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NUGENOB:
The study, the database, and perspectives for integration
Mid Term Review
QLK1-CT-2000-00618
Focus
• The interactions between macronutrient composition of the diet,
and specific genetic variants in human obesity.
• Combining clinical/physiological response to a high-fat test meal
and a long-term hypo-energetic low-fat or medium-fat diet with
knowledge of genetic make up and expression levels of individual
genes.
Aims - I
• Identification and characterisation of novel nutrient-sensitive
candidate genes for obesity.
• Assessment of the effects of the variants and combinations of
variants in nutrient-sensitive genes on the response to a high-fat
test meal
Aims - II
• Identification of predictors of the changes in body weight during a 10week hypo energetic low-fat or medium-fat dietary intervention
programme:
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Anthropometrical characteristics,
Obesity-related life style factors,
Physiological functions observed at the test meal challenge,
Variants or combinations of variants of the nutrient-sensitive genes,
Gene expression in adipose tissue,
Gene-phenotype or gene-environment interactions based on combinations of
these predictors.
To identify predictors of differantial response to the two diets
• Assessment of the combined effects of
– variants of nutrient-sensitive genes
– short- and long-term alterations in dietary fat content
on the differential expression of selected genes in adipose tissue.
WP2
Source populations
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processing of DNA
M2
M3
M4
WP7
WP8
Data:
M1
Samples:
WP1
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Databank and
statistical analysis
Genotypic screening
Contribution to
evidence-based
dietary
guidelines
Nutrient-gene
interaction in
human obesity
Study population
771 obese – 119 lean subjects
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Inclusion criteria for obese subjects
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BMI >= 30
Intention to include white Europeans
Age 20-50 years
Pre-menopausal women
Exclusion criteria for obese subjects
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Medical conditions and medication
Pregnant women
Alcohol or drug abuse
Weight change of > 3 kg within 3 months.
WP2
WP3
M2
M3
M4
Source populations
Recruitment of
study population
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processing of DNA
WP7
WP8
Data:
M1
Samples:
WP1
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Databank and
statistical analysis
Genotypic screening
Contribution to
evidence-based
dietary
guidelines
Nutrient-gene
interaction in
human obesity
Data I - Habitual diet, life style factors
• 3-day, weighed food record
• Questionnaire addressing:
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social variables,
physical activity,
drinking and smoking habits,
weight and dieting history,
health and illness,
family history of obesity and obesity related diseases
SF-36
M1
WP2
Source populations
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processing of DNA
M2
M3
M4
WP7
WP8
Data:
WP1
Samples:
Clinical Investigation day
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Databank and
statistical analysis
Genotypic screening
Contribution to
evidence-based
dietary
guidelines
Nutrient-gene
interaction in
human obesity
10 weeks
Anthropometrics
Body composition
Anthropometrics
Body composition
Fasting blood
Data II – Clinical Investigation Day
Timepoints (fasting since, times for hood measures, vas, biopsies, etc)
Weight, height, waist, hip, FM, FFM, fat%, impedance 5,50,100,200 khz
Fatbiposy in fasting and 3-h post prandial (time, amount, reason for not taking it)
Estimated BMR (WHO 1985: use Weight and Height), basis for energy in test meal (50%)
Amount of cream and butter in test meal
VAS for hunger, fullness, satiety, wish to eat, and thirst at baseline, + 1,2,3 h after test meal
Vo2 and Vco2 at baseline at measurements ending 30, 60, 90, 120, 150, and 180 after test meal
RQ and energy expenditure calcuated from Vo2 and Vco2 (baseline, 30,60,90,120,150,180)
Calculated substrate utilisation for fat and carbohydrates (according to Frayn)
Remarks during test day
Well bening
Subject getting sick
Blood: insulin, glucose, free fatty acids, total glycerol, free glycerol, triglycerides
total cholesterol, HDL cholesterol, LDL cholesterol, Cortisol, VLDL, Leptin, IGF
M1
WP2
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
