Retrospective Analysis of Canadian Adults with Tuberous Sclerosis Complex.

BACKGROUND: Our study goal was to characterize the relative frequencies of molecular and phenotypic traits of tuberous sclerosis complex (TSC) in a Canadian adult population. Previous studies have sought to identify TSC-related genotypic and phenotypic trends in pediatric cohorts, but little is known about clinical manifestations and severity when it presents in adults. METHODS: We conducted a retrospective chart review of adult patients seen at the TSC clinic at the University Health Network genetics clinics (Toronto, Ontario) to compare trends in the relative frequency of TSC manifestations with genotype. RESULTS: Fifty-one patients were eligible for this study. Eight patients had a pathogenic/likely pathogenic variant in the tuberous sclerosis complex 1 (TSC1) gene, 18 had a tuberous sclerosis complex 2 (TSC2) pathogenic/likely pathogenic variant, 6 patients had multiple variants identified in TSC1/TSC2 or TSC2/PKD1, 11 had no mutation identified (NMI) and 8 had no genetic testing done. Patients with a pathogenic/likely pathogenic variant in TSC2 presented with an increased involvement of multiple systems and a higher frequency of TSC-related manifestations relative to the other mutation groups. CONCLUSION: Previous studies comparing the wide phenotypic variability with TSC genotype have mainly comprised pediatric cohorts. With a focus on adults, we found trends to be similar across previous literature. An informed multidisciplinary approach should be taken to ensure proper surveillance and management of adults with TSC until a correlation between genotype and phenotype, especially past infancy, is better understood.

A model for the return and referral of all clinically significant secondary findings of genomic sequencing.

Secondary findings (SFs) identified through genomic sequencing (GS) can offer a wide range of health benefits to patients. Resource and capacity constraints pose a challenge to their clinical management; therefore, clinical workflows are needed to optimise the health benefits of SFs. In this paper, we describe a model we created for the return and referral of all clinically significant SFs, beyond medically actionable results, from GS. As part of a randomised controlled trial evaluating the outcomes and costs of disclosing all clinically significant SFs from GS, we consulted genetics and primary care experts to determine a feasible workflow to manage SFs. Consensus was sought to determine appropriate clinical recommendations for each category of SF and which clinician specialist would provide follow-up care. We developed a communication and referral plan for each category of SFs. This involved referrals to specialised clinics, such as an Adult Genetics clinic, for highly penetrant medically actionable findings. Common and non-urgent SFs, such as pharmacogenomics and carrier status results for non-family planning participants, were directed back to the family physician (FP). SF results and recommendations were communicated directly to participants to respect autonomy and to their FPs to support follow-up of SFs. We describe a model for the return and referral of all clinically significant SFs to facilitate the utility of GS and promote the health benefits of SFs. This may serve as a model for others returning GS results transitioning participants from research to clinical settings.

Complications, reoperations and revisions after proximal interphalangeal joint arthroplasty: a systematic review and meta-analysis.

This systematic review and meta-analysis investigates the prevalence of complications, reoperations (surgeries without implant modifications) and revisions (surgeries with implant modifications) after proximal interphalangeal joint arthroplasty with pyrocarbon, metal-polyethylene and silicone implants. Thirty-four articles investigating 1868 proximal interphalangeal joints were included. Implant-related complications were associated with 14%, 10% and 11% of the pyrocarbon, metal-polyethylene and silicone implants, respectively, yet these rates were not significantly different from one another. Silicone implants showed more finger deviations (3%) and instabilities (2%) compared with the other implants. Reoperations were fewer for silicone arthroplasties (1%) compared with pyrocarbon (7%) and metal-polyethylene implants (10%). The revision rates of 4%, 3% and 2% were similar for pyrocarbon, metal-polyethylene and silicone implants. Our results indicate that silicone implants remain a valuable option for the treatment of stable proximal interphalangeal joints. Surface replacing implants might be better to correct unstable or deviated proximal interphalangeal joints, although they are associated with a higher risk of reoperations.

OTX2 mutations cause autosomal dominant pattern dystrophy of the retinal pigment epithelium.

