NIHR Cambridge Biomedical Research Centre Research Articles

Cortical and Striatal Reward Processing in Parkinson's Disease Psychosis.

Psychotic symptoms frequently occur in Parkinson’s disease (PD), but their pathophysiology is poorly understood. According to the National Institute of Health RDoc programme, the pathophysiological basis of neuropsychiatric symptoms may be better understood in terms of dysfunction of underlying domains of neurocognition in a trans-diagnostic fashion. Abnormal cortico-striatal reward processing has been proposed as a key domain contributing to the pathogenesis of psychotic symptoms in schizophrenia. This theory has received empirical support in the study of schizophrenia spectrum disorders and preclinical models of psychosis, but has not been tested in the psychosis associated with PD. We, therefore, investigated brain responses associated with reward expectation and prediction error signaling during reinforcement learning in PD-associated psychosis. An instrumental learning task with monetary gains and losses was conducted during an fMRI study in PD patients with (n = 12), or without (n = 17), a history of psychotic symptoms, along with a sample of healthy controls (n = 24). We conducted region of interest analyses in the ventral striatum (VS), ventromedial prefrontal and posterior cingulate cortices, and whole-brain analyses. There was reduced activation in PD patients with a history of psychosis, compared to those without, in the posterior cingulate cortex and the VS during reward anticipation (p < 0.05 small volume corrected). The results suggest that cortical and striatal abnormalities in reward processing, a putative pathophysiological mechanism of psychosis in schizophrenia, may also contribute to the pathogenesis of psychotic symptoms in PD. The finding of posterior cingulate dysfunction is in keeping with prior results highlighting cortical dysfunction in the pathogenesis of PD psychosis.

Individualised monitoring of patients with metastatic melanoma using plasma DNA

BackgroundCirculating tumour DNA (ctDNA) is released by cancer cells into the bloodstream, which can be analysed via liquid biopsy. Analysis of liquid biopsy samples provides a real-time snapshot of tumour burden. After treatment, ctDNA concentrations can be low, making detection challenging. To study clonal evolution during treatment in patients with melanoma with high sensitivity, we sought to maximise the number of mutations targeted through individualised next-generation sequencing panel design. Methods72 patients with stage III or IV melanoma were recruited to MelResist, a translational, multicentre research study. Serial plasma samples were taken from patients at monthly intervals during treatment (median 6·7 samples per patient). Clinical events were scored according to Response Evaluation Criteria In Solid Tumors (RECIST) (version 1.1) criteria. Exome and targeted sequencing were carried out on tumour samples at baseline and progression for nine patients, whose identified mutations were used to design an individualised targeted sequencing panel. FindingsMultiple mutations per patient were tracked to monitor response to therapy. The percentage change in ctDNA mutant allele fraction after treatment initiation agreed with RECIST response for seven out of eight evaluable patients. ctDNA concentration strongly correlated with lactate dehydrogenase (LDH) concentration, a currently used measure of melanoma tumour burden (r2=0·64, p=9·93 × 10−7). ctDNA dynamics were compared against rising LDH, which showed a median lead-time to biochemical progression (rising LDH) of 70 days (IQR 28–152·3). Targeting multiple mutations could improve sensitivity compared with individual mutations. InterpretationIn this study, we applied an individualised targeted sequencing panel on ctDNA from patients with melanoma. As tumour sequencing becomes more routine, individualised sequencing panel design might become more feasible, facilitating a more sensitive approach for ctDNA analysis than targeting individual loci. Tracking clonal evolution during therapy non-invasively may facilitate personalised treatment decisions with molecularly targeted agents. FundingCancer Research UK, Lewis Family Charitable Trust, Addenbrooke's Charitable Trust, Cambridge Cancer Trials Centre, NIHR Cambridge Biomedical Research Centre and Human Research Tissue Bank.

Tumour risks and genotype–phenotype–proteotype analysis of patients with germline mutations in the succinate dehydrogenase subunit genes SDHB, SDHC, and SDHD

