Covariate-adjusted analysis of the Phase 3 REFLECT study of lenvatinib versus sorafenib in the treatment of unresectable hepatocellular carcinoma.

BACKGROUND: In the Phase 3 REFLECT trial in patients with unresectable hepatocellular carcinoma (uHCC), the multitargeted tyrosine kinase inhibitor, lenvatinib, was noninferior to sorafenib in the primary outcome of overall survival. Post-hoc review revealed imbalances in prognostic variables between treatment arms. Here, we re-analyse overall survival data from REFLECT to adjust for the imbalance in covariates. METHODS: Univariable and multivariable adjustments were undertaken for a candidate set of covariate values that a physician panel indicated could be prognostically associated with overall survival in uHCC. The values included baseline variables observed pre- and post-randomisation. Univariable analyses were based on a stratified Cox model. The multivariable analysis used a "forwards stepwise" Cox model. RESULTS: Univariable analysis identified alpha-fetoprotein (AFP) as the most influential variable. The chosen multivariable Cox model analysis resulted in an estimated adjusted hazard ratio for lenvatinib of 0.814 (95% CI: 0.699-0.948) when only baseline variables were included. Adjusting for post-randomisation treatment variables further increased the estimated superiority of lenvatinib. CONCLUSIONS: Covariate adjustment of REFLECT suggests that the original noninferiority trial likely underestimated the true effect of lenvatinib on overall survival due to an imbalance in baseline prognostic covariates and the greater use of post-treatment therapies in the sorafenib arm. TRIAL REGISTRATION: Trial number: NCT01761266 (Submitted January 2, 2013).

Defective presynaptic choline transport underlies hereditary motor neuropathy.

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies.

DNA methylation and body-mass index: a genome-wide analysis.

BACKGROUND: Obesity is a major health problem that is determined by interactions between lifestyle and environmental and genetic factors. Although associations between several genetic variants and body-mass index (BMI) have been identified, little is known about epigenetic changes related to BMI. We undertook a genome-wide analysis of methylation at CpG sites in relation to BMI. METHODS: 479 individuals of European origin recruited by the Cardiogenics Consortium formed our discovery cohort. We typed their whole-blood DNA with the Infinium HumanMethylation450 array. After quality control, methylation levels were tested for association with BMI. Methylation sites showing an association with BMI at a false discovery rate q value of 0·05 or less were taken forward for replication in a cohort of 339 unrelated white patients of northern European origin from the MARTHA cohort. Sites that remained significant in this primary replication cohort were tested in a second replication cohort of 1789 white patients of European origin from the KORA cohort. We examined whether methylation levels at identified sites also showed an association with BMI in DNA from adipose tissue (n=635) and skin (n=395) obtained from white female individuals participating in the MuTHER study. Finally, we examined the association of methylation at BMI-associated sites with genetic variants and with gene expression. FINDINGS: 20 individuals from the discovery cohort were excluded from analyses after quality-control checks, leaving 459 participants. After adjustment for covariates, we identified an association (q value ≤0·05) between methylation at five probes across three different genes and BMI. The associations with three of these probes--cg22891070, cg27146050, and cg16672562, all of which are in intron 1 of HIF3A--were confirmed in both the primary and second replication cohorts. For every 0·1 increase in methylation ß value at cg22891070, BMI was 3·6% (95% CI 2·4-4·9) higher in the discovery cohort, 2·7% (1·2-4·2) higher in the primary replication cohort, and 0·8% (0·2-1·4) higher in the second replication cohort. For the MuTHER cohort, methylation at cg22891070 was associated with BMI in adipose tissue (p=1·72 × 10(-5)) but not in skin (p=0·882). We observed a significant inverse correlation (p=0·005) between methylation at cg22891070 and expression of one HIF3A gene-expression probe in adipose tissue. Two single nucleotide polymorphisms--rs8102595 and rs3826795--had independent associations with methylation at cg22891070 in all cohorts. However, these single nucleotide polymorphisms were not significantly associated with BMI. INTERPRETATION: Increased BMI in adults of European origin is associated with increased methylation at the HIF3A locus in blood cells and in adipose tissue. Our findings suggest that perturbation of hypoxia inducible transcription factor pathways could have an important role in the response to increased weight in people. FUNDING: The European Commission, National Institute for Health Research, British Heart Foundation, and Wellcome Trust.

