Environmental versus genetic and epigentic influences on
growth, metabolism and cognitive function in offspring of
mothers with type 1 diabetes
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1. Introduction
1.1 Objective
Health conditions result from a combination of genetic susceptibility and environmental influences. While the
genotype is determined at conception the phenotype is modulated and influenced by environmental and
epigenetic factors throughout life, not only postnatally but already in utero and possibly even at preimplantation
stages 1.
Recent studies have highlighted the possible role of intrauterine exposure to maternal diabetes in the
pathogenesis of overweight, type 2 diabetes and cardiovascular disease1. A hyperglycaemic intrauterine
environment may also affect cognitive function 2, childhood growth and pubertal development 3 in the offspring.
During 1993-99 all pregnancies in women with pre-gestational type 1 diabetes were prospectively reported to a
national registry in the Danish Diabetes Association. This nationwide registry contain detailed information of
maternal demography, pregnancy outcome and diabetes status including HbA1c in a prospective cohort of 900
women and their newborn offspring 6. These data are therefore ideal for a large-scale study on long-term effects
of a diabetic intrauterine environment.
The aim of the present study is to use this cohort to determine potential influence of a hyperglycaemic
intrauterine environment and the genetic and epigentic influences on later health, growth, metabolism and
cognitive function in childhood and adolescence.
1.2 Background
1.3 Animal studies
In animal studies, intrauterine hyperglycaemia increases the risk of abnormal glucose tolerance, diabetes,
overweight and insulin-resistance in the offspring8. Transplantation of islet cells, before the last trimester of
pregnancy, normalizes maternal glycaemia and prevents the harmful effects8;9. In a study of pregnant rats with
streptozotocine-induced diabetes, offspring of poorly controlled mothers showed impaired neurodevelopment
compared with offspring of better controlled mothers as well as un-exposed controls, and arachidonic acid
supplement improved performance in all offspring groups10.
1.4 Human studies
Adults:
Overall, there is little literature addressing the risk of type 2 diabetes, overweight and other cardiovascular risk
factors in adult offspring of women with diabetes during pregnancy11-17 and only one study evaluates cognitive
function in the offspring18. Findings are not fully consistent, and only few papers include data on estimates of
maternal glycaemic control during pregnancy11;12;14;18 The population of Pima Indians in Arizona, has been
followed prospectively with Oral Glucose tolerance tests (OGTT´s) since 196519. Papers have been published
demonstrating diabetes in pregnancy as well as elevated 2-hour blood glucose during OGTT in pregnancy to be
strong predictors of overweight and type 2 diabetes in the offspring13-15, and signs of impaired insulin sensitivity
has been reported in the form of elevated levels of fasting insulin14. However, Pima Indians have a very specific
genetic setup, with a prevalence of overweight and type 2 diabetes each reaching 70% by the age of 25, making it
difficult to apply findings to other populations. In a study of cognitive function18- 227 male offspring of women
with diabetes (un-specified) during pregnancy had a significantly higher army rejection rate and insignificantly
lower test-scores at conscription (corresponding to 3 points on an IQ-scale, P=0.12) than un-exposed controls.
In a subgroup, HbA1c was inversely associated with cognitive performance. When it comes to adult offspring of
women with type 1 diabetes, literature is even sparser11;12;16;17. In a study from Prague, 148 offspring of women
with type 1 diabetes were compared with 31 matched controls of healthy mothers without a family history of
diabetes16. Offspring of women with type 1 diabetes had significantly higher blood glucose and insulin levels
during OGTT as well as higher body mass index (BMI) and blood pressure. Nine percent of the offspring had
IGT or type 2 diabetes and 5% had type 1 diabetes. There was no data on IGT and diabetes in the control
group. A French highly selected study compared 15 offspring of mothers with type 1 diabetes (exposed) with 16
offspring of fathers with type 1 diabetes (un-exposed) and found no difference concerning BMI, fat mass, waisthip ratio or blood pressure17. However, 33% of the exposed offspring compared with none of the un-exposed
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offspring had impaired glucose tolerance (IGT). Finally, a recent Danish study found that the risk of prediabetes/type 2 diabetes, overweight and the metabolic syndrome was 2-4-fold increased in 18-27 years old
offspring of women with type 1 diabetes (160 subjects) compared with offspring of women from the
background population (128 subjects). Maternal hyperglycemia during the last part of pregnancy was associated
with increased risk of offspring pre-diabetes/type 2 diabetes4;5. Furthermore, offspring of women with type 1
diabetes had lower cognitive scores, but after adjusting for gestational age, socioeconomic status and other
possible confounders the groups no longer differed significantly (Clausen et al Diabetic Medicine 2011, in press).