BackgroundGermline mutations in the succinate dehydrogenase subunit genes SDHB, SDHC, and SDHD are the most frequent causes of inherited phaeochromocytomas and paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. Our aim was to provide estimates of penetrance and genotype–phenotype correlations in a large cohort of succinate dehydrogenase mutation carriers. MethodsWe undertook a retrospective survey of 800 individuals in the UK (401 previously reported) with germline mutations in SDHB (620), SDHC (31), and SDHD (149). We estimated and compared tumour risks for each gene according to age using survival analysis and Cox proportional hazards modelling with the statistical programming language R. DUET, a computational approach for predicting the effects of mutations on protein stability, was used to evaluate the functional effects of SDHB and SDHD mutations. FindingsAnalysis of age-related tumour risks provided novel estimates of penetrance. In addition to tumour-specific differences in risk for individual genes, we confirmed that the SDHD p.Pro81Leu mutation had a distinct phenotype, with a low risk of phaeochromocytoma and extra-adrenal paraganglioma (only one case in 55 patients), and found evidence suggesting higher penetrance with SDHB p.Ile127Ser mutations. Comparison of age-dependent penetrance of disease in p.Ile127Ser carriers versus other SDHB missense mutation carriers showed that p.Ile127Ser was associated with a higher overall penetrance of phaeochromocytoma and paraganglioma (χ2=4·49, p=0·034). SDHB p.Ile127Ser was predicted by DUET to be the most destabilising SDHB missense mutation, through disruption of key intramolecular hydrophobic interactions by the introduction of a polar serine. The penetrance in SDHB and SDHD mutation-positive non-probands by age 60 years was 22·1% (95% CI 15·6–28·3) and 47·5% (29·3–61·1), respectively, and the risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4·2% (1·1–7·2). InterpretationIncreased knowledge of the lifetime tumour risks is crucial to long-term surveillance and management. Knowledge of the molecular basis of the phenotypic variability commonly observed in individuals with germline SDHB, SDHC, and SDHD mutations will facilitate the development of personalised management based on gene-specific and mutation-specific tumour risks. FundingEast Anglian Foundation Programme (KAA), University of Cambridge (KAA), NIHR Cambridge Biomedical Research Centre (KAA and ERM), British Heart Foundation (ERM), NHMRC CJ Martin Fellowship (APP1072476) (DBA), Fundação de Amparo a Pesquisa do Estado de Minas Gerais and Centro de Pesquisas René Rachou (CPqRR/FIOCRUZ Minas) (DEVP).

Role of melanocortin signalling in the preference for dietary macronutrients in human beings

BackgroundSignalling though the melanocortin 4 receptor (MC4R), which is widely expressed in the hypothalamus, mediates food intake and macronutrient preference in rodents. Studies in patients with MC4R deficiency can provide insights into the role of this pathway in man. We investigated the role of melanocortin signalling in fat and sucrose preference in human beings by studying patients with loss of function mutations in MC4R. MethodsWe studied 24 obese patients with MC4R deficiency, and 80 healthy controls (40 obese, 40 lean). We used an ad-libitum meal protocol consisting of three meals covertly manipulated to provide 20% (low), 40% (medium), and 60% (high) fat content. We used the same procedure for meals manipulated to provide 8% (low), 26% (medium), and 54% (high) sucrose content. We measured food intake and rated liking for the meals with visual analogue scores. Data were analysed by ANOVA and Tukey's post-hoc tests or a linear mixed-effects model with an interaction term for study group and study meal when appropriate. FindingsAlthough the liking of the three different fat meals did not differ between the three groups, patients with MC4R mutations consumed 95% more of the high fat meal than did lean controls and 65% more of the high fat meal than did obese controls (p=0·0222 for the interaction of group by meal). By contrast, although liking ratings for low and medium sucrose meals were comparable in the individuals with MC4R deficiency, liking ratings for the high sucrose meal were significantly reduced (p=0·0252 in linear mixed-effects model, intercept 57·8, MC4R group factor −26·2, factors in the model for MC4R-low sucrose 27·7, MC4R-medium sucrose 22·6). Similarly, patients with MC4R deficiency consumed less of all three sucrose meals than did healthy controls (p=0·0064). InterpretationOur study shows that the central melanocortin system has divergent effects on macronutrient preference and intake in human beings. FundingWellcome Trust, NIHR Cambridge Biomedical Research Centre, Bernard Wolfe Health Neuroscience Fund, NeuroFAST consortium, which is funded by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 245009.

Unsupervised overnight closed loop insulin delivery during free living: analysis of randomised cross-over home studies in adults and adolescents with type 1 diabetes

