Constitutional mismatch repair-deficiency and whole-exome sequencing as the means of the rapid detection of the causative MSH6 defect.

BACKGROUND: Cases of children with more than one type of cancer either diagnosed simultaneously or successively, rarely occur in pediatric oncology. A second malignant neoplasm may be caused by mutagenic effects of the treatment of the primary malignancy and/or may point towards an underlying genetic cancer susceptibility syndrome. One example of such a syndrome is constitutional mismatch repair-deficiency, (CMMR-D) which carries an increased risk of various tumors including childhood hematologic malignancies and Lynch syndrome associated tumors. Timely diagnosis of CMMR-D is crucial, since this diagnosis has implications for the entire family. PATIENT: We report the case of a 15-year-old girl who was born to consanguineous parents. At the age of 20 months she was diagnosed with a T-cell non-Hodgkin lymphoma. Treatment was given according to NHL-BFM 95. 12 years later, an invasive adenocarcinoma of the colon was surgically removed which relapsed shortly afterwards. METHODS: Whole-exome sequencing of germline DNA was employed to rapidly detect the underlying mutation in this suspected CMMR-D patient. RESULTS: After a short turnaround time of less than 3 weeks, the diagnosis of CMMR-D could be confirmed by the identification of a homozygous 29-bp deletion in MSH6 (exon 6), which was confirmed by independent methods. CONCLUSIONS: We demonstrate that "bed-side" whole-exome sequencing is both feasible and cost-effective and may be the method of choice to rapidly uncover the genetical basis of (inherited) diseases.

The impact of TEL-AML1 (ETV6-RUNX1) expression in precursor B cells and implications for leukaemia using three different genome-wide screening methods.

The reciprocal translocation t(12;21)(p13;q22), the most common structural genomic alteration in B-cell precursor acute lymphoblastic leukaemia in children, results in a chimeric transcription factor TEL-AML1 (ETV6-RUNX1). We identified directly and indirectly regulated target genes utilizing an inducible TEL-AML1 system derived from the murine pro B-cell line BA/F3 and a monoclonal antibody directed against TEL-AML1. By integration of promoter binding identified with chromatin immunoprecipitation (ChIP)-on-chip, gene expression and protein output through microarray technology and stable labelling of amino acids in cell culture, we identified 217 directly and 118 indirectly regulated targets of the TEL-AML1 fusion protein. Directly, but not indirectly, regulated promoters were enriched in AML1-binding sites. The majority of promoter regions were specific for the fusion protein and not bound by native AML1 or TEL. Comparison with gene expression profiles from TEL-AML1-positive patients identified 56 concordantly misregulated genes with negative effects on proliferation and cellular transport mechanisms and positive effects on cellular migration, and stress responses including immunological responses. In summary, this work for the first time gives a comprehensive insight into how TEL-AML1 expression may directly and indirectly contribute to alter cells to become prone for leukemic transformation.

Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases.

The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.

Array-based sequence capture and next-generation sequencing for the identification of primary immunodeficiencies.

Primary immunodeficiencies are genetic disorders in which components of immunological pathways are either missing or dysregulated. With the advent of next-generation sequencing, testing for genes in conditions with a heterogeneous genetic background seems more promising. We designed a custom microarray with 385K probe capacity to capture exons of 395 human genes, known or predicted to be associated with primary immunodeficiency and immune regulation. Enriched target DNA was sequenced using a GS FLX Titanium 454 platform. The patients selected were likely to have an underlying immunodeficiency. In one patient with hepatosplenomegaly, recurrent infections and an elevated IgM level, sequence analysis of the patient and his two unaffected parents identified ATM (ataxia telangiectasia mutated) as the underlying defect. In a second child with a clinical SCID phenotype, we detected a mutation in the ARTEMIS gene after focusing on SCID-associated genes. 454 sequencing yielded 152,000-397,000 high-quality reads per patient. 78-99% of the targeted nucleotides were covered at least one time, 76-82% at least five times. Array-based sequence capture expands our capacities to sequence large targeted DNA regions in a less laborious and time-consuming approach. Our array was capable to find the underlying genetic defect in two patients with suspected primary immunodeficiency. Upcoming whole-exome sequencing definitely will add more valuable data, but bioinformatical analysis and validation of variants already pose major challenges.

Highly skewed T-cell receptor V-beta chain repertoire in the bone marrow is associated with response to immunosuppressive drug therapy in children with very severe aplastic anemia.

One of the major obstacles of immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA) comes from the often months-long unpredictability of bone-marrow (BM) recovery. In this prospective study in children with newly diagnosed very severe AA (n=10), who were enrolled in the therapy study SAA-BFM 94, we found a dramatically reduced diversity of both CD4+ and CD8+ BM cells, as scored by comprehensive V-beta chain T-cell receptor (TCR) analysis. Strongly skewed TCR V-beta pattern was highly predictive for good or at least partial treatment response (n=6, CD8+ complexity scoring median 35.5, range 24-73). In contrast, IST in patients with rather moderate reduction of TCR V-beta diversity (n=4, CD8+ complexity scoring median 109.5, range 82-124) always failed (P=0.0095). If confirmed in a larger series of patients, TCR V-beta repertoire in BM may help to assign children with SAA up-front either to IST or to allogeneic stem-cell transplantation.