Children:
There are a number of follow-up studies in children up to adolescence20-29. Several papers are from studies of the
Pima Indians19, or from the Chicago-group30, but literature covers populations from most parts of the Western
World and Asia21;31, though there are no studies from Africa. However, many studies are small including less
than 100 diabetes-exposed offspring or no internal control-groups. Furthermore some of the studies have
limitations due to analysis including maternal type 1 and type 2 diabetes together26;30 or high numbers lost to
follow-up24. There are two small follow-up studies of children from randomized trials in women with GDM32;33.
All studies of offspring of women with type 1 diabetes include children younger than 12 years 20;24;27;28;34-44 and
only two of these cohorts include more than 200 children27;28. Findings are conflicting, some finding increased
risk of overweight, type 2 diabetes/pre-diabetes and cognitive deficits in offspring of women with type 1
diabetes24;34-36;38;39, others finding either no42-44 or only few of such indications27;28;37;40;41.
Adolescents:
Studies have shown that offspring of women with type 1 diabetes have an increased linear growth and an
increased BMI in childhood. Obesity in childhood may be linked to an earlier onset of puberty and an earlier age
at menarche 45;46, but possible associations between maternal diabetes and pubertal development have not been
investigated directly. Furthermore, polycystic ovary syndrome (PCOS), the most frequent hormonal disease
among women in fertile age, which is characterized by insulin resistance, androgen excess, oligomenorrea and
infertility, has been associated with low birth weight followed by a rapid catch-up growth in infancy 47;48. The
association between hyperglycaemia in pregnancy and subsequent development of PCOS has not been studied
previously.
2. Gene-environment interactions and metabolic memory at birth
Pathophysiological mechanisms behind fetal developmental changes observed in relation to intrauterine
hyperglycaemia are complex and unclear49. Oxidative stress induced by hyperglycaemia and subsequent altered
gene expression and accelerated apoptosis may be a general mechanism behind formation of congenital
malformations. Direct actions of hyperglycaemia and hyperinsulinaemia on adipose tissues, muscles, liver, blood
vessels and pancreas are possible pathogenetic pathways. From previous studies of tissue biopsies from healthy
humans exposed to hyperglycemia50 or exogenous insulin51-53 or suffering from type 1 diabetes54-55, type 2
diabetes56-57, or insulin resistance in high-risk individuals, e.g. PCOS58, and first-degree relatives57 it is clear that
insulin and hyperglycaemia regulates gene expression in human skeletal muscle and adipose tissue50,59, and also
that insulinopenia in type 1 diabetes60 and relative insulinopenia in insulin resistant conditions56,57,58,61 are
associated with specific patterns/signatures of abnormalities. Common characteristics seem to include
disturbances in muscle protein synthesis (down) and proteasomal degradation (proteolysis) (up)54,60,61, reduced
mitochondrial biogenesis55-59,61, and/or ATP synthesis rate55,62, and increased oxidative stress63,64. However, the
epigenetic transgenerational transmission of type 2 diabetes and overweight seems also to involve hypothalamic
regions of the brain through insulin mediated central insulin resistance, induced either by fetal hyperglycaemia or
early neonatal overfeeding.
Hyperglycaemia adversely affects hippocampal regions and cognitive function in adult rats and these changes
may also be seen in offspring exposed to diabetes in utero. In theory, a hyperglycaemic and hyperinsulinaemic
intrauterine environment could enhance leptin and other hormones, increase childhood linear growth and
obesity and thereby modulate onset of puberty and glucose- and fat metabolism in adolescents.
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The role of insulin seems ambiguous; on one hand insulin is needed to prevent direct damage caused by
hyperglycaemia, on the other hand elevated levels of insulin during critical perinatal periods of life may
permanently alter organ functions.
