Challenges and practical recommendations for successfully recruiting inactive, statin-free older adults to clinical trials.

OBJECTIVES: To outline the challenges and provide practical recommendations for recruiting inactive, statin-free older adults to facilitate feasible study designs. Data was obtained from a double-blind randomised-controlled clinical trial investigating the effects of acipimox versus placebo on muscle function and metabolism in older (65-75 years), inactive, statin-free males. The initial recruitment target was 20 volunteers within 12 months (November 2016-November 2017). RESULTS: Recruitment occurred via the Exeter 10,000 database containing 236 'eligible' males, a Facebook campaign reaching > 8000 ≥ 65 years old males, 400 directly-addressed letters to ≥ 66 year old males, > 1500 flyers distributed within the community, > 40 emails to local community groups, 4 recruitment talks, 2 magazine adverts and 1 radio advert. Widespread recruitment efforts reaching > 120,000 people led to the recruitment of 20 volunteers (18 completed the clinical trial) within a 25-month timeframe, highlighting the challenge of the timely recruitment of inactive, statin-free older adults for clinical trials. We recommend recruitment for future clinical trials should take a multi-pronged approach from the outset, prioritising the use of volunteer databases, Facebook campaigns and delivering recruitment talks.

An engaged research study to assess the effect of a 'real-world' dietary intervention on urinary bisphenol A (BPA) levels in teenagers.

OBJECTIVE: Bisphenol A (BPA) has been associated with adverse human health outcomes and exposure to this compound is near-ubiquitous in the Western world. We aimed to examine whether self-moderation of BPA exposure is possible by altering diet in a real-world setting. DESIGN: An Engaged Research dietary intervention study designed, implemented and analysed by healthy teenagers from six schools and undertaken in their own homes. PARTICIPANTS: A total of 94 students aged between 17 and 19 years from schools in the South West of the UK provided diet diaries and urine samples for analysis. INTERVENTION: Researcher participants designed a set of literature-informed guidelines for the reduction of dietary BPA to be followed for 7 days. MAIN OUTCOME MEASURES: Creatinine-adjusted urinary BPA levels were taken before and after the intervention. Information on packaging and food/drink ingested was used to calculate a BPA risk score for anticipated exposure. A qualitative analysis was carried out to identify themes addressing long-term sustainability of the diet. RESULTS: BPA was detected in urine of 86% of participants at baseline at a median value of 1.22 ng/mL (IQR 1.99). No effect of the intervention diet on BPA levels was identified overall (P=0.25), but there was a positive association in those participants who showed a drop in urinary BPA concentration postintervention and their initial BPA level (P=0.003). Qualitative analysis identified themes around feelings of lifestyle restriction and the inadequacy of current labelling practices. CONCLUSIONS: We found no evidence in this self-administered intervention study that it was possible to moderate BPA exposure by diet in a real-world setting. Furthermore, our study participants indicated that they would be unlikely to sustain such a diet long term, due to the difficulty in identifying BPA-free foods.

Recognition and Management of Individuals With Hyperglycemia Because of a Heterozygous Glucokinase Mutation.

Glucokinase-maturity-onset diabetes of the young (GCK-MODY), also known as MODY2, is caused by heterozygous inactivating mutations in the GCK gene. GCK gene mutations are present in ∼1 in 1,000 of the population, but most are not diagnosed. They are common causes of MODY (10-60%): persistent incidental childhood hyperglycemia (10-60%) and gestational diabetes mellitus (1-2%). GCK-MODY has a unique pathophysiology and clinical characteristics, so it is best considered as a discrete genetic subgroup. People with GCK-MODY have a defect in glucose sensing; hence, glucose homeostasis is maintained at a higher set point resulting in mild, asymptomatic fasting hyperglycemia (5.4-8.3 mmol/L, HbA1c range 5.8-7.6% [40-60 mmol/mol]), which is present from birth and shows slight deterioration with age. Even after 50 years of mild hyperglycemia, people with GCK-MODY do not develop significant microvascular complications, and the prevalence of macrovascular complications is probably similar to that in the general population. Treatment is not recommended outside pregnancy because glucose-lowering therapy is ineffective in people with GCK-MODY and there is a lack of long-term complications. In pregnancy, fetal growth is primarily determined by whether the fetus inherits the GCK gene mutation from their mother. Insulin treatment of the mother is only appropriate when increased fetal abdominal growth on scanning suggests the fetus is unaffected. The impact on outcome of maternal insulin treatment is limited owing to the difficulty in altering maternal glycemia in these patients. Making the diagnosis of GCK-MODY through genetic testing is essential to avoid unnecessary treatment and investigations, especially when patients are misdiagnosed with type 1 or type 2 diabetes.

