Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

Five-year prognosis in an incident cohort of people presenting with acute myocardial infarction

Background

Following an AMI, it is important for patients and their physicians to appreciate the subsequent risk of death, and the potential benefits of invasive cardiac procedures and secondary preventive therapy. Studies, to-date, have focused largely on high-risk populations. We wished to determine the risk of death in a population-derived cohort of 2,887 patients after a first acute myocardial infarction (AMI).

Methods

Logistic regression and survival analysis were conducted to investigate the effect of different baseline characteristics, pharmacological therapies and revascularization procedures on coronary heart disease (CHD) and all-cause mortality outcomes.

Results

Within five years 44.4% of patients died (27.1% short-term [<30 days] and 23.7% longer-term [≥30 days]). Percutaneous transluminal coronary angioplasty (Adjusted Hazards Ratio (AHR) = 0.49, 95% Confidence Interval (CI) 0.26–0.93), β-blockers (AHR = 0.58, 95%CI 0.46–0.74) and statins (AHR = 0.60, 95%CI 0.47–0.77) were all associated with significant reductions in longer-term CHD-related mortality. However, not all patients received secondary preventive therapy (8.7%). Diabetes (AHR = 1.83, 95%CI 1.43–2.34), stroke (AHR = 1.73, 95%CI 1.35–2.22), heart failure (AHR = 1.69, 95%CI 1.28–2.22), smoking (AHR = 1.72, 95%CI 1.18–2.51) and obesity (>30 kg/m2; AHR = 1.39, 95%CI 1.01–1.90) increased the risk of longer-term mortality independent of other risk factors.

Conclusions

It is encouraging that the coronary procedure PTCA and pharmacological secondary prevention therapies were found to be strongly associated with an important reduced risk of subsequent death, although not all patients received these interventions. Smoking, being obese and having cardiovascular related disease at baseline were also associated with an increased likelihood of longer-term mortality, independent of other baseline characteristics. Thus, the provision of smoking cessation, advice on diet (for obese patients) and optimal treatment is likely to be crucial for reducing mortality in all patients after AMI.     

Modulation of sulfur mustard induced cell death in human epidermal keratinocytes using IL-10 and TNF-alpha

We compared the effects of overexpressing a tightly regulated anti-inflammatory cytokine, interleukin 10 (IL-10), and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on sulfur mustard induced cytotoxicity in human epidermal keratinocytes. Both cytokines were overexpressed when compared with the cells transfected with the empty vector as determined by quantitative ELISA. Cells overexpressing interleukin 10 suppressed the pro-inflammatory cytokines interleukin 8 and interleukin 6 following exposure to 50-300 microM sulfur mustard. These cells exhibited delayed onset of sulfur mustard induced cell death. On the other hand, cells overexpressing tumor necrosis factor alpha induced a sustained elevation in both interleukin 6 and 8 expression following exposure to 50-300 microM sulfur mustard. These cells were sensitized to the effects of sulfur mustard that resulted in an increased sulfur mustard induced cell death. Normal human epidermal keratinocytes treated with sulfur mustard exhibited elevated levels of tumor necrosis factor alpha expression and increased activity of nuclear factor kappa B. Gene array data indicated that cells overexpressing interleukin 10 induced several genes that are involved in growth promotion and cell-fate determination. We, therefore, identify IL-10 and TNF-alpha signal transduction pathways and their components as possible candidates for early therapeutic intervention against sulfur mustard induced cell injury.     

Physiological responses to single versus double stepping pattern of ascending the stairs

The aim of this study was to compare the physiological responses and energy cost between two ascending patterns, the single-step (SS) and the double-step (DS), in climbing a public staircase. In the SS pattern, a person climbs one step at a time whilst in the double-step (DS) pattern, the individual traverses two steps in a single stride. Advocates of each stepping pattern claimed that their type of ascent is physically more taxing and expends more calories. Thirty subjects (10 males and 20 females) climbed a typical 11-storey flat (each step height of 0.15 m, a total of 180 steps and a vertical displacement of 27.0 m). The subjects climbed using either the SS pattern at a tempo of 100 steps x min(-1) or the DS pattern at 50 steps x min(-1). The prescribed stepping frequencies ensured that an equal amount of total work was performed between the SS and DS patterns. The climbing patterns were performed in random order. Physiological measures during the last 30 s of the climbs were used in the comparative analysis. The results showed that ventilation, oxygen uptake and heart rate values were significantly higher (all p < 0.01) in the SS as compared to the DS pattern. However, the caloric expenditure during the SS pattern was calculated to be only marginally higher than the DS pattern. In conclusion, ascending with the SS pattern led to significantly higher physiological responses compared to the DS pattern. The higher calorie expended with the SS compared to the DS pattern was deemed to be of little practical significance.     

High expression of IMPACT protein promotes resistance to indoleamine 2,3‐dioxygenase‐induced cell death

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO‐induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non‐derepressible‐2(GCN2) kinase stress‐responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO‐induced low tryptophan environment. IDO‐expressing human fibroblasts were co‐cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO‐expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO‐induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co‐cultured with IDO‐expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT‐expressing vector, was rescued significantly in tryptophan‐deficient but not IDO‐expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO‐mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO‐generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non‐immune cells such as skin cells acts as a protective mechanism against IDO‐induced GCN2 activation, therefore, makes them resistant to the amino acid‐deprived environment caused by IDO. J. Cell. Physiol. 225: 196–205, 2010. © 2010 Wiley‐Liss, Inc.     

Assessment of bovine sperm viability by MTT reduction assay

The MTT reduction assay depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.     

Neurophysiological evaluation of healthy human anorectal sensation

Patients with functional gastrointestinal disorders often demonstrate abnormal visceral sensation. Currently, rectal sensation is assessed by manual balloon distension or barostat. However, neither test is adaptable for use in the neurophysiological characterization of visceral afferent pathways by sensory evoked potentials. The aim of this study was to assess the reproducibility and quality of sensation evoked by electrical stimulation (ES) and rapid balloon distension (RBD) in the anorectum and to apply the optimum stimulus to examine the visceral afferent pathway with rectal evoked potentials. Healthy subjects (n = 8, median age 33 yr) were studied on three separate occasions. Variability, tolerance, and stimulus characteristics were assessed with each technique. Overall ES consistently invoked pain and was chosen for measuring rectal evoked potential whereas RBD in all cases induced the strong urge to defecate. Rectal intraclass correlation coefficient (ICC) for ES and RBD (0.82 and 0.72, respectively) demonstrated good reproducibility at pain/maximum tolerated volume but not at sensory threshold. Only sphincter ICC for ES at pain showed acceptable between-study reproducibility (ICC 0.79). Within studies ICC was good (>0.6) for anorectal ES and RBD at both levels of sensation. All subjects reported significantly more unpleasantness during RBD than ES (P < 0.01). This study demonstrates that ES and RBD are similarly reproducible. However, the sensations experienced with each technique differed markedly, probably reflecting differences in peripheral and/or central processing of the sensory input. This is of relevance in interpreting findings of neuroimaging studies of anorectal sensation and may provide insight into the physiological characteristics of visceral afferent pathways in health and disease.