Recent methodologies (single nucleotide polymorphism genotyping, genome wide association studies etc.) have
identified a large number of genes associated with type 1 diabetes. The strongest associations are seen with the
HLA system and the candidate genes: INS (insulin gene), PTPN22 (a functional variant of the lymphoid-specific
protein tyrosine phosphatise) and IL2RA (α-subunit of the IL-2 receptor complex) with odds ratios of 1.616.8065. These candidate genes play important roles in the immune system and therefore also possibly in
autoimmune processes. In this study we will test offspring of mothers with type 1 diabetes with respect to these
genes as a marker of genetic susceptibility of this condition in order to distinguish between the intrauterine
environmental influence and the genetic influence.
3. Aims of the study
Study I
To study long-term effects of a diabetic intrauterine environment in offspring of women with type 1 diabetes
compared to matched controls from the background population with respect to:
A)
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
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Cognitive function
Pubertal development
Diabetes/pre-diabetes
Overweight
B) To study if HbA1c-level in pregnancy is an independent predictor of these outcomes within the study group.
Study II
To study differences in offspring of women with type 1 diabetes compared to offspring from the background
population with respect to:
- morbidity and congenital malformations
- medical treatment
- mortality,
Study III
To study differences in offspring of women with type 1 diabetes compared to compared to matched controls
from the background population without a family history of diabetes with respect to:
A) State-of-the-art metabolic characterization using euglycemic-hyperinsulinemic clamp
B) DNA methylation, RNA transcription and protein quantification in muscle and adipose-tissue
4. Design
The study is a prospective follow-up study. The study group includes the offspring of women with type 1
diabetes from the national diabetes birth registry (1993-99, n=900) with information of HbA1c prior to
conception and/or 1st trimester HbA1c and a control group including offspring of women without diabetes who
delivered during the same period matched with respect to gender and age of offspring and the family’s postcode
as an indirect marker of the socio-economic background.
4.1 Material and Methods
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Clinical characteristics at the time of exposure:
Study group (diabetes-exposed):
The following data were reported to the national diabetes birth registry:
Mothers:
 Demographics (age, parity, height, pre-gestational weight)
 Diabetes status (pregestational /1st trimester HbA1c, 3rd trimester HbA1c, pre-gestational /1st trimester
Urine Albumin Excretion Rate, hypertension, proliferative retinopathy, diabetes duration, pregestational
insulin requirements (IE/d), occurrence of severe hypoglycemic events)
 Pregnancy complications (Preeclampsia, preterm delivery)
Offspring:
 Demographics (gestational age, birth weight)
 Neonatal morbidity (congenital malformations, hypoglycaemia, respiratory distress syndrome, jaundice,
systemic infection)
Control group
Maternal age, parity, height, pre-gestational weight, information on preeclampsia, gestational age, birth weight
and neonatal morbidity will be collected from medical records.
Examination program at follow-up:
The examinations of the participants will be performed in two different locations (Odense and Århus). The
examinations will be performed in two steps (see study design figure 1). First, a clinical examination (Study I)
growth, pubertal development, metabolism and cognitive function will be performed on 900 diabetes-exposed
offspring from the national diabetes birth registry and 450 matched controls. A register-based study (Study II)
on the entire cohort from the national diabetes birth registry will be performed in order to compare the
morbidity, medical treatment and mortality in this cohort with the background population. Finally, a sub-study
(Study III) on a group of 50 diabetes-exposed and 50 un-exposed controls will be performed in order to
characterize epigenetic changes in this population and to perform a thourough state-of-the-art metabolic
characterization of this subgroup (figure 1).
Study I: Clinical study (n450 diabetes-exposed and n450 controls aged 12-18 years)
Clinical examination to determine the effect of intrauterine hyperglycaemia on growth, pubertal development,
metabolism and cognitive function in childhood.
Study population
All 900 children in the national diabetes birth registry will be invited and with an expected participation rate of
50% there will be approximately 450 diabetes-exposed and 450 controls from the Central Person Register
matched according to gender, age and postcode.
Exclusion criteria: Offspring with major handicaps or chronic disease will be registered, but will not be
examined. Multiple pregnancies and recurrent pregnancies will not be included in the study.