BackgroundThe closed-loop system (artificial pancreas) delivers insulin in a glucose-responsive manner by the use of a control algorithm that automatically directs insulin delivery, based on real-time sensor glucose concentrations. Results from hospital-based studies have shown improved overnight glucose control and reduced risk of hypoglycaemia in type 1 diabetes. We aimed to assess whether unsupervised closed-loop systems can provide a realistic treatment option in patients with type 1 diabetes. MethodsWe combined data from two open-label, phase 2, randomised, cross-over, unsupervised home trials of people with type 1 diabetes, one in 24 adults (mean age 43 years [SD 12], HbA1c 8·0% [0·9]) and the other in 16 adolescents (15·6 [3·6], 8·1 [0·8]). In each trial, after training on study devices, participants were allocated to two periods of sensor-augmented pump therapy either with or without overnight closed loop that used a model predictive control algorithm to direct insulin delivery. Allocation sequence was done with a computer-generated random code. Each period lasted 4 weeks in adults and 3 weeks in adolescents. Primary outcome for both trials was time when sensor glucose was in the target range (3·9–8·0 mmol/L). Analysis was by intention to treat. Participants (or parents) gave written informed consent. The trials are registered with ClinicalTrials.gov, numbers NCT01440140 and NCT01221467. FindingsClosed loop was started by participants on their own volition on 866 (89%) of 978 nights. The proportion of time when sensor glucose was in the target range between 0000 h and 0800 h was increased by a mean of 18·4% (95% CI 13·5–23·4, p<0·0001) during closed loop compared with no closed loop. Closed loop significantly reduced mean overnight sensor glucose by 0·9 mmol/L (95% CI 0·4–1·3, p=0·0001), and reduced the proportion of time when sensor glucose values were suggestive of hyperglycaemia (>8·0 mmol/L) (15·9%, 10·7–21·0; p<0·0001) and hypoglycaemia (<3·9 mmol/L) (median 0·9, IQR 0·2–2·2; p=0·014). Lower mean overnight glucose was associated with increased overnight insulin delivery (p<0·0001) without changing total daily insulin amount (p=0·84). InterpretationExtended use of overnight closed loop at home without supervision is feasible in adults and adolescents with type 1 diabetes. Clinically significant reduction in overnight glucose was observed accompanied by reduced time spent by patients in hypoglycaemia. To our knowledge, such combined effect has not been documented with any other means of intensified conventional insulin delivery. Longer term studies are warranted to assess its clinical potential. FundingDiabetes UK, Juvenile Diabetes Research Foundation, NIHR Cambridge Biomedical Research Centre.

Tumour genomic and microenvironmental heterogeneity for integrated prediction of 5-year biochemical recurrence of prostate cancer: a retrospective cohort study

Clinical prognostic groupings for localised prostate cancers are imprecise, with 30-50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1-9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65-0·76]) and radical prostatectomy (4·0 [1·6-9·7]; p=0·0024; AUC 0·57 [0·52-0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2-12]; p=0·019; AUC 0·67 [0·61-0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0-19]; p=0·0015; AUC 0·74 [95% CI 0·65-0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4-6·0]; p=0·0039; AUC 0·68 [95% CI 0·63-0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.

Resistance to thyroid hormone caused by a mutation in thyroid hormone receptor (TR)α1 and TRα2: clinical, biochemical, and genetic analyses of three related patients

The thyroid hormone receptor α gene (THRA) transcript is alternatively spliced to generate either thyroid hormone receptor (TR)α1 or a non-hormone-binding variant protein, TRα2, the function of which is unknown. Here, we describe the first patients identified with a mutation in THRA that affects both TRα1 and TRα2, and compare them with patients who have resistance to thyroid hormone owing to a mutation affecting only TRα1, to delineate the relative roles of TRα1 and TRα2. We did clinical, biochemical, and genetic analyses of an index case and her two sons. We assessed physical and radiological features, thyroid function, physiological and biochemical markers of thyroid hormone action, and THRA sequence. The patients presented in childhood with growth failure, developmental delay, and constipation, which improved after treatment with thyroxine, despite normal concentrations of circulating thyroid hormones. They had similar clinical (macrocephaly, broad faces, skin tags, motor dyspraxia, slow speech), biochemical (subnormal ratio of free thyroxine:free tri-iodothyronine [T3], low concentration of total reverse T3, high concentration of creatine kinase, mild anaemia), and radiological (thickened calvarium) features to patients with TRα1-mediated resistance to thyroid hormone, although our patients had a heterozygous mis-sense mutation (Ala263Val) in both TRα1 and TRα2 proteins. The Ala263Val mutant TRα1 inhibited the transcriptional function of normal receptor in a dominant-negative fashion. By contrast, function of Ala263Val mutant TRα2 matched its normal counterpart. In vitro, high concentrations of T3 restored transcriptional activity of Ala263Val mutant TRα1, and reversed the dominant-negative inhibition of its normal counterpart. High concentrations of T3 restored expression of thyroid hormone-responsive target genes in patient-derived blood cells. TRα1 seems to be the principal functional product of the THRA gene. Thyroxine treatment alleviates hormone resistance in patients with mutations affecting this gene, possibly ameliorating the phenotype. These findings will help the diagnosis and treatment of other patients with resistance to thyroid hormone resulting from mutations in THRA. Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Marie Curie Actions, Foundation for Development of Internal Medicine in Europe.