PURPOSE: To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment epithelium (RPE) in two families. METHODS AND RESULTS: Two families with autosomal-dominant PD were identified. Eight members of family 1 (five affected) were subjected to whole-genome SNP genotyping; multipoint genome-wide linkage analysis identified 7 regions of potential linkage, and genotyping four additional individuals from family 1 resulted in a maximum logarithm of odds score of 2.09 observed across four chromosomal regions. Exome sequencing of two affected family 1 members identified 15 shared non-synonymous rare coding sequence variants within the linked regions; candidate genes were prioritised and further analysed. Sanger sequencing confirmed a novel heterozygous missense variant (E79K) in orthodenticle homeobox 2 (OTX2) that segregated with the disease phenotype. Family 2 with PD (two affected) harboured the same missense variant in OTX2. A shared haplotype of 19.68 cM encompassing OTX2 was identified between affected individuals in the two families. Within the two families, all except one affected demonstrated distinct 'patterns' at the macula. In vivo structural retinal imaging showed discrete areas of RPE-photoreceptor separation at the macula in all cases. Electroretinogram testing showed generalised photoreceptor degeneration in three cases. Mild developmental anomalies were observed, including optic nerve head dysplasia (four cases), microcornea (one case) and Rathke's cleft cyst (one case); pituitary hormone levels were normal. CONCLUSIONS: This is the first report implicating OTX2 to underlie PD. The retinal disease resembles conditional mice models that show slow photoreceptor degeneration secondary to loss of Otx2 function in the adult RPE.

Basal cell signaling by p63 controls luminal progenitor function and lactation via NRG1.

The mammary epithelium is organized as a bilayer of luminal and basal/myoepithelial cells. During pregnancy, the luminal compartment expands for milk production, while basal cells are thought to provide structural and contractile support. Here, we reveal a pregnancy-specific role of basal epithelia as a central coordinator of lactogenesis. We demonstrate that genetic deletion of the transcription factor p63 (Trp63) gene exclusively within basal cells of the adult gland during pregnancy leads to dramatic defects in luminal cell proliferation and differentiation, resulting in lactation failure. This phenotype is explained by direct transcriptional activation of the epidermal growth factor family ligand gene Nrg1 by p63 selectively in basal cells, which is required for luminal ERBB4/STAT5A activation and consequent luminal progenitor cell maturation. Thus, paracrine basal-to-luminal cell signaling, controlled by p63 via NRG1, orchestrates the entire lactation program. Collectively, these findings redefine the paradigm for cellular interactions specifying the functional maturation of the mammary gland.

Mcl-1 and FBW7 control a dominant survival pathway underlying HDAC and Bcl-2 inhibitor synergy in squamous cell carcinoma.

Effective targeted therapeutics for squamous cell carcinoma (SCC) are lacking. Here, we uncover Mcl-1 as a dominant and tissue-specific survival factor in SCC, providing a roadmap for a new therapeutic approach. Treatment with the histone deacetylase (HDAC) inhibitor vorinostat regulates Bcl-2 family member expression to disable the Mcl-1 axis and thereby induce apoptosis in SCC cells. Although Mcl-1 dominance renders SCC cells resistant to the BH3-mimetic ABT-737, vorinostat primes them for sensitivity to ABT-737 by shuttling Bim from Mcl-1 to Bcl-2/Bcl-xl, resulting in dramatic synergy for this combination and sustained tumor regression in vivo. Moreover, somatic FBW7 mutation in SCC is associated with stabilized Mcl-1 and high Bim levels, resulting in a poor response to standard chemotherapy but a robust response to HDAC inhibitors and enhanced synergy with the combination vorinostat/ABT-737. Collectively, our findings provide a biochemical rationale and predictive markers for the application of this therapeutic combination in SCC.

Physical association of HDAC1 and HDAC2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma.

Squamous cell carcinoma (SCC) is a treatment-refractory subtype of human cancer arising from stratified epithelium of the skin, lung, esophagus, oropharynx, and other tissues. A unifying feature of SCC is high-level expression of the p53-related protein p63 (TP63) in 80% of cases. The major protein isoform of p63 expressed in SCC is ΔNp63α, an N-terminally truncated form which functions as a key SCC cell survival factor by mechanisms that are unclear. In this study, we show that ΔNp63α associates with histone deacetylase 1 (HDAC1) and HDAC2 to form an active transcriptional repressor complex that can be targeted to therapeutic advantage. Repression of proapoptotic Bcl-2 family member genes including p53 upregulated modulator of apoptosis (PUMA) by p63/HDAC is required for survival of SCC cells. Cisplatin chemotherapy, a mainstay of SCC treatment, promotes dissociation of p63 and HDAC from the PUMA promoter, leading to increased histone acetylation, PUMA activation, and apoptosis. These effects are recapitulated upon targeting the p63/HDAC complex selectively with class I/II HDAC inhibitors using both in vitro and in vivo models. Sensitivity to HDAC inhibition is directly correlated with p63 expression and is abrogated in tumor cells that overexpress endogenous Bcl-2. Together, our results elucidate a mechanism of p63-mediated transcriptional repression and they identify the ΔNp63α/HDAC complex as an essential tumor maintenance factor in SCC. In addition, our findings offer a rationale to apply HDAC inhibitors for SCC treatment.

A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma.

