A founder mutation in BBS2 is responsible for Bardet‐Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders

Innes AM, Boycott KM, Puffenberger EG, Redl D, MacDonald IM, Chudley AE, Beaulieu C, Perrier R, Gillan T, Wade A, Parboosingh JS. A founder mutation in BBS2 is responsible for Bardet‐Biedl syndrome in the Hutterite population: utility of SNP arrays in genetically heterogeneous disorders. Bardet‐Biedl syndrome (BBS) is a multisystem genetically heterogeneous disorder, the clinical features of which are largely the consequence of ciliary dysfunction. BBS is typically inherited in an autosomal recessive fashion, and mutations in at least 14 genes have been identified. Here, we report the identification of a founder mutation in the BBS2 gene as the cause for the increased incidence of this developmental disorder in the Hutterite population. To ascertain the Hutterite BBS locus, we performed a genome‐wide single nucleotide polymorphism (SNP) analysis on a single patient and his three unaffected siblings from a Hutterite family. The analysis identified two large SNP blocks that were homozygous in the patient but not in his unaffected siblings, one of these regions contained the BBS2 gene. Sequence analysis and subsequent RNA studies identified and confirmed a novel splice site mutation, c.472‐2A>G, in BBS2. This mutation was also found in homozygous form in three subsequently studied Hutterite BBS patients from two different leuts, confirming that this is a founder mutation in the Hutterite population. Further studies are required to determine the frequency of this mutation and its role, if any, in the expression of other ciliopathies in this population.     

Limited Margins Using Modern Radiotherapy Techniques Does Not Increase Marginal Failure Rate of Glioblastoma

PURPOSE: We investigate the patterns of failure in the treatment of glioblastoma(GBM) based on clinical target volume(CTV) margin size,dose delivered to the site of initial failure,and the use of temozolomide and intensity-modulated radiotherapy(IMRT).METHODS: Between August 2000 and May 2010,161 patients with GBM were treated with radiotherapy with or without concurrent temozolomide.Patients were treated with CTV expansions that ranged from 5 to 20 mm using a shrinking field technique.Patterns of failure and time to progression and overall survival were compared based on CTV margin,use of temozolomide,and use of IMRT.Kaplan Meier analysis was used to estimate survival times,and χ test was used for comparison of cohorts.RESULTS: For patients treated with 5-,10-,and 15-to 20-mm CTV,79%,77%,and 86% experienced failures in the 60 Gy volume,respectively.Forty-eight percent,55%,and 66% of patients with 5-,10-,and 15-to 20-mm CTV experienced failures in the 46 Gy volume,respectively.There was no statistical difference between patients treated with 5-,10-,15-to 20-mm margins with regard to 60 Gy failure(P=0.76),46 Gy failure(P=0.51),or marginal failure(P=0.73).Eighty percent of patients receiving temozolomide experienced failures in the 60 Gy volume.There was no increased likelihood of marginal failures in patients receiving IMRT(P =0.97).CONCLUSIONS: Modern treatment techniques including use of concurrent temozolmide,limited CTV margin size,and IMRT have not greatly changed the patterns of failure of GBM.     

Inhibitory synaptogenesis in mouse somatosensory cortex

It is widely believed that inhibitory synapses are not present or present in only small numbers in the rodent cerebral cortex during the early postnatal period when the cortex is being innervated by thalamocortical fibers. Quantitative electron microscopy was carried out on the posteromedial barrel subfield of mouse somatosensory cortex from postnatal day 4 (P4) when thalamocortical innervation of the barrels is becoming established, through to sexual maturity (>P32), and in adulthood. Both asymmetrical (putatively excitatory) and symmetrical (putatively inhibitory) synapses were present in all layers from P4. The symmetrical synapses were immunoreactive for GABA at all ages. There was a progressive increase in both asymmetrical and symmetrical synapses up to P32, density in all layers increasing 16-fold, with the production of asymmetrical synapses leading and greatly outstripping that of symmetrical. From P32 to P120, the oldest age studied, synaptic numbers declined by 18% to 13 times the P4 level, but this affected predominantly layers II/III, IV and V, and mainly involved asymmetrical synapses. The relative percentage of asymmetrical to symmetrical synapses from P4 to P8 was 57%/43% but at P32 it was 89.5%/10.5% and in adulthood 85.4%/14.6%. These data indicate that inhibitory synaptogenesis in the rodent cortex begins earlier than previously thought, a basis for inhibition being present from the earliest period. Pruning of all synapses occurs well after thalamocortical innervation is established and inhibitory synapses are less affected by the pruning process.      