Spectrin mutations cause spinocerebellar ataxia type 5.

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Defective mitochondrial mRNA maturation is associated with spastic ataxia.

In human mitochondria, polyadenylation of mRNA, undertaken by the nuclear-encoded mitochondrial poly(A) RNA polymerase, is essential for maintaining mitochondrial gene expression. Our molecular investigation of an autosomal-recessive spastic ataxia with optic atrophy, present among the Old Order Amish, identified a mutation of MTPAP associated with the disease phenotype. When subjected to poly(A) tail-length assays, mitochondrial mRNAs from affected individuals were shown to have severely truncated poly(A) tails. Although defective mitochondrial DNA maintenance underlies a well-described group of clinical disorders, our findings reveal a defect of mitochondrial mRNA maturation associated with human disease and imply that this disease mechanism should be considered in other complex neurodegenerative disorders.

Pancreatic stone protein point-of-care testing can reduce healthcare expenditure in sepsis.

BACKGROUND: Sepsis is a life-threatening organ dysfunction in response to infection. Early recognition and rapid treatment are critical to patient outcomes and cost savings, but sepsis is difficult to diagnose because of its non-specific symptoms. Biomarkers such as pancreatic stone protein (PSP) offer rapid results with greater sensitivity and specificity than standard laboratory tests. METHODS: This study developed a decision tree model to compare a rapid PSP test to standard of care in the emergency department (ED) and intensive care unit (ICU) to diagnose patients with suspected sepsis. Key model parameters included length of hospital and ICU stay, readmission due to infection, cost of sepsis testing, length of antibiotic treatment, antibiotic resistance, and clostridium difficile infections. Model inputs were determined by review of sepsis literature. RESULTS: The rapid PSP test was found to reduce costs by $1688 per patient in the ED and $3315 per patient in the ICU compared to standard of care. Cost reductions were primarily driven by the specificity of PSP in the ED and the sensitivity of PSP in the ICU. CONCLUSIONS: The results of the model indicate that PSP testing is cost saving compared to standard of care in diagnosis of sepsis. The abundance of sepsis cases in the ED and ICU make these findings important in the clinical field and further support the potential of sensitive and specific markers of sepsis to not only improve patient outcomes but also reduce healthcare expenditures.

Economic Evaluation of FebriDx®: A Novel Rapid, Point-of-Care Test for Differentiation of Viral versus Bacterial Acute Respiratory Infection in the United States.

Background: Acute respiratory infections (ARIs) are commonly treated with antibiotics in outpatient settings, but many infections are caused by viruses and antibiotic treatment is therefore inappropriate. FebriDx®, a rapid point-of-care test that can differentiate viral from bacterial infections, can inform antibiotic treatment decisions. Objectives: The primary aim of this study is to conduct a literature-based US economic evaluation of a novel rapid point-of-care test, FebriDx®, that simultaneously measures two key infection biomarkers, C-reactive protein (CRP) and Myxovirus resistance protein A (MxA), to accurately differentiate viral from bacterial infection. Methods: A budget impact model was developed based on a review of published literature on antibiotic prescribing for ARIs in the United States. The model considers the cost of antibiotic treatment, antibiotic resistant infections, antibiotic-related adverse events, and point-of-care testing. These costs were extrapolated to estimate savings on a national level. Results: The expected national cost to treat ARIs under standard of care was US $8.25 billion, whereas the expected national cost of FebriDx point-of-care-guided ARI treatment was US $5.74 billion. Therefore, the expected national savings associated with FebriDx® rapid point-of-care testing was US $2.51 billion annually. Conclusions: FebriDx, a point of care test that can reliably aid in the differentiation of viral and bacterial infections, can reduce antibiotic misuse and, therefore, antibiotic resistant infections. This results in significant cost savings, driven primarily by the reduction in antibiotic resistant infections.