Examinations
The participants meet fasting in the morning. The following examinations will be performed:
Diabetes/prediabetes:
 Oral glucose tolerance test (OGTT) with glucose, insulin, C-peptide and proinsulin at 0, 30, 60, 120
minutes
 Blood pressure
 HbA1c, cytokines (CRP, IL-6), adiponectin, leptin, inkretins
 GAD-antibodies
 EDTA blood sample for DNA analysis
Obesity:
 BMI, waist/hip circumference
 Lipids (total, HDL-C, LDL-C, triglycerides)
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 Dual energy X-ray absorptiometry (DEXA) scan to determine body composition
Growth and pubertal development:
 Height, sitting height, weight, head circumference
 Pubertal stage (Breast development/testicular size and pubic hair)
 Hirsutism in girls?
 X-ray of the left hand in order to determine bone age (BA).
 Trans-abdominal ultrasound examination of uterus and ovaries in the girls in order to determine size of
the internal genitalia and numbers of follicles in the ovaries
 IGF-I, IGFBP-3, IGFBP-1, free IGF-I
 Testosterone, estradiol, SHGB, FSH, LH, inhibin A, inhibin B, AMH
 Adrenal androgens (4-Adion, DHEAS)
 Thyroid hormones (TSH, T4, fT4)
Cognitive examination including:
 Assessment of global intelligence:
WISC or WAIS for the oldest participants
 Assessment of specific cognitive functions
Attention
Learning and Memory
Psychomotor speed and reaction time
The children and their parents will be asked to fill in a questionnaire addressing psychosocial aspects,
demographics (height, weight) and history of menstrual cycle. Furthermore, the children will be asked to fill in a
questionnaire on Self-reported physical activity (IPAQ-questionnaire)64.
Primary endpoints
 Cognitive function
 Pubertal development
 Diabetes/pre-diabetes
 Overweight (> 85-percentile for age and gender)
Secondary endpoints
 BMI
 Body composition (% body fat)
 Blood pressure
 Dyslipidaemia
 Insulin levels
 Markers of endothelial function
 Markers of autoimmunity (GAD-antibodies)
 PCOS
Study II: Register-based study (n=900 diabetes-exposed; for every case we will sex and age match 100
controls, resulting in n=90,000 controls aged 12-18 years)
A register-based study will be performed to determine the effect of intrauterine hyperglycaemia on congenital
malformations, morbidity and mortality in childhood.
Study population
In the cohort of 900 children from the national diabetes birth cohort, we will study all subjects utilizing register
data concerning morbidity, mortality and use of prescription medicine. For every diabetes-exposed person
Statistics Denmark will identify 100 age, sex and calendar-time matched controls from the background
population from the Central Person Register (Statistics Denmark).
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Morbidity and mortality - Operational strategy
a) In the National Registry of Patients (NRP) and the National Cancer Registry all diagnoses from identified
diabetes-exposed and controls will be identified.
b) In the National Drug Prescription Database all information concerning prescription drugs on identified
diabetes-exposed and controls will be identified.
c) In the National Registry of Death all diagnoses from identified diabetes-exposed and controls will be
identified.
Endpoints
Morbidity: diabetes-exposed vs. controls from the background population
Medical treatment: diabetes-exposed vs. controls from the background population
Mortality: diabetes-exposed vs. controls from the background population
Statistics
Incidence rates will be calculated as new cases per 100.000 per year and analyzed by Possion regression. Via
Statistics Denmark controls will be identified and matched appropriately.
Morbidity and mortality will be analyzed by the Kaplan-Meier statistic, with log-rank test and Cox regression
with relevant covariates.
Study III: Sub-study (n=50 diabetes-exposed and n=50 un-exposed controls aged 18-19 years)
A selected subgroup of offspring of mothers with type 1 diabetes and matched controls will be investigated by
state-of-the-art metabolic characterization using euglycemic-hyperinsulinemic clamp with tracer glucose
combined with indirect calorimetry allowing reliable estimates of glucose disposal rates, endogenous glucose
production and glucose and lipid oxidation. Moreover assessment of physical activity level using the IPAQquestionnaire will be complemented by studies of maximal oxygen consumption (VO2max). During the clamp
studies, tissue biopsies from subcutaneous abdominal fat and thigh muscle (m. vastus lateralis) will be obtained
for studies of potential long-term molecular consequences of intrauterine exposure to hyperglycemia – metabolic
memory of birth. This will include application of several discovery-mode (hypothesis free), global approaches
such as DNA methylation and transcriptional profiling using microarray-based technologies, quantitative
proteomics, bioinformatics including pathway analysis and subsequent validation of observed abnormalities using
qRT-PCR, immunoblotting and other more classical protein technologies.
