Epilepsy genetics: Current knowledge, applications, and future directions.

The rapid pace of disease gene discovery has resulted in tremendous advances in the field of epilepsy genetics. Clinical testing with comprehensive gene panels, exomes, and genomes are now available and have led to higher diagnostic rates and insights into the underlying disease processes. As such, the contribution to the care of patients by medical geneticists, neurogeneticists and genetic counselors are significant; the dysmorphic examination, the necessary pre- and post-test counseling, the selection of the appropriate next-generation sequencing-based test(s), and the interpretation of sequencing results require a care provider to have a comprehensive working knowledge of the strengths and limitations of the available testing technologies. As the underlying mechanisms of the encephalopathies and epilepsies are better understood, there may be opportunities for the development of novel therapies based on an individual's own specific genotype. Drug screening with in vitro and in vivo models of epilepsy can potentially facilitate new treatment strategies. The future of epilepsy genetics will also probably include other-omic approaches such as transcriptomes, metabolomes, and the expanded use of whole genome sequencing to further improve our understanding of epilepsy and provide better care for those with the disease.

Circulating tumor DNA predicts survival in patients with resected high-risk stage II/III melanoma.

Background: Patients with high-risk stage II/III resected melanoma commonly develop distant metastases. At present, we cannot differentiate between patients who will recur or those who are cured by surgery. We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma. Patients and methods: We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial. Results: Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples. Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79-5.47; P < 0.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80-5.79; P < 0.0001) versus those with undetectable ctDNA. Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83-5.83, P < 0.0001; DMFI: HR 3.45, 95% CI 1.88-6.34, P < 0.0001). Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%-55%) versus 65% (95% CI 56%-72%) for those with undetectable ctDNA. Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40-4.96); P = 0.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32-4.74, P = 0.005). Conclusion: ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy. Clinical trial number: ISRCTN 81261306.

Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial.

Background: Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers. We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence. Patients and methods: Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation. The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI). Tumour and blood were analysed for prognostic and predictive markers. Results: Patients (n=1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18-88 years). With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation]. OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82-1.16, P = 0.78). At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74-0.99, P = 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78-1.07, P = 0.25). Forty four percent of 682 melanomas assessed had a BRAFV600 mutation. In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P = 0.06). BRAF mutation positivity trended towards better OS with bevacizumab (P = 0.21). Conclusions: Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS. BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab. Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64.

Late-onset Leclercia adecarboxylata bacteraemia in a premature infant in the NICU.

UNLABELLED: Late-onset sepsis is a unique entity in the neonatal intensive care unit (NICU), as organisms involved are, by definition, nosocomial. As such, a limited number of microbes are characteristically involved. Leclercia adecarboxylata is a gram-negative bacillus rarely cultured in a clinical context, with the few published cases primarily involving immunocompromised adults. We present an ex-26-week newborn girl who developed late-onset sepsis with Leclercia adecarboxylata bacteraemia in the NICU. The infection was successfully treated with gentamicin and cefotaxime. This is the fifth paediatric report of Leclercia adecarboxylata infection, and the first in a neonate. The case raises the possibility that prior courses of antibiotics may have predisposed this individual to a rare infection essentially limited to immunocompromised individuals. CONCLUSION: Leclercia adecarboxylata is a rare infection, particularly in immunocompetent individuals. In neonates, the clinical course can be good with timely initiation of appropriate antibiotics.

Outcome of endovenous laser therapy for saphenous reflux and varicose veins: medium-term results assessed by ultrasound surveillance.

OBJECTIVE: To assess the efficacy of endovenous laser therapy (EVLT) for treating saphenous reflux associated with varicose veins. DESIGN: Out-patient treatment by EVLT with an 810nm laser wavelength with results assessed by ultrasound surveillance. PATIENTS: 361 patients who received EVLT for 509 incompetent saphenous veins over a five-year period. METHODS: EVLT was used for proximal saphenous veins and ultrasound-guided sclerotherapy (UGS) for distal saphenous veins and tributaries. Control of reflux and occlusion or obliteration of the saphenous veins was assessed by serial ultrasound studies. Univariate Kaplan-Meier life table analysis showed cumulative primary and secondary success rates, and multivariate Cox regression analysis assessed covariates that could be associated with increased risk of ultrasound failure. RESULTS: Life table analysis showed primary success at four years in 76% (95% CI 56-87%) and secondary success at four years after further treatment of recurrence by UGS in 97% (95% CI 93-99%). Cox regression analysis showed a non-significant trend towards worse primary success in male patients and worse results for older patients and limbs with clinical CEAP categories C4-6. Cox regression showed significantly worse secondary success for limbs with clinical CEAP C4-6. CONCLUSIONS: EVLT effectively controls saphenous reflux particularly with ultrasound surveillance to detect early recurrence that can be treated by UGS. Modifications in technique may be required to improve the late primary success rate.