Interarm blood pressure difference in people with diabetes: measurement and vascular and mortality implications: a cohort study.

OBJECTIVE: Differences in blood pressure between arms are associated with vascular disease and increased mortality; this has not been reported in diabetes. We explored these associations, and assessed reference standard and pragmatic measurement techniques, in people with diabetes and in nondiabetic controls. RESEARCH DESIGN AND METHODS: A prospective cohort study in Devon, England, recruited 727 people with type 1 or type 2 diabetes and 285 nondiabetic controls. Simultaneous repeated measurements of bilateral blood pressure were made at recruitment. Data were used to inform a pragmatic measurement strategy. Interarm differences were examined for cross-sectional associations with target organ disease and prospective mortality associations (median follow-up 52 months). RESULTS: We found 8.6% of participants with diabetes and 2.9% of controls had systolic interarm differences ≥10 mmHg. Single pairs of blood pressure measurements had high negative predictive values (97-99%) for excluding interarm differences. Systolic interarm differences ≥10 mmHg in diabetes were associated with peripheral arterial disease (odds ratio [OR] 3.4 [95% CI 1.2-9.3]). Differences ≥15 mmHg were associated with diabetic retinopathy (OR 5.7 [1.5-21.6]) and chronic kidney disease (OR 7.0 [1.7-29.8]). Systolic interarm differences were associated prospectively with increased cardiovascular mortality: hazard ratios 3.5 (1.0-13.0) for ≥10 mmHg and 9.0 (2.0-41.0) for ≥15 mmHg. CONCLUSIONS: Blood pressure should be measured in both arms during initial assessment in diabetes. Systolic interarm differences can be excluded with a single pair of measurements. In the population with diabetes, systolic differences may be associated with an increased risk of morbidity and mortality.

Prevalence of vascular complications among patients with glucokinase mutations and prolonged, mild hyperglycemia.