Study population:
Fifty offspring of mothers with type 1 diabetes will be matched to 50 healthy subjects from the former control
group according to BMI, gender, age, level of physical activity. All participants should be drug-naive and healthy
with a BMI between 20 and 30, and controls with no family history of diabetes. All participants will be over the
age of 18 and all participants should be able to provide informed written consent.
Exclusion criteria: 1) Any unknown disease or need for medication that occurs after inclusion, 2) Abnormal
ECG, screening blood tests and/or severe hypertension, and 3). Impaired glucose tolerance in non-diabetic
subjects.
Examinations:
Physical activity
Self-reported physical activity (IPAQ-questionnaire)64. Determination of VO2max by a graded maximal test
(VO2peak) on a cycle ergometer using indirect calorimetry64.
State-of-the-art metabolic characterization:
Euglycemic-hyperinsulinemic clamp
Subjects are admitted after a 12-h overnight fast to the centre at Odense University Hospital. They are instructed
to consume a standardized diet, and to refrain from physical activity for 48-h before the experiments. The study
subjects are examined by a euglycemic-hyperinsulinemic clamp (insulin 40mU/min/m2 for 4-h) using tracer
technology as described55. The studies are combined with indirect calorimetry allowing estimates of glucose
disposal rates, endogenous glucose production, glucose and lipid oxidation, and non-oxidative glucose
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metabolism as described55. Blood samples are drawn every 5-20 min during the clamp for assessment of plasma
glucose, FFA, adipokines, and serum insulin and C-peptide. During the basal and insulin-stimulated states, tissue
biopsies are taken from m. vastus lateralis and subcutaneous abdominal fat using a modified Bergström needle
with suction under local anesthesia. Each biopsy is rapidly frozen in liquid nitrogen within 30 s and stored at 130°C for later analysis as described below. Body fat (%) is determined by the bioimpedance method.
Transcriptomic and proteomic analysis of skeletal muscle and adipose tissue
Microarray-based transcriptional profiling and biological pathway analysis:
Total RNA is extracted from skeletal muscle and adipose tissue using the TRIzol protocol (Life Technologies,
Gaithersburg, MD) and prepared according to the Affymetrix manual (Affymetrix, Santa Clara, CA) for
microarray analysis using the Affymetrix HGU133 Plus 2.0 chips as described previously55. The R statistical
software was applied for data preprocessing (www.bioconductor.org) and statistical analysis as described55.
Global pathway analysis will be performed using two recognized different approaches for global pathway analysis
as described. Thus, the gene map annotator and pathway profiler (GenMAPP 2.1), including the integrated tool
MAPPFinder 2.1, and gene set enrichment analysis (GSEA 2.0.1), were employed to assess significantly regulated
pathways, gene ontology (GO) terms, and gene sets in the two data sets as described in details previously.
Discovery-mode quantitative proteomics of tissue biopsies:
For a comprehensive unbiased investigation of differences in protein abundance we will employ state-of-the-art
nanospray tandem mass spectrometry (MS/MS) technology in combination with an approach termed ”isobaric
tag for relative and absolute quantification” (iTRAQ)66. The iTRAQ methodology allows quantification of
differential abundance of proteins in skeletal muscle and adipose tissue from the 2 study groups. It is expected
that this discovery-mode quantitative proteomic approach will lead to the identification of critical pathways
characterizing the molecular and metabolic abnormalities in muscle and fat of offspring of mothers with type 1
diabetes.
Targeted quantitative proteomics:
The results obtained by transcriptional profiling, RT-PCR and discovery-mode phosphoproteomics will be used
to create a strategy for validation of these findings by targeted quantification of selected sets of up to several
hundreds of proteins. We will take advantage of a novel targeted phosphoproteomic approach called multiple
reaction monitoring (MRM)67 using a new high-sensitivity LC-MS/MS system, the Thermo TSQ Vantage, which
has been installed at the Protein Research Group at University of Southern Denmark. Interesting differences in
protein abundance will be further validated by Western blot analysis using commercial antibodies and kinase
activity assays.