Factors affecting the risk of deep venous occlusion after ultrasound-guided sclerotherapy for varicose veins.

OBJECTIVE: To determine which covariates predisposed to deep venous occlusion (DVO) after ultrasound-guided sclerotherapy (UGS) for varicose veins. DESIGN: Ultrasound scans before and at 3 to 7 days after UGS to detect post-procedure deep venous occlusion. MATERIALS: A consecutive series of 1931 treatment sessions in 852 patients treated by a technique for UGS. METHODS: Ultrasound examination of the full length of axial deep veins above and below knee before and soon after every procedure. Crude chi(2) analysis of all covariates allowed selection of those that showed apparent significant influence. Logistic regression analysis of these then determined which independently predisposed to post-procedure deep venous occlusion. RESULTS: Deep venous occlusion was only observed after UGS using foamed sclerosant and occurred following 28 treatment sessions. No significant difference for risk of deep venous occlusion was observed for patient characteristics or which veins were treated. Logistic regression analysis showed significant independent increased risk in a limb from using highly diluted or undiluted sclerosant (OR 0.55; 95% CI 0.19 to 1.59 for 0.6-1.0% solution, OR 10.45; 95% CI 3.12 to 34.99 for 2-2.3% and OR 0.36; 95% CI 0.07 to 1.74 for 3% solution), treating veins >or=5mm diameter (OR 3.70; 95% CI 1.23 to 11.13) and injecting >or=10 ml of foamed sclerosant for a limb (OR 3.64; 95% CI 1.21 to 10.90). CONCLUSIONS: The risk of deep venous occlusion after UGS in this series was lower when using highly diluted or undiluted sclerosant, when treating veins less than 5mm in diameter and when restricting the volume of foam injected to less than 10 ml.

Biomechanical implications of the negative heel rocker sole shoe: gait kinematics and kinetics.

Rocker sole shoes are commonly prescribed to diabetic patients with insensate feet. Recent passage of the therapeutic shoe bill has drawn an increased focus to prescription rehabilitative footwear. The purpose of this work is to investigate the dynamics of lower extremity joints (hip, knee and ankle) with the application of a negative heel rocker sole shoe under controlled lab conditions. Forty normal adults volunteered for gait evaluations using controlled baseline and prescription negative heel rocker sole shoes. Three-dimensional motion analysis techniques were used to acquire kinematic and kinetic data using a six-camera Vicon 370 motion system and two AMTI force plates. No significant change in walking speed or stride length was seen with the negative heel rocker shoe, although cadence was increased. The most significant kinematic changes with the application of the negative heel shoe occurred at the ankle in the sagittal plane with increased plantarflexion at terminal stance. Significant hip and knee changes were also noted with increased mid-stance hip extension and knee flexion. The most significant kinetic effects were seen in the transverse plane followed by changes in the sagittal and coronal planes. Changes in power were mostly noted in the sagittal plane. Other statistically significant changes in gait kinematics and kinetics were observed, although the magnitudes and durations were limited and as a result were not considered clinically significant. The study results indicated the negative heel rocker shoe significantly altered proximal joint metrics (hip and knee). The most significant distal joint alterations were seen in sagittal plane ankle kinetics. These kinematic and kinetic changes, along with previously studied effects of pressure relief at the metatarsal heads, should aid medical professionals in prescribing prophylactic footwear.

Organisation of the mouse and human 5T4 oncofoetal leucine-rich glycoprotein genes and expression in foetal and adult murine tissues.