The p53 tumor suppressor, a central mediator of chemosensitivity in normal cells, is functionally inactivated in many human cancers. Therefore, a central challenge in human cancer therapy is the identification of pathways that control tumor cell survival and chemosensitivity in the absence of functional p53. The p53-related transcription factors p63 and p73 exhibit distinct functions­p73 mediates chemosensitivity while p63 promotes proliferation and cell survival­and are both overexpressed in squamous cell carcinomas (SCCs). However, how p63 and p73 interact functionally and govern the balance between prosurvival and proapoptotic programs in SCC remains elusive. Here, we identify a microRNA-dependent mechanism of p63/p73 crosstalk that regulates p53-independent survival of both human and murine SCC. We first discovered that a subset of p63-regulated microRNAs target p73 for inhibition. One of these, miR-193a-5p, expression of which was repressed by p63, was activated by proapoptotic p73 isoforms in both normal cells and tumor cells in vivo. Chemotherapy caused p63/p73-dependent induction of this microRNA, thereby limiting chemosensitivity due to microRNA-mediated feedback inhibition of p73. Importantly, inhibiting miR-193a interrupted this feedback and thereby suppressed tumor cell viability and induced dramatic chemosensitivity both in vitro and in vivo. Thus, we have identified a direct, microRNA-dependent regulatory circuit mediating inducible chemoresistance, whose inhibition may provide a new therapeutic opportunity in p53-deficient tumors.

The integrin alpha(v)beta(3-5) ligand MFG-E8 is a p63/p73 target gene in triple-negative breast cancers but exhibits suppressive functions in ER(+) and erbB2(+) breast cancers.

The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified downregulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LCM) tissues. We first identified MFG-E8 downregulation in invasive lesions in transgenic mammary tumor models, which were confirmed in LCM-isolated human invasive ductal carcinomas compared with patient-matched normal tissues. In situ analyses of MFG-E8 expression in estrogen receptor (ER) positive cases confirmed its downregulation during breast cancer progression and small inhibitory MFG-E8 RNAs accelerated ER(+) breast cancer cell proliferation. MFG-E8 also decreased in erbB2(+) human cancers and erbB2 transgenic mice lacking MFG-E8 showed accelerated tumor formation. In contrast, MFG-E8 expression was present at high levels in triple-negative (ER(-), PgR(-), erbB2(-)) breast cancers, cell lines, and patient sera. Knockdown, chromatin immunoprecipitation, and reporter assays all showed that p63 regulates MFG-E8 expression, and MFG-E8 knockdowns sensitized triple-negative breast cancers to cisplatin treatment. Taken together, our results show that MFG-E8 is expressed in triple-negative breast cancers as a target gene of the p63 pathway, but may serve a suppressive function in ER(+) and erbB2(+) breast cancers. Its potential use as a serum biomarker that contributes to the pathogenesis of triple-negative breast cancers urges continued evaluation of its differential functions.

Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation.

In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and represses expression of P21CIP1 as part of a heterodimeric complex with Miz1. In vivo, expression of ZBTB4 is downregulated in advanced stages of multiple human tumours. In cell culture, depletion of ZBTB4 promotes cell cycle arrest in response to activation of p53 and suppresses apoptosis through regulation of P21CIP1, thereby promoting long-term cell survival. Our data suggest that Zbtb4 is a critical determinant of the cellular response to p53 activation and reinforce the notion that p21Cip1 can provide an essential survival signal in cells with activated p53.

Post-SELEX chemical optimization of a trypanosome-specific RNA aptamer.

African trypanosomes are the causative agent of sleeping sickness. The therapeutics used to control and treat the disease are very ineffective and thus, the development of improved drugs is urgently needed. Recently, new strategies for the design of novel trypanocidals have been put forward. Among them are techniques that rely on parasite-specific RNA aptamers. One approach involves the aptamer-directed transport of lytic compounds to the lysosome of the parasite. The aptamer has been termed 2-16 RNA and here we report the optimization of the RNA for its applications in vivo. To convert aptamer 2-16 into a serum-stable reagent 2'-deoxy-2'-F- and/or 2'-deoxy-2'-NH(2)-uridine- and cytidine-substituted RNAs were generated. While 2'-NH(2)-dC/dU-modified RNAs were RNase-resistant, they were functionally inactive. By contrast, 2'-F-dC/dU-substituted 2-16 RNA retained its ability to bind to live trypanosomes (K(d)=45 nM) and was routed to the lysosome identically to unmodified RNA. 2'-F-dC/dU-substituted 2-16 RNA is thermostable (T(m)=75 degrees C) and has a serum half-life of 3.4 days. Furthermore, aptamer 2-16 was site-specifically PEGylated to increase its serum retention time. Conjugation with PEG polymers < or = 10 kDa only marginally impacted the binding characteristics of the RNA, while the addition of higher molecular mass PEG molecules resulted in non-functional aptamers. Together, the data provide optimized conjugation chemistries for the large-scale production of substituted aptamer 2-16 preparations with improved in vivo functionality.

p300 protein acetyltransferase activity suppresses systemic lupus erythematosus-like autoimmune disease in mice.