WP6
M4
Recruitment of
study population
WP3
M2
M3
Source populations
WP7
WP8
Data:
WP1
Samples:
Hypo-energetic 10 week
low or high fat diet
Co ntributio n to
evidence-ba sed
dieta ry
guidelines
Hypoenergetic 10-week
low- or high-fat diet
Sampling and
processin g of DNA
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Genotypic screening
Databank and
statistical analysis
Nutrient-gene
intera ction in
human obes ity
Results of WP 5
Weight loss by diet
Weight loss intervention
• 10 weeks
• - 600 kcal per day
• Either low fat or medium fat diet
Data III – Dietary intervention
Weight for week 0 (either CID weight or new fasting weight enetered) corresponding body composition
Weight at week 1-10 (week 0 and week 10 are fasting weights)
Variables indicating whether weight from week 1-9 were self reported
Compliance assessed by subject and dietitian at week 1-10 ( categori - 5 categories)
Notes at week 0-10
Dropout: time, reason from CRF, reasons summarised into overall categories
Food recording: week 2,5,7,10
M1
WP2
Source populations
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processing of DNA
M2
M3
M4
WP7
WP8
Data:
WP1
Samples:
Identification of
candidate genes and gene
variants
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Databank and
statistical analysis
Genotypic screening
Contribution to
evidence-based
dietary
guidelines
Nutrient-gene
interaction in
human obesity
Publiched results
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GAD2 on Chromosome 10p12 Is a Candidate Gene for Human
Obesity. Boutin P et al. PLoS Biol. 2003 Dec;1(3):E68. Epub 2003 Nov
03
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Association studies between microsatellite markers within the gene
encoding human 11 beta-hydroxysteroid dehydrogenase type 1 and
body mass index, waist to hip ratio, and glucocorticoid metabolism.
Draper Net al. Journal of Clinical Endocrinology and Metabolism, Vol.
87 (11) pp. 4984-4990 (2002)
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Mutational analysis of the UCP2 core promoter and relationships of
variants with obesity. Dalgaard LT et al. Obes Res 2003;11:1420-1427
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A genome-wide scan for childhood obesity-associated traits in French
families shows significant linkage on chromosome 6q22.31-q23.2.
Meyre et al.Diabetes. 2004 Mar;53(3):803-811.
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The FOXC2 -512C>T variant is associated with hypertriglyceridemia
and elevated serum C-peptide in glucose tolerant subjects Yanagisawa
et al. Diabetologia, 2003 Nov;46(11):1576-80
M1
WP2
Source populations
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-wee k
low- or high-fat diet
WP6
Sampling and
processing of DNA
M2
M3
M4
WP7
WP8
Data:
WP1
Samples:
Genotypic screening
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Databank and
statistical analysis
Genotypic screening
Co ntributio n to
evidence-ba sed
dieta ry
guidelines
Nutrient-gene
intera ction in
human obesity
Data IV – Genotyping
43 SNPs in 27 genes
Adipocyte secretory peptides
Regulation of adiposcyte differantiation
Mitocondrial proteins
Nuclear receptors and transcription factors
Hormones and receptors involved in appetite regulation
Insulin signalling and glucose regulation
Lipoprotein metabolism
M1
WP2
M4
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processin g of DNA
M2
M3
Source populations
WP7
WP8
Data:
WP1
Samples:
Quantitative gene
expression
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Genotypic screening
Databank and
statistical analysis
Co ntributio n to
evidence-ba sed
dieta ry
guidelines
Nutrient-gene
intera ction in
human obes ity
Data V – Quantitative gene expression
Quantitative gene expression analyses for 40 candidate genes
in 2 x 25 subjects (25 from each diet group)
Gene Expression data (before and after weight loss, fasting biopsies)
Genes related to:
Signal transduction
Adipocyte secretion
Glucose metabolism
Lipid metabolism
Mitochondria/ Energy metabolism
Transcription (co-)factors
Reference mRNAs
M1
WP2
Source populations
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processing of DNA
M2
M3
M4
WP7
WP8
Data:
WP1
Samples:
Gene expression
profiling
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Databank and
statistical analysis
Genotypic screening
Contribution to
evidence-based
dietary
guidelines
Nutrient-gene
interaction in
human obesity
Data VI – Gene expression profiling
• Affymetrix chip analyses on 13+10 women from a single center