Epigentic changes
These latter tissues will be studied using DNA and RNA methodology enabling profiling of methylation status
(DNA) of 450,000 CpG islands68 and the effect this has on expression (RNA) and compared with the
phenotypic outcome (see above). We will utilize new methodology such as next generation sequencing (NGS)69,70
which will enable us to analyze large amounts of genetic data.
Analysis of DNA methylation
Genomic DNA is extracted from white mononuclear cells, adipose and muscle tissue using standard methods.
One microgram of DNA from each tissue is bisulfite modified using EpiTect Bisulfite Kit (Qiagen, Copenhagen,
Denmark) for MS-HRM, and using EZ-96 DNA Methylation D5004 (Zymo Research, Orange, CA) for
microarrays and bisulfite sequencing.
Bisulfite sequencing. Bisulfite modified DNA is amplified using primers designed with MethPrimer
(http://www.urogene.org/methprimer/index1.html) and TEMPase DNA Polymerase (Ampliqon, Skovlunde,
Denmark). PCR products is gel-purified and cloned using TOPO TA Cloning ® Kit for Sequencing
(Invitrogen,Taastrup, Denmark). PCR amplification for sequencing is performed directly on the colonies with
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M13 primers (DNA Technology, Risskov, Denmark) and TEMPase DNA Polymerase (Ampliqon). For each
gene, 10 clones were randomly selected and sequenced using BigDye terminator cycle sequencing kit and a
3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA).
Whole genome methylation analysis. Bisulfite modified DNA is whole genome amplified and hybridized to Infinium
HumanMethylation450 BeadChips (Illumina, San Diego, CA) overnight as described by the manufacturer.
BeadChips were scanned with a BeadXpress Reader instrument (Illumina) and data analyzed using Genome
Analyzer IIx Methylation Module Software (Illumina). Methylation levels will be provided in beta values, with a
beta value of 0 corresponding to no methylation, and 1 corresponding to full methylation.
Methylation-Sensitive High Resolution Melting analysis (MS-HRM). The validation set will consist of samples from 200400 additional offspring from the initial cohort. Amplification of bisulfite modified DNA is performed in
triplicates with primers designed according to guidelines published by Wojdacz et al.71
Gene Expression Analysis
RNA is extracted from white blood cells, adipose and muscle tissue standard methods. For gene expression
analysis, 100 ng of total RNA is labeled using GeneChip Whole Transcript (WT) Sense Target Labeling Assay
(Affymetrix, Santa Clara, CA) and hybridized overnight to Human Exon 1.0 ST Arrays (Affymetrix) according to
the manufacturer’s instructions. Arrays are scanned in an Affymetrix GCS 3000 7G scanner. To avoid batch
effects, all samples will be labeled and scanned in random order. Data analysis is performed using GeneSpring
GX 10 software (Agilent Technologies, Naerum, Denmark). Samples are quantile-normalized using iterPLIER16
with transcript level core (17881 transcripts) and by using antigenomic background probes.
Quantitative RT-PCR
Quantitative real-time RT–PCR (qRT–PCR) is performed in triplicates on a ABI 7500 Fast Real Time System
(Applied Biosystems) using the relevant (TaqMan or SYBR Green) Master Mix (Applied Biosystems). For
normalization, the gene Ubiquitin C (UBC) is employed. The suitability of UBC as a normalization gene for
analysis of normal mucosa and CRC specimen sample sets and the UBC primer sequences have been published
earlier.54
Sequencing
After purification using QIAquick PCR Purification Kit (Qiagen), sequencing of selected samples will be
performed using BigDye terminator cycle sequencing kit and a 3130xl Genetic Analyzer (Applied Biosystems)
and primers used for MS-HRM analysis.