Mendelian randomization: estimation of inpatient hospital costs attributable to obesity.

BACKGROUND: Mendelian Randomization is a type of instrumental variable (IV) analysis that uses inherited genetic variants as instruments to estimate causal effects attributable to genetic factors. This study aims to estimate the impact of obesity on annual inpatient healthcare costs in the UK using linked data from the UK Biobank and Hospital Episode Statistics (HES). METHODS: UK Biobank data for 482,127 subjects was linked with HES inpatient admission records, and costs were assigned to episodes of care. A two-stage least squares (TSLS) IV model and a TSLS two-part cost model were compared to a naïve regression of inpatient healthcare costs on body mass index (BMI). RESULTS: The naïve analysis of annual cost on continuous BMI predicted an annual cost of £21.61 [95% CI £20.33 - £22.89] greater cost per unit increase in BMI. The TSLS IV model predicted an annual cost of £14.36 [95% CI £0.31 - £28.42] greater cost per unit increase in BMI. Modelled with a binary obesity variable, the naïve analysis predicted that obese subjects incurred £205.53 [95% CI £191.45 - £219.60] greater costs than non-obese subjects. The TSLS model predicted a cost £201.58 [95% CI £4.32 - £398.84] greater for obese subjects compared to non-obese subjects. CONCLUSIONS: The IV models provide evidence for a causal relationship between obesity and higher inpatient healthcare costs. Compared to the naïve models, the binary IV model found a slightly smaller marginal effect of obesity, and the continuous IV model found a slightly smaller marginal effect of a single unit increase in BMI.

Mutation of FA2H underlies a complicated form of hereditary spastic paraplegia (SPG35).

Hereditary spastic paraplegia (HSP) describes a heterogeneous group of inherited neurodegenerative disorders in which the cardinal pathological feature is upper motor neurone degeneration leading to progressive spasticity and weakness of the lower limbs. Using samples from a large Omani family we recently mapped a gene for a novel autosomal recessive form of HSP (SPG35) in which the spastic paraplegia was associated with intellectual disability and seizures. Magnetic resonance imaging of the brain of SPG35 patients showed white matter abnormalities suggestive of a leukodystrophy. Here we report homozygous mutations in the fatty acid 2-hydroxylase gene (FA2H) in the original family used to define the SPG35 locus (p.Arg235Cys) as well as in a previously unreported Pakistani family with a similar phenotype (p.Arg53_Ile58del). Measurement of enzyme activity in vitro revealed significantly reduced enzymatic function of FA2H associated with these mutations. These results demonstrate that mutations in FA2H are associated with SPG35, and that abnormal hydroxylation of myelin galactocerebroside lipid components can lead to a severe progressive phenotype, with a clinical presentation of complicated HSP and radiological features of leukodystrophy. (c) 2010 Wiley-Liss, Inc.

Application of a Mapping Function to Estimate Utilities for Ragweed Allergy Immunotherapy Trials.

BACKGROUND: Ragweed pollen sensitivity is a common cause of allergic rhinitis (AR) worldwide. AR symptoms include itchy and runny eyes, sneezing, blocked nose, impaired sleep and social and emotional problems, which can have a significant impact on quality of life. OBJECTIVE: The objective of this analysis was to estimate utilities for two pooled standardised quality (SQ) ragweed sublingual immunotherapy (SLIT) tablet trials by applying a previously developed mapping algorithm. This study validated the algorithm and extended its application to ragweed seasonal allergy trials. The mapping algorithm relates disease-specific quality-of-life scores to preference-based utilities that may be used to calculate quality-adjusted life-years (QALYs) in cost-effectiveness studies. METHODS: A mapping algorithm based on a grass pollen allergy immunotherapy trial, GT-08 (EudraCT no. 2004-000083-27) was applied to pooled data from two ragweed pollen immunotherapy trials, P05233 (EudraCT 2008-003863-38) and P05234 (EudraCT 2008-003864-20) to generate EuroQoL 5-Dimensions, 3-Levels (EQ-5D-3L) utilities from Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) data. RESULTS: The mean utility difference between the SQ ragweed SLIT tablet and placebo was 0.025 [95% confidence interval (CI) 0.011-0.038]. The SQ ragweed SLIT tablet showed an incremental quality-adjusted life-days (QALDs) benefit of 1.900 (95% CI 0.835-2.916) over 75 days. CONCLUSIONS: Application of a previously developed mapping function allowed for the calculation of QALDs associated with the SQ ragweed SLIT tablet. The results showed a QALD benefit of the SQ ragweed SLIT tablet in P05233 and P05234 trials in the treatment of ragweed pollen-induced AR.