Tailored hormonal therapy in secretory adrenocortical cancer

Mitotane is often considered the front-line hormonal therapyof adrenocortical carcinoma (ACC). An illustrative case concerningthis issue and the rationale to ponder other alternatives isreported. A 69 year-old woman, diagnosed with ACC was admittedwith hypertensive crisis, supraventricular tachycardia, congestiveheart-failure, diarrhoea and rabdomyolisis. Two years earlier,she had undergone     

Inhibitor of BRAFV600E Mutation as a Treatment Option for Hairy Cell Leukemia With Deep Neutropenia and Infectious Complications

Standard therapy in hairy cell leukemia (HCL) is often impossible at the time of deep neutropenia/agranulocytosis with or without infectious complications; it is thus a complex therapeutic problem. Vemurafenib has been used to treat resistant HCL since 2012. Because vemurafenib does not have a myelotoxic effect, we thought that it could be used to treat HCL associated with deep neutropenia/agranulocytosis with or without the development of infectious complications as a preliminary stage before treatment with cladribine. We conducted a retrospective analysis of treatment with vemurafenib followed by a standard course of cladribine provided to 22 patients with deep neutropenia/agranulocytosis with or without infectious complications at diagnosis. Vemurafenib was provided to 22 patients with HCL. The response to therapy was evaluated by complete blood cell count (absolute neutrophil count [ANC], hemoglobin concentration, platelet count, absence of hairy cells), spleen size (assessed by ultrasound), and reduce infectious complications. After that, a standard course of cladribine was provided. Among the 22 patients, the male/female sex ratio was 2:1, and median (range) age was 52 (24-78) years. There were 7 patients with severe infectious manifestations admitted to the intensive care unit, including 1 patient during extracorporeal membrane oxygenation. The median (range) ANC at diagnosis was 0.3 (0.04-0.7) × 10⁹/L. Vemurafenib was provided at a dosage of 240 mg 1 or 2 times a day. In 20 patients, vemurafenib was provided for 3 months or more. In 1 case, the effect was not obtained during 1 month of treatment, and the patient died from severe infectious complications during prolonged agranulocytosis. In 21 patients treated with vemurafenib, an increase of ANC was observed and the infectious complications resolved, thus allowing the application of cladribine therapy. After a standard course (0.1 mg/kg per day for 7 days) of cladribine chemotherapy, 18 patients (90%) experienced complete clinical remission and 2 patients (10%) experienced partial remission with residual splenomegaly. In 1 patient, vemurafenib therapy was still ongoing 2 months after initiating therapy. In cases of proven BRAF^V600E mutation, vemurafenib can be successfully used as an effective preliminary therapy in patients with deep neutropenia/agranulocytosis with or without infectious complications before standard therapy with purine analogs.     

Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells

Human endothelial cells release two forms of a plasminogen activator- specific inhibitor: an active form that readily binds to and inhibits plasminogen activators and an inactive or latent form that has no anti- activator activity but which can be activated by denaturation. Latent and active forms of plasminogen activator-specific inhibitor were measured in cultures of human umbilical vein endothelial cells. Latent inhibitor was activated by treatment with 1% sodium dodecyl sulfate (SDS), and both forms were assayed by the 125I-fibrin plate method. After 16 hours, the conditioned medium contained 104.6 U/mL latent inhibitor activity and 2.6 U/mL active inhibitor. The level of each form in the culture medium increased with time although the activity associated with the latent form rose more rapidly: the ratio of latent to active inhibitor activity was 12 at four hours (10.3 U/mL v 0.86 U/mL) and reached 56 at 24 hours (155.3 U/mL v 2.80 U/mL). Intracellular inhibitor activity was associated with the active form only; no additional inhibitor activity was observed following SDS treatment of cell extracts. A decline in active inhibitor activity occurred during incubation at 37 degrees C with a 50% reduction in activity occurring in two hours. Treatment of conditioned medium with 10 U/mL thrombin also resulted in a loss of active inhibitor activity. The latent inhibitor, however, was not affected by either of these conditions. The inhibitor activity lost during incubation at 37 degrees C or thrombin treatment could be regenerated by SDS treatment, suggesting that the loss of the active inhibitor activity represented a conversion of this form to its latent counterpart. Thus, the concentration, stability, and regulation of these two forms of plasminogen activator inhibitor in human endothelial cell cultures differ significantly.     