Statistical Analysis
Mann-Whitney U test will be used to assess for each CpG, whether two groups had the same distribution of
methylation. To eliminate probes that did not work and other diverging values, beta values in each group will be
trimmed for extremes prior to the calculation of the average beta value. Next, only genes with the highest 5% tail
of the absolute average beta value differences between the two groups will be selected. Thus CpGs with a pvalue below 0.005 and with the highest beta value differences between the two groups will be used for
identification of genes with statistically significant diabetes-specific changes in methylation. Comparison of array
methylation results with MS-HRM results will be performed with a Mann-Whitney U test and a Chi2 test for
trend. Spearman coefficients will be calculated to assess the correlation between methylation and gene
expression.
5. Statistical power and sample size
Study I:
With a power of 0.8 and a type 1 error of 0.05 (two-sided test) the following sample size in each group was
calculated:
 Cognitive function (cognitive deficits (IQ <80): Expected proportion in study group vs. control group:
20% vs. 8% - ie. Sample size 131
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 Diabetes/pre-diabetes: 11% vs. 4% - ie sample size 222
 Pubertel development: No available data.
 Obesity: 10% vs. 5% - ie. Sample size 435
On the basis of these calculations a sample size of about 450 participants in each group seems justified.
Study III:
With a power of 0.9 and a type 1 error of 0.05 (two-sided test) the following sample size in each group was
calculated:
 Euglycemic-hyperinsulinemic clamp (Insulin stimuleret Rd) 15% difference between diabetes-exposed
cases and controls –ie. Sample size 100
On the basis of these calculations a sample size of 50 participants in each group seems justified.
6. Ethical considerations
The study will be carried out in accordance with the Helsinki Declaration. Acceptance from ethical committees
will be obtained. Written informed consent will be obtained from the patients and their parents. Subject
information and informed consent form will be provided as required by local regulations.
7. Novelty and importance
The rapidly increasing burden of overweight and cardiovascular disease is becoming a threat to both the
individual and global economy. It is therefore essential to identify risk groups to target preventive strategies.
Exposure to intrauterine hyperglycemia contributes to the epidemic through a vicious-cycle passing increased
susceptibility on to the next generation via pathways that are only sparsely understood.
This study includes a very large cohort of diabetes-exposed offspring of well-characterized prospectively studied
women with type 1 diabetes. It includes information on maternal glycemia during pregnancy, which is imperative
to assess possible associations with estimates of offspring metabolism and cognitive function. The register-based
study is unique and enables collection of information on morbidity and mortality in the whole cohort. The
specific molecular signature of intrauterine exposure to hyperglycemia in human tissues has to our knowledge
not been examined before, and although the above-mentioned studies suggest the possible involvement of
certain pathways in skeletal muscle and adipose tissue, it is important to investigate the metabolic memory of
birth using hypothesis-free, discovery-mode global approaches to discover potential perturbations in both
expected and unexpected pathways
8. Facilities available
Facilities available
The study will be performed in two highly specialised endocrine units in Odense and Århus.
The clinical unit in Odense processes both inpatient and outpatient, and more than 3,000 patients with
diabetes are in regular control. The Diabetes Research Centre at Odense University Hospital is led by
professor Henning Beck-Nielsen. There is a long tradition for investigations of glucose and fat metabolism in
skeletal muscles, which has resulted in up to hundred publications within this area. The core facility is the
Diabetic Research Center, a metabolic unit set up for whole body investigations; euglycemic-hyperinsulinemic
clamp, indirect calorimetry, tracer kinetics, and muscle and fat biopsies. In this unit two technicians are
serving together with 2 research nurses, and ad hoc ph.d.-students.
Microarray-based transcriptional profiling, qRT-PCR and immunoblotting of tissue biopsies will take place at
our newly established section for Molecular Diabetes & Metabolism directed by Kurt Højlund. At present,
one post-doc and two ph.d.-students
The facilities for discovery-mode and targeted quantitative proteome analysis and mass spectrometry are
located at the Department of Biochemistry and Molecular Biology at the University of Southern Denmark
led by associate professor Ole Nørregaard Jensen. In addition to technicians, one ph.d.-students will be partly
dedicated to the project. The facilities are considered to be among the leading centers in the world for that
purpose.