A quality-of-life mapping function developed from a grass pollen sublingual immunotherapy trial to a tree pollen sublingual immunotherapy trial.

Aims: Allergic rhinitis is caused by sensitivity to environmental allergens that can significantly impact quality-of-life. The objective of this analysis was to estimate health state utilities and quality-adjusted life days (QALDs) for a tree allergy immunotherapy trial, TT-04 (EudraCT No.2015-004821-15). Health-state utilities are a measure of patient preference for health states and are necessary to derive QALDs for cost-utility analysis. Preference-based utilities were not collected in the TT-04 trial, so a mapping algorithm was developed based on a similar grass allergy immunotherapy trial, GT-08 (EudraCT No. 2004-000083-27), to estimate utilities.Methods: A two-part model was developed to predict utilities for the GT-08 trial and applied to the TT-04 trial to estimate the difference in mean utility and QALDs between SQ tree sublingual immunotherapy (SLIT)-tablet and placebo.Results: Mean utility difference between SQ tree SLIT-tablet and placebo was 0.030 [95% CI = 0.015-0.046] during the birch pollen season (BPS), 0.019 [95% CI = 0.007-0.030] during the tree pollen season (TPS) and 0.018 [95% CI = 0.007-0.030] during the full trial. The treatment showed a QALD benefit of 1.26 [95% CI = 0.619-1.917] during the BPS, 1.90 [95% CI = 0.692-3.047] during the TPS, and 2.47 [95% CI = 0.930-4.101] during the full trial.Limitations: The generalizability of this algorithm is limited to allergy trials containing the same covariates as those present in the model. The analysis also assumes that grass and tree pollen allergy have the same relationship with EQ5D utilities, which is supported by the fact that both grass and tree pollen induce similar symptoms.Conclusions: Application of the mapping function enabled the calculation of QALDs associated with the treatment, with the caveat that data were extrapolated from grass seasonal allergy to tree seasonal allergy. The results showed a significant QALD benefit of the treatment over placebo in treatment of tree pollen-induced rhinoconjunctivitis.

Cigarette smoking reduces DNA methylation levels at multiple genomic loci but the effect is partially reversible upon cessation.

Smoking is a major risk factor in many diseases. Genome wide association studies have linked genes for nicotine dependence and smoking behavior to increased risk of cardiovascular, pulmonary, and malignant diseases. We conducted an epigenome wide association study in peripheral-blood DNA in 464 individuals (22 current smokers and 263 ex-smokers), using the Human Methylation 450 K array. Upon replication in an independent sample of 356 twins (41 current and 104 ex-smokers), we identified 30 probes in 15 distinct loci, all of which reached genome-wide significance in the combined analysis P < 5 × 10(-8). All but one probe (cg17024919) remained significant after adjusting for blood cell counts. We replicated all 9 known loci and found an independent signal at CPOX near GPR15. In addition, we found 6 new loci at PRSS23, AVPR1B, PSEN2, LINC00299, RPS6KA2, and KIAA0087. Most of the lead probes (13 out of 15) associated with cigarette smoking, overlapped regions of open chromatin (FAIRE and DNaseI hypersensitive sites) or/and H3K27Ac peaks (ENCODE data set), which mark regulatory elements. The effect of smoking on DNA methylation was partially reversible upon smoking cessation for longer than 3 months. We report the first statistically significant interaction between a SNP (rs2697768) and cigarette smoking on DNA methylation (cg03329539). We provide evidence that the metSNP for cg03329539 regulates expression of the CHRND gene located circa 95 Kb downstream of the methylation site. Our findings suggest the existence of dynamic, reversible site-specific methylation changes in response to cigarette smoking , which may contribute to the extended health risks associated with cigarette smoking.