Linear transport equations valid for arbitrary collisionality: comparison with the Chapman-Enskog expansion.

Recently we proposed a method to solve the perturbed Boltzmann equation modeled by the Bhatnagar-Gross-Krook operator [Phys. Rev. E 74, 041204 (2006)]. In this work we use this method to derive linear transport equations in the whole collisionality range. A comparison of the closure relations derived up to the third order in the Knudsen number (super-Burnett) yields the same results as the Chapman-Enskog expansion. The contribution of the projection operators to the transport is investigated. It is pointed out that their contributions are not negligible in the super-Burnett equations and very significant in the collisionless range. The test of stability of the super-Burnett equations is also performed. It is shown that the stability problem can be related to the positivity of the generalized transport coefficients. Using the Padé approximants, nonlocal transport coefficients are proposed which present the desirable stability properties.

Local-field-correction effects on the electron response functions and on the electrical conductivity in a hydrogen plasma.

In a one-component plasma constituted by electrons with a uniform positive background, the correlations are studied in the framework of the Singwi, Tosi, Land, and Sjölander (STLS) model. The local-field corrections (LFCs) are calculated with electron density ranging from 10;{19}to10;{26}cm;{-3} and with a temperature of 10;{4}K . Then, a dielectric formalism is used to deduce the potential energy of a positive test charge (a proton) imbedded in the electron medium. A significant departure of this potential from the random phase approximation (RPA) one has been found. In particular, as the density increases, the discrepancy between the two potentials grows up to reach a maximum correlated to the maximum of the electron coupling parameter. In addition, it is found that in both high and low density limits, the STLS and RPA approaches yield similar results. On the other hand, the effect of the LFC on the electrical conductivity is also estimated in hydrogen plasmas.

Generalized linear transport theory in dilute neutral gases and dispersion relation of sound waves.

The transport processes in dilute neutral gases are studied by using the kinetic equation with a collision relaxation model that meets all conservation requirements. The kinetic equation is solved keeping the whole anisotropic part of the distribution function with the use of the continued fractions. The conservative laws of the collision operator are taken into account with the projection operator techniques. The generalized heat flux and stress tensor are calculated in the linear approximation, as functions of the lower moments, i.e., the density, the flow velocity and the temperature. The results obtained are valid for arbitrary collision frequency nu with the respect to kv(t) and the characteristic frequency omega, where k(-1) is the characteristic length scale of the system and v(t) is the thermal velocity. The transport coefficients constitute accurate closure relations for the generalized hydrodynamic equations. An application to the dispersion and the attenuation of sound waves in the whole collisionality regime is presented. The results obtained are in very good agreement with the experimental data.

A population pharmacokinetic analysis of the penetration of the prostate by levofloxacin.

Prostatitis has remained a pathological entity that is difficult to treat. Part of the difficulty revolves about the putative offending pathogens. For acute prostatitis, members of the Enterobacteriaceae, particularly Escherichia coli, play a central role, while intracellular pathogens such as Chlamydia are more frequently seen in chronic prostatitis. Consequently, a drug needs to be able to penetrate to this specialized site in both the acute and chronic infection forms of the disease and also have potent activity against the most common causative pathogens, both intracellular and extracellular. Levofloxacin has such an activity profile. We wished to document its ability to penetrate to the site of infection. Patients undergoing prostatectomies were administered 500 mg of levofloxacin orally every 24 h for 2 days prior to surgery, and then on the day of surgery, 500 mg was administered as an hour-long, constant-rate intravenous (i.v.) infusion. A set of blood samples was obtained as guided by stochastic optimal design theory. Prostate biopsy times were determined by randomizing subjects into one of four groups, based on the interval after the i.v. dose. All plasma and prostate drug concentrations were comodeled by a population modeling program, BigNPEM, implemented on the Cray T3E Supercomputer housed at the Supercomputer Center at the University of California at San Diego. Penetration was determined as the ratio of the area under the concentration-time curve (AUC) of levofloxacin in the prostate to the plasma levofloxacin AUC. When calculated from the mean population parameters, this penetration ratio was 2.96. We also performed a 1,000-subject Monte Carlo simulation from the mean parameter vector and covariance matrix. The mean penetration ratio here was 4.14 with a 95% confidence interval of 0.20 to 19.6. Over 70% of the population had a penetration ratio in excess of 1.0. Levofloxacin adequately penetrates a noninflamed prostate and should be evaluated for the therapy of prostatitis.

Penetration of topically applied ciprofloxacin, norfloxacin, and ofloxacin into the aqueous humor.

