobial prophylaxis. There were no differences between the two groups in rates of serious infection, minor wound infection, standard febrile morbidity, duration of hospitalization, proportion receiving other postoperative antibiotics, or rates of rehospitalization. Women who received moxalactam had significantly more urinary tract infections, 87% of which were caused by the enterococcus. It is concluded that perioperative prophylaxis with third-generation cephalosporins is not justified at this time.

Separation of random fragments of DNA according to properties of their sequences.

The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule.

DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory.

DNA fragments 536 base pairs long differing by single base-pair substitutions were clearly separated in denaturing gradient gel electrophoresis. Transversions as well as transitions were detected. The correspondence between the gradient gel measurements and the sequence-specific statistical mechanical theory of melting shows that mutations affecting final gradient penetration lie within the first cooperatively melting sequence. Fragments carrying substitutions in domains melting at a higher temperature reach final gel positions indistinguishable from wild type. The gradient data and the sites of substitution bracket the boundary between the first domain and its neighboring higher-melting domain within eight base pairs or fewer, in agreement with the calculated boundary. The correspondence between the gradient displacement of the mutants and the calculated change in helix stability permits substantial inference as to the type of substitution. Excision of the lowest melting domain allows recognition of mutants in the next ranking domain.

Cell cycle changes in Physarum polycephalum histone H1 phosphate: relationship to deoxyribonucleic acid binding and chromosome condensation.

We have examined the relationship of phosphate content in histone H1 of Physarum polycephalum to mitotic chromosome condensation and affinity for deoxyribonucleic acid (DNA). H1 undergoes a series of posttranslational phosphorylations which increase its apparent molecular weight on NaDodSO4-polyacrylamide gels. Our studies confirm the observation by Bradbury and co-workers [Bradbury, E. M., Inglis, R. J., Matthews, H. R., & Sarner, N (1973) Eur. J. Biochem. 33, 131-139; Bradbury, E. M., Inglis, R. J., & Matthews, H. R. (1974) Nature (London) 247, 257-261] that the accumulation of phosphate in H1 increases markedly shortly before the onset of mitosis. However, we show in pulse-chase experiments with both 32PO4H1 and [14C]lysine H1 that there is no significant dephosphorylation of the histone either during or shortly after mitosis, suggesting that nonspecific postmitotic dephosphorylation of H1 is not a prerequisite for chromosome decondensation. We also show that both phosphorylated and unphosphorylated forms of H1 bind with somewhat greater affinity to single-stranded DNA-cellulose than to native DNA-cellulose and that phosphorylation weakens the affinity of H1 to both forms of DNA-cellulose.

Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis.

When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60 degrees C in a concentration gradient of a urea-formamide mixture, each Eco RI fragment of lambda or E. coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment has been retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a two-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the Eco RI digest of E. coli DNA. Corresponding fragments from an isogenic lambda lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.

Detection of single base-pair mismatches in DNA by chemical modification followed by electrophoresis in 15% polyacrylamide gel.

We have developed a method for distinguishing fragments of DNA that contain single-base mismatches from their perfectly paired homologues. Single-stranded regions within a duplex fragment are accessible to 1-cyclohexyl-3-(2-[4-(4-methyl)morpholinyl]ethyl)carbodiimide, which reacts with unpaired guanidylate and thymidylate residues in DNA. Intact linear duplex DNA molecules do not react with carbodiimide, whereas DNA molecules containing single-base mismatches react quantitatively. After carbodiimide reaction, the DNA molecules are electrophoresed in high-percentage polyacrylamide gels so that modified and unmodified fragments can be resolved. Application of this technique should make it possible to locate and purify DNA fragments that exhibit sequence differences from those that do not; these might be used to signal phenotypic variation as well as to diagnose inherited disease.

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.