1. Genetics of diabetes Genetic factors influencing onset of diabetes
Pat Scott
Clinical approaches to controlling diabetes
Krisa Christian

2. What is diabetes?
Diabetes is a metabolic disease in which the body is unable
to use glucose because of failure to produce or inability to use insulin.
Two principle forms of diabetes are Type 1 Diabetes
and Type 2 Diabetes.
Impact of diabetes:
Affects 7.8% of US population at this time.
Incidence is growing.
Leading cause of end-stage renal disease,
adult-onset blindness, non-traumatic leg amputations.
Major risk factor for coronary artery disease
and cerebrovascular disease.
National Diabetes Information Clearinghouse

3. Overview: Genetic factors influencing onset of diabetes
Introduction
Key role of insulin in glucose metabolism
Overview of genetics of diabetes
Type 1 Diabetes
Etiology
Genetic contribution- MHCII polymorphisms
Type 2 Diabetes
Etiology
Genetic contribution- monogenetic diseases
Genetic contribution- multifactorial disease

4. Why is insulin important?
Glucose metabolism in healthy individual
HIGH blood glucose triggers actions to use glucose:
Release of insulin from pancreas
Insulin acts on liver to stimulate glycogen synthesis
enzymes, inhibit gluconeogenic enzymes.
Insulin acts on skeletal muscle and adipose cells
to promote uptake of glucose. Glucose is used to generate
energy, to synthesize lipids, proteins
LOW blood glucose triggers actions that release glucose:
Release of glucagon from pancreas
Effect of insulin on insulin responsive tissues:
Liver-
Glucose is taken up by Glut2 glucose transporters which are not insulin dependent.
Insulin stimulates glycogen synthesis by stimulating glycogen synthase, inhibits glycogen phosphorylase.
Effect iof insulin stimulation of glycogen synthesis is to pull glucose into cell because it is used up as it is brought in.
Skeletal muscle-
Promotes uptake of glucose by promoting translocation of Glut4 glucose transporter to the cell surface.
Glucose is used to generate energy, synthesize glycogen and protein.
Adipose tissue-
Promotes lipogenesis by increasing lipoprotein lipase activity.
Inhibits lipid breakdown by inhibiting hormone sensitive lipase.
Net effect decreased levels of fatty acid in blood.
Glucagon acts on liver to promote glucose production via
gluconeogenesis and breakdown of glycogen
Insulin is key to utilization of glucose and glucose homeostasis.

5. How does insulin regulate glucose homeostasis?
Insulin regulation of glucose levels requires:
Pancreatic beta cells to make insulin.
Regulatory system in pancreatic beta cells to
stimulate insulin release in response to blood
glucose levels
Pancreatic beta cell
Pancreatic beta cells: specialized cell type in pancreas that synthesizes and secretes insulin.
In healthy individuals beta cells have the capacity to increase in size and number if necessary.
This is mediated by various transcription factors.
Regulatory system:
Insulin secretion is regulated primarily by blood glucose levels.
Insulin secretion pathway is commonly disrupted in Type 2 diabetes.
Glucose is taken up by Glut2 transporter.
Glucose is phosphorylated by glucokinase to glucose-6-phosphate.
Glucose-6-phosphate is substrate for glycolysis and oxidative phosphorylation which generate ATP.
Increased ATP inhibits potassium channel, causes accumulation of potassium in cell, which causes depolarization of the membrane.
Depolarization activates a voltage-dependent calcium channel.
Influx of calcium stimulates the release of insulin.
Insulin signaling:
Insulin binds insulin receptor, activates tyrosine kinase activity of receptor.
Activated receptor autophosphorylates, creates site sfor binding of signaling molecules, initiates signaling cascade.
Signaling cascade results in actions that promote utilization of glucose.
Intact insulin signaling system in target cells
to utilize glucose
Liver
Adipose tissue and skeletal muscle

6. How is insulin function defective in diabetes?
In diabetes one or more of these elements is impaired.
Type 1 Diabetes
~5%
Pancreatic beta cells destroyed by
autoimmune process.
Pancreatic beta cell
Type 2 Diabetes
~95%
Defects in both pancreatic beta cells and target cells
Intracellular signaling system of target cells
no longer responds to insulin. (Insulin resistance)
Beta cells can no longer increase insulin
output in response to glucose
Liver
Adipose tissue and
skeletal muscle