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The clinical unit in Århus processes both inpatient and outpatient, and more than 3,000 patients with diabetes
are in regular control. We have a long research tradition for investigations of glucose and fat metabolism in
skeletal muscles. The core facility is the Medical Research Laboratories at Aarhus University Hospital, a
metabolic unit set up for whole body investigations; euglycemic-hyperinsulinemic clamp, indirect calorimetry,
tracer kinetics, and muscle and fat biopsies. In this unit 13 technicians are serving together with 3 research
nurses, and 20 ph.d.-students.
The facilities for epigenetic studies are located at the Medical Molecular Laboratory at Aarhus University
Hospital led by professor Torben Ørntoft. The laboratory is set up to provide high-throughput sequencing
using next-generation sequencing (NGS) techniques with the cutting edge technology, together with a host of
other techniques. In this laboratory 6 post-docs, 20 phd-students and 15 technicians are serving. The
facilities are considered to be among the leading centers in the world for that purpose.
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Environmental versus genetic and epigentic influences on growth, metabolism and cognitive function
in offspring of mothers with type 1 diabetes
Introduction
The rapidly increasing burden of overweight and cardiovascular disease is becoming a threat to both the
individual and global economy. It is therefore essential to identify risk groups to target preventive strategies.
Exposure to intrauterine hyperglycemia contributes to the epidemic through a vicious-cycle passing increased
susceptibility on to the next generation via pathways that are only sparsely understood.
Aim
The aim of this study is to examine the long-term consequences of a diabetic intrauterine environment in
offspring of women with type 1 diabetes compared to matched controls from the background population
without a family history of diabetes. The study will focus on the influence of a diabetic intrauterine environment
and the genetic and epigenetic influences on later health, growth, metabolism and cognitive function in
childhood and adolescence.
Methodology
The study is a prospective follow-up study of a large cohort of offspring of women with type 1 diabetes from the
national diabetes birth registry (1993-99, n=900). The national diabetes birth registry includes detailed
information of the pregnancies of women with type 1 diabetes, which has been published previously. The
registry contains detailed information on maternal age, parity, pre-gestational weight, diabetes status during
pregnancy and pregnancy complications. All 900 offspring will be invited to participate in the follow-up study in
2011 where they will be aged 12-18 years. In addition, a group of controls will be invited. This group consists of
offspring of women without diabetes who delivered during the same period matched with respect to gender and
age of offspring and the family’s postcode as an indirect marker of the socio-economic background.
The study will be divided into three different studies:
Study I: Clinical study (n450 diabetes-exposed cases and n450 matched controls)
A clinical examination performed in order to determine the effect of intrauterine hyperglycaemia on growth,
pubertal development, body composition, metabolism and cognitive function in childhood and adolescence.
Clinical examination, fasting blood samples, oral glucose tolerance test, DEXA scan and cognitive tests will be
performed.
Study II: Register-based study (n=900 diabetes-exposed; for every case we will sex and age match 100
controls, resulting in n=90,000 controls aged 12-18 years)
A register-based study will be performed to determine the effect of intrauterine hyperglycaemia on congenital
malformations, morbidity and mortality in childhood.
Study III: Substudy (n=50 diabetes-exposed and n=50 un-exposed controls aged 18-19 years)
A selected subgroup of offspring of mothers with type 1 diabetes and matched controls will be investigated by
state-of-the-art metabolic characterization using euglycemic-hyperinsulinemic clamp and examination of maximal
oxygen consumption (VO2max). Tissue biopsies from subcutaneous abdominal fat and thigh muscle will be
obtained for studies of potential long-term molecular consequences of intrauterine exposure to hyperglycemia –
metabolic memory of birth. This include application of several discovery-mode, global approaches such as DNA
methylation and transcriptional profiling using microarray-based technologies, quantitative proteomics,
bioinformatics including pathway analysis and subsequent validation of observed abnormalities using qRT-PCR,
immunoblotting and other more classical protein technologies.
This large cohort with detailed information on diabetic pregnancies collected prospectively gives us a unique
opportunity to examine the long-term consequences of a hyperglycaemic intrauterine environment on growth,
pubertal development, metabolism and cognitive function in childhood and adolescence. The register-based
study is unique and enables collection of information on morbidity and mortality in the whole cohort.
Examinations of the specific molecular signature of intrauterine exposure to hyperglycemia in human tissues
have to our knowledge not been done before.
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Figure 1: Study design
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