Spinocerebellar ataxia type 5.

In 1994, Ranum and colleagues identified a ten-generation American kindred with a relatively mild autosomal dominant form of spinocerebellar ataxia (Ranum et al., 1994). The mutation was mapped to the centromeric region of chromosome 11, and the disorder designated SCA5 (Ranum et al., 1994). Using a multifaceted mapping approach, Ikeda et al. (2006) discovered that ß-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in the American kindred and two additional independently reported SCA5 families. The American and French families have separate in-frame deletions of 39 and 15 bp, respectively, in the third of 17 spectrin repeat motifs. A third mutation, found in a German family, is located in the second calponin homology domain, a region known to bind actin and Arp1. Consistent with Purkinje cell degeneration in SCA5, ß-III spectrin is highly expressed in cerebellar Purkinje cells. TIRF microscopy performed on cell lines transiently transfected with mutant or wild-type spectrin shows that mutant ß-III spectrin fails to stabilize the glutamate transporter EAAT4 at the plasma membrane. Additionally, marked differences in EAAT4 and GluRδ2 were found by protein blot and cell fractionation in SCA5 autopsy tissue. This review summarizes data showing that ß-III spectrin mutations are a novel cause of neurodegenerative disease, which may affect the stabilization or trafficking of membrane proteins.

Alterations in the ankyrin domain of TRPV4 cause congenital distal SMA, scapuloperoneal SMA and HMSN2C.

Spinal muscular atrophies (SMA, also known as hereditary motor neuropathies) and hereditary motor and sensory neuropathies (HMSN) are clinically and genetically heterogeneous disorders of the peripheral nervous system. Here we report that mutations in the TRPV4 gene cause congenital distal SMA, scapuloperoneal SMA, HMSN 2C. We identified three missense substitutions (R269H, R315W and R316C) affecting the intracellular N-terminal ankyrin domain of the TRPV4 ion channel in five families. Expression of mutant TRPV4 constructs in cells from the HeLa line revealed diminished surface localization of mutant proteins. In addition, TRPV4-regulated Ca(2+) influx was substantially reduced even after stimulation with 4alphaPDD, a TRPV4 channel-specific agonist, and with hypo-osmotic solution. In summary, we describe a new hereditary channelopathy caused by mutations in TRPV4 and present evidence that the resulting substitutions in the N-terminal ankyrin domain affect channel maturation, leading to reduced surface expression of functional TRPV4 channels.

Refinement of the locus for distal hereditary motor neuronopathy VII (dHMN-VII) and exclusion of candidate genes.

Distal hereditary motor neuronopathy type seven (dHMN-VII) is an autosomal dominant condition characterized by distal muscular atrophy associated with unilateral or bilateral vocal cord paralysis. We previously mapped the dHMN-VII locus to chromosome 2q14 using a genome-wide linkage scan in a single large pedigree. Here we have performed more detailed microsatellite saturation analysis and also evaluated two new affected individuals not described in the original study. We have significantly refined the extent of the disease locus and show that two distinct regions of chromosome 2q14.2, comprising 9.2 Mb and 4.3 Mb separated by an unusual double recombination event, cosegregate with the disease phenotype. The proximal linked region is now defined by markers D2S3038-D2S160, and the distal region by D2S2970-D2S2969. Sequencing of 15 candidate genes within the critical interval has not yet revealed any pathogenic mutations. Inspection of genomic databases indicates that this refinement of the critical interval by 8.4 Mb reduces the number of candidate genes from approximately 400 to approximately 100.