The human 5T4 oncotrophoblast leucine-rich glycoprotein may contribute to the process of placentation or metastasis by modulating cell adhesion, shape and motility. To understand better the role of 5T4 in development and cancer, the gene structure has been elucidated from both human and mouse genomic clones and mRNA expression has been studied in foetal and adult mouse tissues. The protein coding region is located within the second of two exons, the first exon comprising solely of 5'-untranslated region. Upstream there are no TATA or CAAT boxes, but there are a number of potential Sp1 binding sites. The murine and human proteins show a homologous domain organisation of the leucine rich repeats (LRR) and associated N- and C-terminal flanking regions, although the hydrophilic sequence which intervenes between the two LRR domains contains six additional amino acids in the mouse. The signal peptide, transmembrane region and cytoplasmic tail sequences are identical as are 6 out of the 7 potential N-linked glycosylation sites. Mouse 5T4 transcripts are abundant in placenta and also highly expressed in embryos while in adult tissues transcripts are restricted to brain and ovary. These patterns of expression and the genomic organisation are discussed in relation to possible function and other recently described LRR containing proteins.

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Putative determinants of arterial wall compliance in NIDDM.

Aortoiliac arterial wall compliance was measured in apparently healthy subjects and in patients with non-insulin-dependent diabetes (NIDDM), on diet alone, who had no clinical evidence of peripheral arterial disease. Compliance was significantly lower in patients with diabetes. The two clinical groups were combined to provide as wide a range of blood glucose values as possible. After allowing for the influence of age, there were significant negative correlations between compliance and free fatty acid and insulin levels. These were almost completely accounted for by differences in blood glucose levels. Therefore, arterial compliance was best predicted on the basis of age and the area under the blood glucose curve.

Doppler ultrasound recognition of preclinical changes in arterial wall in diabetic subjects: compliance and pulse-wave damping.

Doppler ultrasound was used to detect early changes in arteries of the legs by two independent techniques. Pulse-wave velocity was measured to calculate arterial wall compliance and Fourier analysis was used to measure damping of the pulse-wave forms. Ten non-insulin-dependent diabetic men with no clinical evidence of peripheral arterial disease had significantly lower compliance and greater pulse-wave damping than 10 matched nondiabetic control subjects. There was a good correlation between the results for the two different techniques.

Evaluating clinical teachers: does the learning environment matter?

Teaching evaluations are an important part of promotion reviews. This study of the effect of learning environment on evaluations found ratings from students in ambulatory settings were higher than were those from inpatient settings.

Prevention of venous thrombosis with elastic stockings during long-haul flights: the LONFLIT 5 JAP study.

The aim of this study was to evaluate deep venous thrombosis (DVT) prophylaxis with specific elastic stockings in long-haul flights (11-13 hours), in high-risk subjects. A group of 300 subjects was included; 76 were excluded for several problems including concomitant treatments; 224 were randomized into two groups (stockings vs. controls) to evaluate prophylaxis with below-knee stockings. An exercise program was used in both groups. Scholl (UK) Flight Socks (14-17 mmHg of pressure at the ankle) were used. DVT was diagnosed with ultrasound scanning. The femoral, popliteal, and tibia] veins were scanned before and within 90 minutes after the flights. Of the 205 included subjects, 102 controls and 103 treated subjects completed the study. Drop-outs were due to flight connection problems. Age, gender, and risk distributions were comparable in the two groups. In the treatment group (103 subjects; mean age, 42; SD 9; M:F, 55:48), one limited, distal DVT was observed (0.97%). In the control group (102 subjects; mean age, 42.1; SD 10.3; M:F, 56:46), six subjects (5.8%) had a DVT. There were no superficial thromboses. The difference in DVT incidence is significant (p<0.0025; six times greater in the control group). Intention-to-treat analysis counts 18 failures in the control group (12 lost to follow-up + six thromboses) of 112 subjects (15.8%) versus eight failures (7.3%) in the treatment group (p<0.05). The tolerability of the stockings was very good and there were no complaints or side effects. All events were asymptomatic. Considering these observations, Scholl Flight Socks are effective in reducing the incidence of DVT in high-risk subjects.

Semi-closed, ex-situ, non-reversed or reversed autogenous vein grafting. A technique for femoro-distal arterial bypass.

A technique is described for femoro-distal grafting in which vein is removed through very small incisions using the Mayo vein stripper, valves are ablated with the vein "ex-situ", and the graft is inserted non-reversed or reversed according to arbitrary criteria as to its diameter, with the distal anastomosis performed under tourniquet control. Early experience with 35 consecutive vein grafts showed no serious technical problems, satisfactory results and excellent patient acceptance.