IMPORTANCE: Glycemic targets in diabetes have been developed to minimize complication risk. Patients with heterozygous, inactivating glucokinase (GCK) mutations have mild fasting hyperglycemia from birth, resulting in an elevated glycated hemoglobin (HbA1c) level that mimics recommended levels for type 1 and type 2 diabetes. OBJECTIVE: To assess the association between chronic, mild hyperglycemia and complication prevalence and severity in patients with GCK mutations. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study in the United Kingdom between August 2008 and December 2010. Assessment of microvascular and macrovascular complications in participants 35 years or older was conducted in 99 GCK mutation carriers (median age, 48.6 years), 91 nondiabetic, familial, nonmutation carriers (control) (median age, 52.2 years), and 83 individuals with young-onset type 2 diabetes (YT2D), diagnosed at age 45 years or younger (median age, 54.7 years). MAIN OUTCOMES AND MEASURES: Prevalence and severity of nephropathy, retinopathy, peripheral neuropathy, peripheral vascular disease, and cardiovascular disease. RESULTS: Median HbA1c was 6.9% in patients with the GCK mutation, 5.8% in controls, and 7.8% in patients with YT2D. Patients with GCK had a low prevalence of clinically significant microvascular complications (1% [95% CI, 0%-5%]) that was not significantly different from controls (2% [95% CI, 0.3%-8%], P=.52) and lower than in patients with YT2D (36% [95% CI, 25%-47%], P<.001). Thirty percent of patients with GCK had retinopathy (95% CI, 21%-41%) compared with 14% of controls (95% CI, 7%-23%, P=.007) and 63% of patients with YT2D (95% CI, 51%-73%, P<.001). Neither patients with GCK nor controls required laser therapy for retinopathy compared with 28% (95% CI, 18%-39%) of patients with YT2D (P<.001). Neither patients with GCK patients nor controls had proteinuria and microalbuminuria was rare (GCK, 1% [95% CI, 0.2%-6%]; controls, 2% [95% CI, 0.2%-8%]), whereas 10% (95% CI, 4%-19%) of YT2D patients had proteinuria (P<.001 vs GCK) and 21% (95% CI, 13%-32%) had microalbuminuria (P<.001). Neuropathy was rare in patients with GCK (2% [95% CI, 0.3%-8%]) and controls (95% CI, 0% [0%-4%]) but present in 29% (95% CI, 20%-50%) of YT2D patients (P<.001). Patients with GCK had a low prevalence of clinically significant macrovascular complications (4% [95% CI, 1%-10%]) that was not significantly different from controls (11% [95% CI, 5%-19%]; P=.09), and lower in prevalence than patients with YT2D (30% [95% CI, 21%-41%], P<.001). CONCLUSIONS AND RELEVANCE: Despite a median duration of 48.6 years of hyperglycemia, patients with a GCK mutation had low prevalence of microvascular and macrovascular complications. These findings may provide insights into the risks associated with isolated, mild hyperglycemia.

Use of HbA1c in the identification of patients with hyperglycaemia caused by a glucokinase mutation: observational case control studies.

AIMS: HaemoglobinA1c (HbA1c) is recommended for diabetes diagnosis but fasting plasma glucose (FPG) has been useful for identifying patients with glucokinase (GCK) mutations which cause lifelong persistent fasting hyperglycaemia. We aimed to derive age-related HbA1c reference ranges for these patients to determine how well HbA1c can discriminate patients with a GCK mutation from unaffected family members and young-onset type 1 (T1D) and type 2 diabetes (T2D) and to investigate the proportion of GCK mutation carriers diagnosed with diabetes using HbA1c and/or FPG diagnostic criteria. METHODS: Individuals with inactivating GCK mutations (n = 129), familial controls (n = 100), T1D (n = 278) and T2D (n = 319) aged ≥18years were recruited. Receiver Operating Characteristic (ROC) analysis determined effectiveness of HbA1c and FPG to discriminate between groups. RESULTS: HbA1c reference ranges in subjects with GCK mutations were: 38-56 mmol/mol (5.6-7.3%) if aged ≤40years; 41-60 mmol/mol (5.9-7.6%) if >40years. All patients (123/123) with a GCK mutation were above the lower limit of the HbA1c age-appropriate reference ranges. 69% (31/99) of controls were below these lower limits. HbA1c was also effective in discriminating those with a GCK mutation from those with T1D/T2D. Using the upper limit of the age-appropriate reference ranges to discriminate those with a mutation from those with T1D/T2D correctly identified 97% of subjects with a mutation. The majority (438/597 (73%)) with other types of young-onset diabetes had an HbA1c above the upper limit of the age-appropriate GCK reference range. HbA1c ≥48 mmol/mol classified more people with GCK mutations as having diabetes than FPG ≥7 mmol/l (68% vs. 48%, p = 0.0009). CONCLUSIONS: Current HbA1c diagnostic criteria increase diabetes diagnosis in patients with a GCK mutation. We have derived age-related HbA1c reference ranges that can be used for discriminating hyperglycaemia likely to be caused by a GCK mutation and aid identification of probands and family members for genetic testing.

Insights into the pathogenicity of rare missense GCK variants from the identification and functional characterization of compound heterozygous and double mutations inherited in cis.