PURPOSE: To determine the intraocular penetration of topically applied fluoroquinolone antibiotics into aqueous humor. METHODS: Thirty-two patients undergoing cataract extraction received either 0.3% ciprofloxacin, 0.3% norfloxacin, or 0.3% ofloxacin topical drops. The patients were given two drops 90 minutes preoperatively and two drops 30 minutes preoperatively. At the time of surgery, 0.1 ml aqueous fluid was aspirated from the anterior chamber and immediately stored at -70 degrees C. RESULTS: Concentrations of ciprofloxacin, norfloxacin, and ofloxacin were determined using a broth dilution bioassay. Morganella morganii with a known minimal inhibitory concentration was used to assay ciprofloxacin and norfloxacin levels. Salmonella enteritidis with a known minimal inhibitory concentration was used to assay ofloxacin levels. Topically applied ciprofloxacin achieved a mean aqueous level of 0.072 microgram/ml (range, 0.02-0.153 microgram/ml). One sample was below the sensitivity of the bioassay. Topical norfloxacin achieved a mean aqueous level of 0.0570 microgram/ml (range, 0.046-0.10 microgram/ml). Seven samples did not reach the sensitivity of the bioassay. Topical ofloxacin achieved a mean level in the aqueous humor of 0.338 microgram/ml (range, 0.078-0.625 microgram/ml). There was no statistically significant difference in intraocular aqueous humor levels of ciprofloxacin versus norfloxacin (P > 0.05). Topical ofloxacin achieved aqueous humor levels significantly higher than either ciprofloxacin or norfloxacin (P < 0.004). CONCLUSION: Of the currently available topical fluoroquinolone antibiotics, ofloxacin achieves the highest aqueous humor concentrations.

Therapy of pulmonary nocardiosis in immunocompromised mice.

We compared the bactericidal efficacies of various antimicrobial agents and combinations thereof in experimentally induced Nocardia asteroides pneumonia in immunocompromised mice. Cortisone acetate treatment, which produced impaired cell-mediated immune function, was followed by nasal inoculation of 5 x 10(4) CFU of N. asteroides into each mouse. Therapy was begun 24 h after inoculation and continued for the next 96 h. Dosages of antimicrobial agents resulted in concentrations approximating levels in human serum. Animals from each of nine treatment groups were sacrificed every 24 h. The pulmonary tissue obtained was homogenized and quantitatively cultured. Results were calculated to indicate the number of CFU per gram of lung tissue. Amikacin and imipenem were the two most effective single agents studied. Sulfadiazine and ciprofloxacin were ineffective, and ceftriaxone reduced bacterial counts modestly. Combination therapy did not enhance the bactericidal activities of the agents tested. We conclude that amikacin and imipenem, as well as select broad-spectrum cephalosporins, represent therapy superior to the sulfonamides in this experimental model and may represent alternative treatment for patients who cannot tolerate sulfa agents (e.g., human immunodeficiency virus-infected patients) or who fail primary treatment.

Strategies for the management of varicella-susceptible healthcare workers after a known exposure.

Three different sequentially applied post-varicella zoster virus (VZV) exposure management strategies were employed over a 43-month period. We began by using a standard post-exposure protocol in which 50 susceptible healthcare workers (HCW) involved in hospital exposures were furloughed from work at a loss to the hospital of 424 workdays and $46,000. Of the eight nosocomial cases of VZV infection in HCWs, four (50%) caused future HCW and patient exposure. In trial I, we substituted a post-exposure screening procedure for the standard work furlough procedure. We screened 77 exposed staff resulting in one nosocomial VZV infection that was the source of another exposure incident. No secondary cases of varicella resulted from this exposure and only 20 days of furlough time were used during trial I. As VZV resulting from a home exposure source was responsible for most hospital exposures in which HCWs were the source, our trial II protocol added the Centers for Disease Control's (CDC) off-duty procedure, but limited its use to susceptibles exposed at home. The 43-month overall attack rate of nosocomial varicella was 4.7%, while the true home exposure attack rate was 79% (p less than .00001). There was an average of 42.4 lost workdays charged to the hospital per incident under the standard protocol and three days per incident in the combined experience of trials I and II (p less than .0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrobial synergism in the therapy of experimental cerebral nocardiosis.

A mouse model of cerebral nocardiosis was used to determine the efficacy of synergistic antimicrobial combinations in reducing bacterial colony counts per gram of brain tissue. The combinations of imipenem-cefotaxime and imipenem-trimethoprim/sulphamethoxazole (TMP/SMP) were compared with each other and with each agent used alone. A saline treated control group was also included. At the completion of 72 h of therapy the combinations of imipenem-cefotaxime and imipenem-TMP/SMX were the most effective in reducing bacterial colony counts. These were statistically superior to cefotaxime and TMP/SMX used alone but not statistically superior to imipenem alone. TMP/SMX was not effective in this model and was inferior to all other antibiotic treatments.

Susceptibility of Nocardia asteroides to new quinolones and beta-lactams.

The susceptibility of 31 strains of Nocardia asteroides to various quinolones and beta-lactams, as well as coumermycin, amikacin, and minocycline, was determined by the agar dilution technique. Ciprofloxacin was the most active fluoroquinolone tested on a weight basis, as it inhibited approximately 50% of the isolates at achievable drug levels in serum. Ceftriaxone and cefpirome were the most active cephalosporins in this system with MICs of 8 micrograms/ml for 80% of strains tested. Imipenem, amikacin, and minocycline were the most effective agents tested.