Detailed molecular characterization of a novel IDS exonic mutation associated with multiple pseudoexon activation.

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

[Detection of 1,3-beta-D glucan for diagnosis of invasive fungal infections in hematooncological patients: usefulness for screening of invasive mycosis and for confirmation of galactomannan positive results]. / Detekce 1,3-beta-D glukanu v diagnostice invazivních mykotických infekcí u nemocných s hematologickou malignitou: prínos metody ve skríningu invazivních mykóz a v konfirmaci galaktomanan pozitivních vzorku.

BACKGROUND AND AIMS: 1,3-beta-D glucan (BG) -- the antigen of fungal cell wall can be detected by a commercially available test for early detection of invasive fungal infections (IFI). The main advantage of this test is its broad coverage of fungal species. The aim of our study was to evaluate usefulness of BG detection for screening of IFI and for confirmation of galactomannan (GM) positive blood samples. Combination of the results of both tests could lead to correct and early diagnosis of invasive aspergillosis (IA). PATIENTS AND METHODS: Between January 2005 and July 2007 blood samples were collected in patients from intermediate to high risk of IFI. Moreover, between February and October 2007 all patients that had consecutive positive results of GM had their positive symplex tested also for BG. RESULTS: In BG screening study, 1154 of blood samples from 104 treatment cycles were tested for BG. The incidence of IFI was 17.3 % (n = 18) and probable or proven IFI was detected in 9 cases (8.6%). The highest sensitivity, specificity, PPV and NPV (88.9 %, 40.7 %, 13.6 % and 97.2 %) were obtained when as criteria for positivity cut off 80 pg/ml and one positive result were used. When consecutive positivity of the test was applied as criterium, cut off 60 pg/ml was found more useful (sensitivity 66.7 %, specificity 47.7 %, PPV 11.8 % and NPV 93.2 %). Low PPV, caused by frequent false positive results, was identified as main limitation of this assay. 65 treatment cycles were positive if 1 sample above 80 pg/ml was used as a cut of for positivity. If consecutive positivity with cut off 60 pg/ml was used, 58 treatment cycles were positive. But in 51 (78.4 %) and 45 (77.5 %) cases, respectively, the positivity was not associated with IFI (false positivity). We did not find any correlation between positive BG assay result and frequency of empirical antifungal treatment, mucositis, yeast colonization, administration of selected antibiotics or infusion solutions or bacteriaemia. In our confirmation study, 40 GM positive episodes in 39 patients were identified. In 31 (78 %) GM positivity was false and was not associated with clinical signs and symptoms of IA. Sensitivity of GM detection in IA was 100 % but PPV only 18 %. Confirmation of consecutive GM positive samples (using cut off index positivity 0,5) by consecutive positivity of BG (with cut off 60 pg/ml) was found very useful for diagnosis of IA -- most of GM false positive results were eliminated and PPV increased to 88 %. CONCLUSIONS: Our analysis focused on routine use of BG test for panfungal screening of IFI in patients with hematological malignancy and confirmed limited usefulness of this test in such setting. Low sensitivity together with low PPV are major limits of this test. On the other hand, BG testing seems to be a promising tool for confirmation of consecutive GM positive result in serum in patients with IA. Positivity of both tests could increase their PPV of tests and eliminate false positive results.

[Macular area in glaucoma patients]. / Makulární oblast u pacientu s glaukomem.

PURPOSE: To evaluate the possible use of HRT II and OCT 3 for detection and monitoring of glaucoma changes in the macular area. METHODS: In the retrospective study 65 eyes (13 healthy and 52 with primary open angle glaucoma) of 36 patients have been monitored. All patients underwent complete ophthalmologic examination including visual acuity testing, biomicroscopy of fundus, computer perimetry, HRT II and eleven patients were examined using OCT 3. The visual field has been tested on HFA (Humphrey Field Analyzer) using the full threshold test 30-2. Foveolar sensitivity and foveolar sensitivity compared to the age have been evaluated. The optic nerve head has been examined and subsequently evaluated using HRT II. The retinal nerve fiber layer (t-RNFL) and Cup Shape Measurement (t-CSM) in the temporal area have been monitored. The macula has been examined on OCT 3. Foveolar thickness, inner and outer macular thickness, average macular thickness and aggregate macular volume have been monitored. RESULTS: Decrease of the visual acuity and foveolar sensitivity have been registered with the deterioration of the visual field. Further general decrease of the value of t-RNFL and increase of the t-CSM index have been registered from the results of the HRT II examinations. This corresponds to decrease of the nerve fibers and excavation deepening in the temporal area. Average macular thickness and total macular volume examined on OCT 3 demonstrate aggregate decrease of values with the progressive changes in the visual field. CONCLUSION: With the progressive visual field changes with the open angle glaucoma patients occurs also deterioration of finding in the macular area. Changes discovered using the subjective examination and expressed as a slight decrease of the visual acuity as well as decrease of the foveolar sensitivity on HFA can be objectively proved using HRT II and OCT 3.