The coding sequence of POMC and obesity and appetite in Labrador retriever dogs

BackgroundIn human beings, the heritability of obesity is estimated at 40–70%, but only a small proportion (<3%) of the genetic variants that account for naturally occurring obesity have been discovered. In dogs, selective breeding from small founder populations has led to trait-associated genes being concentrated within breeding populations, making dogs an appealing model for genetic study. Labrador retriever dogs are commonly prone to obesity and are highly food motivated; such a high prevalence within the breed suggests a genetic predisposition. The aim of this study was to discover the genetic basis of obesity in Labrador retriever dogs. MethodsPet dogs were prospectively selected for study. Body condition score was used to initially group them as lean or obese. They were further divided into two groups—obese, highly food-motivated dogs; and lean, non-food-motivated dogs—by a combination of a standardised questionnaire to owners and a veterinary surgeon taking a clinical and behavioural history. The coding sequence of AGRP, POMC, and MC4R were sequenced in several dogs from each group. Findings35 dogs (15 obese, 20 lean) were studied. The coding sequence of AGRP was normal in all dogs. Six single nucleotide polymorphisms (SNPs) were identified in MC4R, and eight SNPs and two deletions (one coding, one intronic) were found in POMC. There was no significant difference in the distribution of these variants between lean and obese groups for any of the variants except the coding deletion in POMC. Of 15 obese, food-motivated dogs, that deletion was heterozygous in eight and homozygous in two; the deletion was heterozygous in two of the 20 lean, non-food-motivated dogs. The association with obesity and high food motivation was significant (p=0·001). InterpretationMelanocortin signalling has a key role in normal energy homoeostasis; POMC is a precursor protein whose cleavage products act on MC4 and MC3 receptors to suppress appetite and increase energy expenditure. The deletion of 14 base pairs in exon 3 of POMC is predicted to disrupt the POMC protein, stopping production of its cleavage products β-melanocyte-stimulating hormone and β-endorphin and is therefore a strong candidate for causing much or all of the obesity susceptibility in Labrador retriever dogs. Further genetic studies are in progress to assess the prevalence of the deletion in larger cohorts of dogs from the same and different breeds, with functional studies also planned. FundingWellcome Trust, UK Medical Research Council, NIHR Cambridge Biomedical Research Centre.

Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study

SummaryBackgroundIncreasing numbers of individuals with cystic fibrosis are becoming infected with the multidrug-resistant non-tuberculous mycobacterium (NTM) Mycobacterium abscessus, which causes progressive lung damage and is extremely challenging to treat. How this organism is acquired is not currently known, but there is growing concern that person-to-person transmission could occur. We aimed to define the mechanisms of acquisition of M abscessus in individuals with cystic fibrosis.MethodWhole genome sequencing and antimicrobial susceptibility testing were done on 168 consecutive isolates of M abscessus from 31 patients attending an adult cystic fibrosis centre in the UK between 2007 and 2011. In parallel, we undertook detailed environmental testing for NTM and defined potential opportunities for transmission between patients both in and out of hospital using epidemiological data and social network analysis.FindingsPhylogenetic analysis revealed two clustered outbreaks of near-identical isolates of the M abscessus subspecies massiliense (from 11 patients), differing by less than ten base pairs. This variation represents less diversity than that seen within isolates from a single individual, strongly indicating between-patient transmission. All patients within these clusters had numerous opportunities for within-hospital transmission from other individuals, while comprehensive environmental sampling, initiated during the outbreak, failed to detect any potential point source of NTM infection. The clusters of M abscessus subspecies massiliense showed evidence of transmission of mutations acquired during infection of an individual to other patients. Thus, isolates with constitutive resistance to amikacin and clarithromycin were isolated from several individuals never previously exposed to long-term macrolides or aminoglycosides, further indicating cross-infection.InterpretationWhole genome sequencing has revealed frequent transmission of multidrug resistant NTM between patients with cystic fibrosis despite conventional cross-infection measures. Although the exact transmission route is yet to be established, our epidemiological analysis suggests that it could be indirect.FundingThe Wellcome Trust, Papworth Hospital, NIHR Cambridge Biomedical Research Centre, UK Health Protection Agency, Medical Research Council, and the UKCRC Translational Infection Research Initiative.

Detection of weak receptor–ligand interactions using IgM and J‐chain‐based fusion proteins

Detection of weak receptor–ligand interactions using <scp>I</scp>g<scp>M</scp> and <scp>J</scp>‐chain‐based fusion proteins