• Custom made metabolic chip in 25+25 selected from all centres
• Data stored in local centres
M1
WP2
Source populations
Recruitment of
study population
WP3
Habitual and run-in diet,
life style factors
WP4
Clinical
investigation day
WP5
Hypoenergetic 10-week
low- or high-fat diet
WP6
Sampling and
processing of DNA
M2
M3
M4
WP7
WP8
Data:
WP1
Samples:
Databank and statistical
Analyses
Identification of
candidate genes
Analysis of
candidate genes
M5
WP9
M6
WP10
Adipose tissue
mRNA analysis
M7
WP11
Adipose tissue
cDNA analysis
M8
WP12
Genotypic screening
Databank and
statistical analysis
Contribution to
evidence-based
dietary
guidelines
Nutrient-gene
interaction in
human obesity
The NUGENOB database – formant
• The database has a simple flat format with 1 row per
subject and one column per variable
• SPSS, STATA, SAS...... (Not Excel)
The NUGENOB database – Content
Raw data
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Habitual diet (not ’raw’)
Questionnaire
Anthropometrics
Appetite
Blood parameters
Vo2 and Vco2
Weight during weight loss
Diet during weight loss
Genotypes
Quantitative gene
expression
• Gene expression profiling
Derived measures
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Percentage and avarage
Summary scores
BMI, W/H
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AUC, AUCi
RQ, EE, macronutrien oxidation
Weight loss from week 0 to 10
Percentage and avarage
BMI, W/H
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Ratios
The NUGENOB database –
Selection of sub-populations
• Subjects with high versus low increase in fat oxidation following the
high-fat test meal
• Subjects completing the weight loss intervention, and following the
diet prescriptions
• Subjects being successful or un-successful in loosing weight when
following the dietary intervention
• Subjects with no versus high improvement in insulin sensitivity
following weight loss
Samples in the NUGENOB biobank
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Serum and plasma samples taken in the fasting state, and every hour during a
3-hour period following a high-fat test meal. In obese subjects fasting serum
and plasma samples were obtained following the 10-week intervention.
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Purified DNA in 96-well master plates (two sets) from all but 10 of the 771
obese and 119 lean subjects.
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Buffy coat samples from all but ~120 of the 771 obese and 119 lean subjects
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Extracted mRNA from abdominal subcutaneous adipose tissue biopsies
obtained in fasting before and after the 10-week dietary intervention in ~ 700
obese subjects. mRNA from biopsies obtained 3 hours after the test meal in a
small group of obese subjects and mRNA from biopsies obtained in a small
group of lean subjects.
SOPs of NUGENOB
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Anthropometrical measurements
Questionnaire (Translated)
Blood sampling (obtaining, packing, storing, shipment)
Dietary Intervention
Needle subcutaneous abdominal adipose tissue biopsy
Preparation of the high fat liquid test meal.
Validating, calibrating and conducting indirect calorimetry (Hood).
Clinical investigation day, including blood sampling, energy and substrate
metabolism in fasting and flowing a high fat test meal.
Randomisation.
Data entry and data transfer into the freeware programme EpiData
Delivery of data for analyses
Database on nutrient-sensitive genes
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Database on genes for which there is a known nutrient-gene interaction
and a putative role in obesity
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Info on genes for which gene variatns determine response to diet
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Info on genes for which the expression is changed in responds to calorie
restriction, overfeeding or changes in diet composition (animal and
human).
– Data from quantitative gene expression
– Data from micro array studies.
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Animal studies on obesity, DIO or protection from DIO in KO, TG and
mutation models
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Human gene variants affecting gene expression
(with or without in vitro functional effects)
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Gene-gene interaction for nutrient sensitive genes
For further information see:
www.nugenob.org