The Verona Newly Diagnosed Type 2 Diabetes Study

Study Overview
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Brief Summary
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Type 2 diabetes mellitus is a complex disease whose clinical phenotype results from the variable combination of genetic and nongenetic factors. The aim of the present study is to investigate the network linking phenotypes and genotypes in patients with newly diagnosed type 2 diabetes mellitus. In selected cases, in which clinical evidence hints at possible monogenic basis of the disease, the genotype and the phenotype of relatives also will be assessed to elucidate further the etiology of the disease.

Detailed Description
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The Verona Newly Diagnosed Type 2 Diabetes Study (VNDS) is an ongoing study aiming at building a biobank of patients with newly diagnosed type 2 diabetes mellitus. All patients referred to the Division of Endocrinology and Metabolic Diseases of University of Verona School of Medicine, whose diabetes has been diagnosed in the last six months, are asked to participate in this research. The clinical evidence on which the diagnosis of type 2 diabetes has been made is reviewed and the diagnosis confirmed, according to the current criteria of American Diabetes Association. Patients already treated with antidiabetic drugs undergo a treatment washout of at least one week before metabolic tests are performed. Among the exclusion criteria are age > 75 years, non-Italian ancestry, insulin treatment, presence of anti-GAD antibodies, malignancies, and any condition severely impairing liver and/or kidney function. All subjects consume a weight-maintaining diet containing 200-250 g of carbohydrate/day for at least three days before studies. Body weight must be stable in all subjects for at least 1 month before studies. No subject should participate in any heavy exercise. Each subject gives informed written consent before participating in the research, which was approved by the Human Investigation Committee of the Verona City Hospital. Measurements of standard clinical phenotypes are collected in all patients. Other diabetes related phenotypes may be collected if their determination is available. Metabolic tests are carried out on two separate days in random order. On both days, patients are admitted to the Metabolic Clinic Research Center at 07:30 after an overnight fast. All studies are carried out in a quiet, temperature controlled (22° C) room. On one day an oral glucose tolerance test (OGTT) (75 g) is performed to assess beta cell function. On a separate day, a euglycemic insulin clamp is performed to assess insulin sensitivity. When age of onset and distribution of the disease in the pedigree suggest a potentially monogenic disorder, the relatives of the proband are asked to participate in the study by allowing the collection of standard clinical information and of a fasting blood sample for genetic and phenotypic determinations. -OGTT: For ethical reasons, the OGTT cannot performed in patients presenting with fasting plasma glucose higher than 15 mmol/l. During the entire test patients are sitting in a comfortable cardiac chair. One teflon (21 g) venous catheter is inserted into an antecubital vein for blood sampling and kept patent with heparinized normal saline solution. After a 30' rest to establish baseline and after collecting a 20 cc blood sample for leukocyte DNA extraction, at time = 0' subjects ingest 75 g of glucose in 300 ml of water over 5 min. Blood samples to measure glucose, C-peptide and insulin concentrations are collected at times -10', 0', +15', +30', +45', +60', +90', +120', +150', +180', +210' and +240', +270' and +300'. Urines are collected to measure glycosuria. -Euglycemic Insulin Clamp: During the entire test patients are lying in bed. One teflon catheter is introduced into an antecubital vein for the infusion of test substances. Another teflon catheter is placed retrogradely into a wrist vein for sampling arterialized venous blood, according to the hot box technique. After a 30' rest in bed to establish baseline, indirect calorimetry (at least 40') is performed. At the end of calorimetric measures, baseline blood samples are collected and a standard euglycemic insulin clamp is carried out. After an insulin intravenous prime of 4.8 pmol/min/m^2 BSA and a subsequent continuous infusion of 240 pmol/min/m^2 BSA, plasma glucose is allowed to decline until it reaches 5.5 mmol/l, after which glucose clamping starts with a glucose concentration goal of 5 mmol/l. The duration of the glucose clamp is at least of 120', but it is prolonged, if and as needed, to ensure at least 60' of insulin clamp at euglycemia in each patient. Timed blood samples were collected to measure hormone and substrate levels. In the last 45' of the clamp indirect calorimetry is repeated to assess substrate oxidation and energy production rates. Urine is collected to measure urea excretion rate. In both metabolic tests, all blood samples are collected in pre-chilled tubes and readily spun at 1,500 g. Plasma and serum specimens are stored at -80° C. -Analytical procedures: Plasma glucose concentration is measured in duplicate at bedside. Serum C-peptide and insulin concentrations are measured by chemiluminescence. Glycated hemoglobin and serum lipids were measured by standard in-house methods. GAD-antibodies are measured by immunoradiometry (CentAK, Medipan, Germany), according to manufacturer's instructions. -Genotyping: A leukocyte DNA sample is collected in each subject and the DNA is extracted through standard salting out method. Genotyping is performed by RFLP (Restriction Fragment Length Polymorphism), which consists in a PCR (Polymerase Chain Reaction) followed by proper enzymatic digestion and resolution on agarose gel. Alternatively, it is performed were assessed by the high-throughput genotyping Veracode technique (Illumina Inc, CA), applying the GoldenGate Genotyping Assay according to manufacturer's instructions.

Official Title
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The Verona Newly Diagnosed Type 2 Diabetes Study. Construction of a Biobank of Diabetes Related Genotypes and Phenotypes

Conditions
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Type 2 Diabetes Mellitus

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Type 2 diabetes mellitus, whose diagnosis has been made in the last six months before the first access to the Division of Endocrinology and Metabolic Diseases of University of Verona School of Medicine; Relatives of patients with potentially monogenic newly diagnosed type 2 diabetes Exclusion Criteria: Age > 75 years Non-Italian ancestry Insulin treatment Presence of anti-GAD antibodies Malignancies Any condition severely impairing liver and/or kidney function

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Group A: Diabetic<br>Newly diagnosed type 2 diabetic patients (i.e. diagnosis made no more than 6 months before recruitment) | |
| Group B: Relatives<br>Relatives of patients with potentially monogenic newly diagnosed type 2 diabetes | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Genetic basis of beta cell function | Investigators genotype diabetes risk loci and relate them to two main beta cell metrics, after correction for insulin sensitivity: Derivative control of beta cell function: it is the amount of insulin secreted in response to a rate of glucose increase of 1 mmol/l per min which lasts for 1 minute; Proportional control of beta cell function: it is the stimulus-response curve linking glucose concentration (x-axis) to insulin secretion rate (y-axis) at the preselected glucose concentrations of 5.5, 8.0, 11.0, 15.0, and 20.0 mmol/liter. | Baseline and during the 75 g oral glucose challenge. Subjects will be followed for the duration of their stay in the Metabolic Clinical Research Center (average expected stay: 6 hours) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Genetic basis of insulin sensitivity | Investigators genotype diabetes risk loci and relate them, after correcting for the influence of other genetic and nongenetic modifiers, to one metric of clamp derived insulin sensitivity: 1. M value, which quantifies whole body net glucose disposal during euglycemic hyperinsulinemia | Baseline and during the euglycemic insulin clamp. Subjects will be followed for the duration of their stay in the Metabolic Clinical Research Center (average stay: 4 hours) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Type 2 diabetes mellitus, Beta cell function, Insulin sensitivity, Genetics of diabetes