Early post-operative ankle pressure indices help predict late patency rates after femoro-distal vein bypass grafting.

A prospective graft surveillance programme included 115 femorodistal autogenous vein bypass grafts that were patent when studied by measurements of ankle/brachial pressure indices (ABIs) at 3-5 weeks after operation. The grafts were followed for up to 3 years to determine whether early ABIs helped to predict subsequent failures, defined as graft thrombosis or graft or anastomotic stenosis causing diameter reduction greater than 75% treated by surgery or balloon dilatation. Receiving operating characteristics curves showed that resting ABIs predicted failure better than post-exercise ABIs or the differences between the two, and that an early resting ABI less than 0.85 was the optimal value to predict failure. Primary patency rates at 2 years were 88% for resting ABI greater than or equal to 0.85 and 36% for resting ABI less than 0.85 (p less than 0.0005). Most stenoses occurred in the grafts and not at anastomoses. Arteriographic grading of crural artery outflow was not significantly different for successful or failed grafts or for grafts with resting ABI greater than or equal to 0.85 or less than 0.85. The observation that late graft failure was 3-4 times more likely if the early post-operative ABI was low suggests that most graft failures result from faults relating to the operation.

In vivo heparin response and consumption in patients before and during arterial reconstructive surgery.

This study investigated the effects of heparin in patients with peripheral arterial disease, before and during operation. The relation between heparin dosage and heparin response and consumption were calculated from changes in the activated clotting time (ACT). Before operation, 38 patients were each given 10,000 units of heparin by intravenous injection and ACT was measured after 5, 10, 20, 30 and 60 minutes. Assuming a linear relation between heparin dosage and ACT, a dose of heparin was calculated which should double the normal ACT. This was given during operation in 21 patients while a standard dose of 7,500 units of heparin was given to another 10 patients. ACT was measured during operation at the same time intervals as before operation. ACT was always at its highest level at the 5 minute test and was always still raised at 60 minutes, both before and during operation. No significant correlation was found between ACT, heparin response or heparin consumption, and the patients' ages, sex, body weights and creatinine levels, either before or during operation. It was not possible to predict the intraoperative response from the preoperative test. It is concluded that the heparin effect should be monitored during operation in each patient if the best dose response is to be obtained.

Giacomini's observations on the superficial veins of the abdominal limb and principally the external saphenous.

Carlo Giacomini, later Professor of Anatomy at the University of Turin, Italy, presented a thesis on superficial and deep lower limb venous anatomy in July 1873. This resulted in his name being associated with a vein that he described in detail that passes up deep to the fascia on the back of the thigh. However, the precise nature of his detailed and insightful observations have not previously been presented, at least for the past century. The Authors were able to find and translate the original manuscript, and the first section on the superficial veins is presented here. Giacomini documented the several variations in the origin and terminations, and anterograde and retrograde flow in this vein that have only recently been rediscovered by duplex ultrasound scanning. Much can be learned from his descriptions by all who are involved in treating chronic venous disease.

Venous thromboembolism from air travel: the LONFLIT study.

The LONFLIT study was planned to evaluate the incidence of deep venous thrombosis (DVT) occurring as a consequence of long flights. In the Lonflit study 355 subjects at low-risk for DVT and 389 at high-risk were studied. Low-risk subjects had no cardiovascular disease and used no drugs. All flights were in economy class. The average flight duration was 12.4 hours (range, 10-15 hr). The mean age of the studied subjects was 46 years (range 20-80 yr, SD 11; 56% males). DVT diagnosis was made by ultrasound scans after the flights (within 24 hours). In low-risk subjects no events were recorded while in high-risk subjects 11 had DVT (2.8%) with 13 thromboses in 11 subjects and 6 superficial thromboses (total of 19 thrombotic events in 389 patients [4.9%]). In the Lonflit2 study the authors studied 833 subjects (randomized into 422 control subjects and 411 using below-knee stockings). Mean age was 44.8 years (range, 20-80 yr, SD 12; 57% males). The average flight duration was 12.4 hours. Scans were made before and after the flights. In the control group there were 4.5% of subjects with DVT while only 0.24% of subjects had DVT in the stockings group. The difference was significant. The incidence of DVT observed when subjects were wearing stockings was 18.75 times lower than in controls. Long-haul flights are associated to DVT in some 4-5% of high-risk subjects. Below-knee stockings are beneficial in reducing the incidence of DVT.