7. Why is genetics of diabetes important?
Diabetes has a significant genetic component
Different frequency among ethnic groups
Familial aggregation- close relatives have greater risk
Monozygotic twins have higher risk than dizygotic
Identifying genes with variants (mutations or polymorphisms) that increase risk of diabetes is difficult
because Type 1 and Type 2 Diabetes are complex diseases.
Both environment/lifestyle and genetics contribute.
Polygenic- multiple genes contribute
Heterogenous- different sets of genetic variants can cause disease in different individuals
Characteristics of inheritance of complex diseases-
No simple Mendelian pattern because a given genetic variant can have different disease outcomes, different genetic variants can have same disease outcome depending on environment, other genetic variation in genome.
But do show familial aggregation.
Identifying genetic variants that contribute to development of diabetes
has led to understanding pathophysiology, targets for prevention and treatment.
Diabetes remains a serious health problem so hope is that
a more detailed understanding will advance prevention, treatment and cure.

8. Type 1 Diabetes: Etiology
Pancreatic beta cells destroyed by autoimmune process.
1. T cell receptors (TCR) recognizing self antigens of pancreatic beta cells are
not weeded out because of genetic defects in immune system.
2. Specific viral infections lead to Inflammatory response.
(B)
Epitopes of some viruses mimic beta cell proteins.
Viruses promote inflammatory cytokine production.
3. Autoimmune response-
T cells expressing TCR which recognize beta cell peptides are amplified,
mount immune response against beta cells.
4. Individuals become symptomatic when ~80% of beta cells
are destroyed. Only recourse is insulin.
Mouse pancreas stained
for insulin which identifies beta
Cells. (A) Normal mouse, beta cells
Intact. (B) Diabetic mouse, most
beta cells destroyed

9. Type 1 Diabetes: Genetic contribution
Type 1 Diabetes does not show Mendelian pattern of inheritance, but does show familial aggregation.
Familial Aggregation
Relationship
Proportion of shared risk alleles
Risk 1
40%
Monozygotic twin
0.5 7%
Sibling
------
~0.3%
unrelated
Conclusions:
1. Genetic contribution because greater risk with greater genetic identity
2. Environmental contribution because risk for identical twin less than 100%
3. Polygenetic because not Mendelian inheritance

10. The major genetic variants associated with Type 1 Diabetes are polymorphisms in MHCII genes.
WHY?????
Chromosome 6
HLA (human leukocyte antigen) locus
MHCII genes
DP, DQ, DR
MHCII genes are
highly polymorphic
# of alleles for each gene
MHCII = major histocompatibility complex II
Specific MHCII alleles are strongly associated with
Type 1 Diabetes.
DR3/DQ1*0201
DR4/DQ1*0302

11. Role of MHCII proteins MHCII proteins:
Present antigen peptides to T cell with cognate TCR.
TCR interaction with complex of particular MHCII + peptide activates T cell.
Immune response:
MHCII presents foreign peptide to TH cell.
Initiates adaptive immune response.
Tolerance to “self” antigens:
During T cell development MHCII proteins present “self” peptides on cell surface.
T cells expressing TCR that strongly bind to specific MHCII + “self” peptide complex are targeted for apoptosis.
Tolerance requires that individual’s MHCII proteins be able to bind self antigens.
T cell
apoptosis
MHCII proteins:
Expressed on the surface of antigen presenting cells (APC)
Present antigen peptides to T cell with cognate TCR (T cell receptor)
TCR recognizes complex of particular MHCII variant + peptide.
This interaction activates T cell.
Note-
TCR binding is determined by both MHCII protein and peptide.
Because there are multiple MHCII genes and multiple alleles of each gene, there are several different MHCII proteins available to present peptides in each individual.
Also, individuals vary even more widely in repertoire of MHCII proteins expressed.
MHCII restriction:
Polymorphisms in MHCII genes determine individual repertoire of MHCII proteins.
Repertoire of MHCII proteins determines which peptides will be presented to TCR.
Repertoire influences immune response and tolerance.

12. MHCII polymorphisms linked to Type 1 Diabetes have altered binding site sequence.
Specific MHCII alleles are strongly associated with Type 1 Diabetes:
DR3/DQ1*0201
DR4/DQ1*0302
DQ1 alleles associated with diabetes have altered amino acid in binding site.
Normal Asp57
High risk polymorphisms Asp57Ala
Asp57Val
Asp57Ser
Transgenic mice carry high risk alleles display diabetic characteristics.
Janeway, 2001
Model:
High risk variants of MHCII can not bind pancreatic beta cell “self” peptides strongly.
T cells carrying TCR for these peptides are not weeded out during development.
These T cells have the potential to mount an autoimmune attack when the right environmental trigger occurs.