Conditional knock-in mice expressing a histone acetyltransferase-deficient version of the transcriptional coregulator p300 exclusively in B lymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestations, such as splenomegaly, glomerulonephritis, vasculitis, deposition of immune complexes, and production of autoantibodies against dsDNA. Aged mice show a severe reduction of transitional and marginal zone B cells and generate aberrant mature B cells. These B cells show diminished proliferation in response to stimulation of the BCR, but respond normally to other stimuli. Yet, the mice mount a normal primary immune response against a T-dependent Ag. In contrast, the memory response is impaired. In addition, serum Ig levels, in particular IgG2b, are increased. We conclude that p300 acetyltransferase activity is essential for maintaining self-tolerance of B lymphocytes. These findings support the concept of treating lupus with inhibitors of protein deacetylases and point to B cells as a critical target of these drugs.

The influence of injection rate on the hypnotic effect of propofol during anesthesia: a randomized trial.

OBJECTIVE: Previous studies suggested that slow injection of propofol may increase the hypnotic effect during induction of anesthesia. The aim of the present study was therefore to investigate whether injection rate of propofol has an influence on its maximum effect. DESIGN: Randomized, single-blind trial. SETTING: This study has been carried out in the operating rooms of a university hospital. An anesthesiologist and a resident performed the study with the aid of changing nursing staff. PARTICIPANTS: We investigated 99 unpremedicated patients aged 18 to 60 years with American Society of Anesthesiologists (ASA) physical status 1-3. INTERVENTIONS: Anesthesia was induced by intravenous injection of propofol (2 mg/kg). Propofol was manually injected in group 1 over a period of 5 s; in group 2 (120-s injection interval), and in group 3 (240-s injection interval), propofol was administered by an injection pump. After loss of consciousness, mask ventilation was performed with 100% oxygen. Bispectral index (BIS) was used to measure the hypnotic effect of propofol. After the decrease of BIS to the minimum value (i.e., maximum hypnotic effect) and the following increase of BIS to 60, the study period was finished and anesthesia was performed according to clinical criteria. OUTCOME MEASURES: We analyzed whether injection speed has an influence on the maximum hypnotic effect of a given dose of propofol (2 mg/kg). RESULTS: BIS(min) marks the maximum electroencephalogram (EEG) effect of the propofol bolus as measured by the BIS. The lowest mean BIS(min) was measured in group 1 (28.7 +/- 10.3). In group 2, BIS(min) was 33.0 (+/-13.9), and in group 3, BIS(min) was 36.4 (+/-11.0). There were no significant differences between group 2 and groups 1 or 3, but there were significant differences between groups 1 and 3. In group 1, BIS(min) was reached after 102.91 s (+/-44.20), in group 2 after 172.33 s (+/-29.76), and in group 3 after 274.21 s (+/-45.40). These differences were statistically significant for all comparisons. In summary, the lowest value for BIS(min) was achieved in the group with the fastest rate of propofol injection (group1, 5 s). The highest BIS(min) was obtained in the group with the slowest rate of injection (group 3, 240 s). The hemodynamic parameters were not significantly different among groups. CONCLUSIONS: The hypnotic peak effect of propofol is lower with extremely slow injection (240 s versus 5 s). For clinically usual injection rates (5 s and 120 s), there was no significant difference in propofol peak effect.

Managing elevated intracranial pressure.

PURPOSE OF REVIEW: Elevated intracranial pressure is one of the major deteriorating factors in patients with intracerebral lesions. Therefore, every year many experimental and clinical studies are performed to identify the best method for managing elevated intracranial pressure in head-injured patients. The current review summarizes the most important recent findings for the treatment of increased intracranial pressure. RECENT FINDINGS: The currently most discussed treatments of elevated intracranial pressure are the use of hypertonic saline, which seems to be equal to mannitol, the use of hypothermia, and the performance of decompressive craniectomy. SUMMARY: The treatment strategy to manage increased intracranial pressure includes decisions about anaesthetics, ventilation, head and body position, hypothermia, the use of osmotic drugs and surgical procedures. Propofol seems to be suitable for the sedation of patients with elevated intracranial pressure. Sudden increases in intracranial pressure can be treated using hyperosmotic agents, high-dose thiopental, or short episodes of mild hyperventilation. Surgical decompression of the cranium seems to improve the outcome in patients below the age of 50 years, especially children.