OBJECTIVE: To demonstrate the importance of using a combined genetic and functional approach to correctly interpret a genetic test for monogenic diabetes. RESEARCH DESIGN AND METHODS: We identified three probands with a phenotype consistent with maturity-onset diabetes of the young (MODY) subtype GCK-MODY, in whom two potential pathogenic mutations were identified: [R43H/G68D], [E248 K/I225M], or [G261R/D217N]. Allele-specific PCR and cosegregation were used to determine phase. Single and double mutations were kinetically characterized. RESULTS: The mutations occurred in cis (double mutants) in two probands and in trans in one proband. Functional studies of all double mutants revealed inactivating kinetics. The previously reported GCK-MODY mutations R43H and G68D were inherited from an affected father and unaffected mother, respectively. Both our functional and genetic studies support R43H as the cause of GCK-MODY and G68D as a neutral rare variant. CONCLUSIONS: These data highlight the need for family/functional studies, even for previously reported pathogenic mutations.

Macrosomia and hyperinsulinaemic hypoglycaemia in patients with heterozygous mutations in the HNF4A gene.

BACKGROUND: Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4alpha) and HNF1A/TCF1 (encoding HNF-1alpha), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice. METHODS AND FINDINGS: We examined birthweight and hypoglycaemia in 108 patients from families with diabetes due to HNF4A mutations, and 134 patients from families with HNF1A mutations. Birthweight was increased by a median of 790 g in HNF4A-mutation carriers compared to non-mutation family members (p < 0.001); 56% (30/54) of HNF4A-mutation carriers were macrosomic compared with 13% (7/54) of non-mutation family members (p < 0.001). Transient hypoglycaemia was reported in 8/54 infants with heterozygous HNF4A mutations, but was reported in none of 54 non-mutation carriers (p = 0.003). There was documented hyperinsulinaemia in three cases. Birthweight and prevalence of neonatal hypoglycaemia were not increased in HNF1A-mutation carriers. Mice with pancreatic beta-cell deletion of Hnf4a had hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia at birth. CONCLUSIONS: HNF4A mutations are associated with a considerable increase in birthweight and macrosomia, and are a novel cause of neonatal hypoglycaemia. This study establishes a key role for HNF4A in determining foetal birthweight, and uncovers an unanticipated feature of the natural history of HNF4A-deficient diabetes, with hyperinsulinaemia at birth evolving to decreased insulin secretion and diabetes later in life.

The impact of the angiotensin-converting enzyme insertion/deletion polymorphism on severe hypoglycemia in Type 2 diabetes.

The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene (ACE) is associated with altered serum ACE activity. Raised ACE levels and the ACE DD genotype are associated with a 3.2 to 6.8-fold increased risk of severe hypoglycemia in type 1 diabetes. This relationship has not been assessed in type 2 diabetes. We aimed to test for association of the ACE I/D polymorphism with severe hypoglycemia in type 2 diabetes. Patients with type 2 diabetes (n = 308), treated with insulin (n = 124) or sulphonylureas (n = 184), were classified according to whether or not they had previously experienced severe hypoglycemia. Samples of DNA were genotyped for the ACE I/D polymorphism using two alternative polymerase chain reactions to prevent mistyping due to preferential amplification of the D allele. Overall, 12% of patients had previously experienced one or more episodes of severe hypoglycemia. This proportion did not differ between genotype groups (odds ratio (95% confidence limits) for carriers of D allele relative to II homozygotes: 0.79 (0.35-1.78)). This study found no evidence for association of the ACE I/D polymorphism with severe hypoglycemia frequency in patients with type 2 diabetes. However, we cannot rule out a smaller effect (odds ratio

Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young.

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.

Implementing a research project on the development of diabetes.

The Exeter Research Alliance for Diabetes is a research project recently implemented at the Royal Devon and Exeter Hospital. The project investigates the genetic and environmental influences on the development of diabetes and its associated complications. This article describes the process of instigating the study from an initial research idea through to the implementation of the project.