13. Can Type 1 Diabetes be prevented?
Insulin deficiency does not become clinically apparent until 80% of beta cells are destroyed.
Too late to reverse.
Autoimmune process starts much earlier.
Has pre-clinical phase identifiable by immune changes.
Current research aims at:
1. identifying individuals at earliest stage of autoimmune process
2. Blocking process to prevent beta cell destruction
Natural history study:
For close relatives of individuals with type 1 Diabetes
Screen for autoantibodies to beta cell proteins as early indicator of autoimmune response.
Follow to determine if antibodies predict outcome.
2. Oral insulin study.
For close relatives.
Autoantibodies to insulin are the among the most common in type 1 Diabetes.
Hope to restore tolerance by increasing exposure to insulin.

14. ????????

15. Type 2 Diabetes: Etiology
Involves defects in both insulin responding target tissues
and insulin producing pancreatic beta cells
Insulin signaling pathway in target tissues is inhibited.
Insulin is made, binds to receptor but cell no longer responds. (Insulin resistance)
Main risk factor is obesity.
Liver
Adipose tissue and skeletal muscle
Pancreatic beta cells lose capability to make sufficient insulin.
Defects in glucose-mediated regulation of secretion.
Impaired ability to increase mass and cell number.
In prediabetic stages one or other may predominate.
But ultimately these defects amplify each other.
End result is Type 2 Diabetes, clinical inability to utilize glucose.

16. What causes inhibition of insulin signaling?
Model:
Excess nutrients create stress in adipose tissues.
Trigger release of inflammatory cytokines.
Trigger increase in circulating free fatty acids.
Inflammatory cytokine such as TNF alpha and free fatty acids bind to receptors on target cell surface.
Activate intracellular kinases, especiallyJNK.
Kinases phosphorylate and inactivate IRS1, critical hub for insulin-mediated signaling.
Net result: Glucose is not removed from blood,
Hyperglycemia.
adipocytes
Free Fatty Acids
Inflammatory
cytokines
Insulin
Responsive
tissue
IRS1
JNK
Glycogen synthesis
GLUT4 translocation
Glucose uptake

17. In Type 2 Diabetes beta cells lose capability to secrete insulin.
Normal pancreatic beta cells can compensate for
prolonged hyperglycemia/insulin resistance by
Increased insulin secretion and increased beta cell
growth.
In Type 2 Diabetes beta cells are unable to
compensate for hyperglycemia.
Excess glucose and free fatty acids have toxic effects
on beta cells.
Excess nutrients overload mitochondria and
endoplasmic reticulum.
Create stress responses leading to suppression of
insulin secretion and apoptosis.

18. Type 2 Diabetes: Genetics
Type 2 Diabetes makes up ~95% of all diabetes.
5% monogenetic:
single gene defect causes disease
Mendelian, autosomal dominant inheritance
90% multifactorial:
Strong genetic contribution
Environment (obesity) also important
Polygenic: multiple genes contribute
Heterogenous: different combinations of high risk alleles in different individuals

19. Monogenetic Type 2 Diabetes
Includes Maturity Onset Diabetes of the Young (MODY), insulin resistance syndromes,
mitochondrial diabetes and neonatal diabetes.
Because of Mendelian inheritance individual disease genes have been mapped using classical methods
such as linkage analysis.
Classical linkage mapping
Purpose: Identify genetic variant responsible for a disease
Individual with disease differs from healthy individual at
site of disease allele and throughout genome.
Genetic variant that causes disease can be identified by
tracking nearby polymorphic markers of affected individual.
Region of chromosome found in individuals with disease, not in those without disease identifies location of genetic variant responsible for disease.

20. Genes that cause monogenetic forms of Type 2 Diabetes have added to our understanding and treatment of typical Type 2 Diabetes as well.
Glucokinase:
Enzyme that converts glucose to glucose-6-phosphate,
Initial step in glucose regulation of insulin secretion
Kcnj11:
Encodes subunit of potassium channel.
Inhibition of this channel is a key step in glucose-mediated
regulation of insulin secretion.
ABCC8:
Encodes SUR1, sulfonylurea receptor 1,
the other subunit of the potassium channel.
Binding of sulfonylurea to this receptor inhibits receptor activity,
promotes insulin secretion.
Hnf4A, TCF1, TCF2:
Transcription factors involved in beta cell proliferation and differentiation
PPAR gamma:
Promotes differentiation of adipocytes.
Promotes mitochondrial biogenesis.
Promotes insulin sensitivity in insulin target cells.
PPAR gamma agonists are used in treatment of Type 2 Diabetes

21. Typical Type 2 Diabetes is multifactorial
Kahn, 2008
Apparent strong genetic component. Monozygotic twin of individual with diabetes has
70% risk of developing disease.
No single gene identified has having strong effect.
Genomewide association (GWA) mapping used to identify multiple genes that each
contribute a small amount to phenotype.