Recombinant human interleukin-6 induces a rapid and reversible anemia in cancer patients

Initial studies have shown that recombinant human interleukin-6 (rhIL- 6) induces anemia. Until now, the pathophysiologic mechanism of this induced anemia has been unknown. To unravel the underlying mechanism, we examined 15 cancer patients receiving rhIL-6 as an antitumor immunotherapy in a phase II study. rhIL-6 was administered subcutaneously at 150 micrograms once daily for 6 consecutive weeks. Various hematologic and biochemical parameters were measured weekly during rhIL-6 treatment and 4 weeks after rhIL-6 discontinuation. To determine plasma volume and red blood cell (RBC) volume, radioisotope dilution assays with labeled autologous RBCs and with human serum albumin were performed before rhIL-6 administration and on day 8 of rhIL-6 therapy. Hemoglobin levels decreased (mean change +/- SE) 7% +/- 1.5% within 3 days after the start of rhIL-6 therapy (P < .0001) and 19% +/- 2% at week 4. Levels had normalized at follow-up. The plasma volume increased 18% +/- 5% during the first week of rhIL-6 administration (P < .003), whereas RBC volume remained unaffected. The mean RBC corpuscular volume remained unchanged for 2 weeks and then began to decrease slowly, reaching its nadir at week 6 (5% +/- 1%; P < .01). Serum iron levels decreased 65% +/- 12% at week 4 (P < .002) and then returned to initial baseline values. Erythropoietin levels increased rapidly up to 68% at week 3 (P < .0001) and had normalized 4 weeks after rhIL-6 therapy. Levels of serum albumin, prealbumin, and transferrin decreased (P < .0001, P < .003, and P < .0001, respectively), whereas levels of serum amyloid A (P < .003), C-reactive protein, haptoglobin, and alpha-1-antitrypsin (P < .0001) increased during rhIL-6 treatment. All levels returned to pretreatment values after discontinuation of rhIL-6. No alterations in reticulocyte counts, serum lactic dehydrogenase levels, and bilirubin levels were observed. A 6-week regimen of subcutaneous rhIL-6 results in a rapid dilution anemia, caused by an acute and significant increase in plasma volume and followed by hypoferremia. This anemia is reversible after the cessation of rhIL-6 treatment.     

Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane

Fibrinolytic activity was found to be associated with sonicated platelet membranes after separation from cytosol by differential centrifugation. This fibrinolytic activity was attributed to the presence of a plasminogen activator, which was immunochemically identified as urinary-type plasminogen activator (uPA) by antibody neutralization assay, immunoblotting, and immunofluorescence. The molecular weight (mol wt) of this uPA was 54,000 and was present as the single chain form, although a small amount was detected in a higher mol wt complex indicative of a uPA-inhibitor complex. Treatment of membrane preparations with Triton X-100, 3 mol/L KCl, and 0.1 mol/L glycine, (pH 2.3), but not 10 mmol/L ethylenediamine tetraacetic acid (EDTA), removed the uPA from the membrane. This suggests that uPA is a peripheral membrane protein and that metal ions do not mediate protein- membrane association. Immunofluorescent staining revealed the presence of uPA on the outer surface of the platelet in preparations of intact unstimulated platelets. Thus, uPA is associated with the outer leaflet of the platelet membrane and may be involved with the acceleration of thrombus degradation observed with platelet-rich thrombi.     

A clinical,randomized study on the influence of dental whitening on Streptococcus mutans population

Background

Dental whitening with peroxides has been popularized through the at‐home technique, which employs low concentrations of peroxide applied in individual trays. However, there are few clinical trials reporting the effects of its continuous use on oral microbiota. Thus, the purpose of the present clinical, randomized study was to evaluate the influence of at‐home whitening treatment on Streptococcus mutans in saliva, buccal mucosa, and subgingival and supragingival plaque.

Methods

Thirty volunteers were randomly divided into two study groups (N = 15) according to the whitening therapy: G CP, whitening using 10% carbamide peroxide 4 h daily for 21 days; and G HP, whitening using 6% hydrogen peroxide 1.5 h daily for 21 days. Samples from the predetermined locations were collected at three evaluation periods: T1, before; T2, immediately after; and T3, 30 days after the beginning of the treatment. The microbiological evaluation was made using conventional and molecular methods.

Results

Student's t‐test demonstrated a statistically significant decrease in S. mutans population in the subgingival and supragingival plaque for HP samples between T1 and T2 no difference was found between T1 and T3 regardless of the location and the whitening product used (α = 0.05).

Conclusions

Although HP reduced S. mutans during treatment, the levels returned to baseline when assessed 30 days after the treatment.