22. Genomewide Association Mapping
Genetic variants that contribute to Type 2 Diabetes are identified by statistical analysis
of how frequently a region of a chromosome is associated with Type 2 Diabetes.
Chromosome regions are identified by single nucleotide polymorphisms (SNPs).
100,000s of SNPs over entire genome can be interrogated using chip arrays or barcoded beads.
Each study compares individuals with Type 2 Diabetes to the same number of controls
to identify statistical differences in association with SNPs.
GWA approach is unbiased- do not need any prior knowledge of genes.
Can identify multiple genetic variants that each make a small contribution to risk.

23. Starting point for GWA mapping is SNP map of human genome.
SNP map of chromosome 20, each dot represents 25 SNPs
Each SNP is a site, position on DNA sequence of genome, where one base pair of DNA sequence commonly differs between individuals. The SNP sequence consists of the single base pair difference and short flanking sequence.
SNPs can be easily identified by DNA sequencing or hybridization
One million single nucleotide polymorphisms have been identified placed along human genome sequence to create SNP map.
Chromosomal location of high risk genetic variant can be determined by
statistical determination of association of Type 2 Diabetes phenotype
with specific SNPs of known location.

24. X X X X Mapping by SNP association1 2 3 4 X
X = allele of gene X that confers susceptibility to Type 2
Diabetes
(1) 1 2 3 4
X = allele of gene x that confers no risk X
(2)
SNPS 1, 2, 3, 4 are found on the same chromosome as X.
SNPS 1, 2, 3, 4 are found on the same chromosome as X
SNPs 1, 2, 3, 4 are located at the same positions as SNPs 1, 2, 3, 4
but have a different sequences.
recombination X 1 2 3 4
Many generations
with recombination X 1 2 3 4
After many generations X is still always associated with SNPs 1 and 2 of
original chromosome (1) but not always with more distant SNPS 3 and 4.
SNPs 1 and 2 will be found more often in the genomes
of individuals with Type 2 Diabetes than any of the other SNPs.
SNPs 1 and 2 identify a chromosomal region that may contain an
genetic variant conferring susceptibility to Type 2 Diabetes.

25. X X X GWA analysis 1. SNP array: SNP sequences are arranged in grid.2 3 4 1 2 3 4 X 1 2 3 4 X 1 2 3 4
1. SNP array: SNP sequences are arranged in grid.
Sequence, position on grid and genomic location are known
2. Hybridize genomic DNA from one individual to each chip
3. If SNP is present in genome then genomic DNA will hybridize at site of that SNP, not other SNPS at
same genomic position
4. Analyze which SNPs are present more frequently in individuals with Type 2 Diabetes
These SNPs identify region which likely includes a susceptibility gene 1 2 3 4 1 2 3 4 1 2 3 4
Example: X

26. Summary of GWA studies as of September 2008
Total of five independent screens
7000 cases and 12,000 controls screened in initial phase
SNPs represent ~80% of genome
15 genetic loci identified as consistently associated with Type 2 Diabetes
Loci have relatively low odds ratios
Examples of candidate genes at these loci show complexity of this disease:
SLC0A8:
Zinc membrane transporter.
Highly expressed in beta cells
May transport zinc needed for insulin structure
into insulin secretory vesicles
IGFBP2:
Regulates translation of insulin-like growth factor 2
(IGF2).
IGF2 is a signaling molecule that contributes to
beta cell proliferation
Odds ratio: odds of individual carrying high risk allele developing disease vs. odds of noncarrier developing disease
Caveats:
GWA mapping identifies small region of genome. This region may contain several candidate genes. Candidate genes chosen representthose most likely to contribute to diabetes phenotype based on function.
Most GWA studies have been done on Caucasian/European populations. Most recent studies have been done on other populations, especially Japanese, have identified additional candidate genes.
FTO:
FTO polymorphisms are associated with obesity.
Effect on Type 2 diabetes risk is thought to be
through effect on obesity.

27. Goal is to develop high resolution picture of causes of Type 2 Diabetes
to identify means to prevent, treat and cure.

28. ??????