Genetics of diabetes

Genetic factors influencing onset of diabetes

Clinical approaches to controlling diabetes

Pat Scott

Krisa Christian

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.

Affects 7.8% of US population at this time.Incidence is growing.

National Diabetes Information Clearinghouse

Two principle forms of diabetes are Type 1 Diabetes and Type 2 Diabetes.

Impact of diabetes:

Leading cause of end-stage renal disease, adult-onset blindness, non-traumatic leg amputations.

Major risk factor for coronary artery disease and cerebrovascular disease.

Overview: Genetic factors influencing onset of diabetes

Key role of insulin in glucose metabolism

Overview of genetics of diabetes

Introduction

Type 1 Diabetes

Etiology

Genetic contribution- MHCII polymorphisms

Type 2 Diabetes

Etiology

Genetic contribution- monogenetic diseases

Genetic contribution- multifactorial disease

Why is insulin important?

Insulin is key to utilization of glucose and glucose homeostasis.

Glucose metabolism in healthy individual

HIGH blood glucose triggers actions to use glucose:

• Insulin acts on liver to stimulate glycogen synthesis enzymes, inhibit gluconeogenic enzymes.

• Release of insulin from pancreas

• 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

• Glucagon acts on liver to promote glucose production via gluconeogenesis and breakdown of glycogen

Insulin regulation of glucose levels requires:

Regulatory system in pancreatic beta cells to stimulate insulin release in response to blood glucose levels

Pancreatic beta cells to make insulin.

Intact insulin signaling system in target cells to utilize glucose

How does insulin regulate glucose homeostasis?

Liver Adipose tissue and skeletal muscle

Pancreatic beta cell

In diabetes one or more of these elements is impaired.

Pancreatic beta cells destroyed by autoimmune process.

Type 1 Diabetes

Type 2 Diabetes

~5%

~95%

Defects in both pancreatic beta cells and target cells

How is insulin function defective in diabetes?

• Intracellular signaling system of target cellsno longer responds to insulin. (Insulin resistance)

• Beta cells can no longer increase insulin output in response to glucose

Liver Adipose tissue and skeletal muscle

Pancreatic beta cell

Identifying genes with variants (mutations or polymorphisms) that increase risk of diabetes is difficultbecause Type 1 and Type 2 Diabetes are complex diseases.

Diabetes has a significant genetic component

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.

• Polygenic- multiple genes contribute

Why is genetics of diabetes important?

• Heterogenous- different sets of genetic variants can cause disease in different individuals

• Different frequency among ethnic groups

• Familial aggregation- close relatives have greater risk

• Monozygotic twins have higher risk than dizygotic

• Both environment/lifestyle and genetics contribute.

Mouse pancreas stained for insulin which identifies betaCells. (A) Normal mouse, beta cellsIntact. (B) Diabetic mouse, most beta cells destroyed

(A)

(B)

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.

3. Autoimmune response-

4. Individuals become symptomatic when ~80% of beta cells are destroyed. Only recourse is insulin.

T cells expressing TCR which recognize beta cell peptides are amplified,mount immune response against beta cells.

• Epitopes of some viruses mimic beta cell proteins.

• Viruses promote inflammatory cytokine production.

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

Monozygotic twin 1 40%

Sibling0.5 7%

1. Genetic contribution because greater risk with greater genetic identity

unrelated ------ ~0.3%

2. Environmental contribution because risk for identical twin less than 100%

Conclusions:

3. Polygenetic because not Mendelian inheritance

The major genetic variants associated with Type 1 Diabetes are polymorphisms in MHCII genes.

Chromosome 6

HLA (human leukocyte antigen) locus

Specific MHCII alleles are strongly associated with Type 1 Diabetes.

MHCII genes are highly polymorphic

# of alleles for each gene

MHCII genesDP, DQ, DR

DR3/DQ1*0201DR4/DQ1*0302

WHY?????

Immune response: • MHCII presents foreign peptide to TH cell.• Initiates adaptive immune response.

T cell apoptosis

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.

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.

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.

MHCII polymorphisms linked to Type 1 Diabetes have altered binding site sequence.

Specific MHCII alleles are strongly associated with Type 1 Diabetes: DR3/DQ1*0201DR4/DQ1*0302

DQ1 alleles associated with diabetes have altered amino acid in binding site.

Normal Asp57

High risk polymorphisms Asp57AlaAsp57ValAsp57Ser

Transgenic mice carry high risk alleles display diabetic characteristics.

Janeway, 2001

• 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.

Model:

Can Type 1 Diabetes be prevented?

• Insulin deficiency does not become clinically apparent until 80% of beta cells are destroyed.

• Autoimmune process starts much earlier.

• Current research aims at: 1. identifying individuals at earliest stage of autoimmune process

2. Blocking process to prevent beta cell destruction

www.DiabetesTrialNet.org

• Has pre-clinical phase identifiable by immune changes.

• Too late to reverse.

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Pancreatic beta cells lose capability to make sufficient insulin.• Defects in glucose-mediated regulation of secretion.• Impaired ability to increase mass and cell number.

• 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.

Type 2 Diabetes: Etiology

Liver Adipose tissue and skeletal muscle

Involves defects in both insulin responding target tissues and insulin producing pancreatic beta cells

• 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.

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.

Free Fatty Acids Inflammatory cytokines

JNK

adipocytes

IRS1

InsulinResponsivetissue

GLUT4 translocationGlucose uptake

Glycogen synthesis

Net result: Glucose is not removed from blood, Hyperglycemia.

• 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.

In Type 2 Diabetes beta cells lose capability to secrete insulin.

Normal pancreatic beta cells can compensate for prolonged hyperglycemia/insulin resistance byIncreased insulin secretion and increased beta cellgrowth.

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.

Type 2 Diabetes: Genetics

5% monogenetic:

90% multifactorial:

Type 2 Diabetes makes up ~95% of all diabetes.

Mendelian, autosomal dominant inheritance

Strong genetic contribution

Environment (obesity) also important

Polygenic: multiple genes contribute

Heterogenous: different combinations of high risk alleles in different individuals

single gene defect causes disease

Monogenetic Type 2 Diabetes

Because of Mendelian inheritance individual disease genes have been mapped using classical methods such as linkage analysis.

Includes Maturity Onset Diabetes of the Young (MODY), insulin resistance syndromes, mitochondrial diabetes and neonatal diabetes.

Classical linkage mapping

Region of chromosome found in individuals with disease, not in those without disease identifies location of genetic variant responsible for disease.

Purpose: Identify genetic variant responsible for a disease

Genetic variant that causes disease can be identified by tracking nearby polymorphic markers of affected individual.

Individual with disease differs from healthy individual at site of disease allele and throughout genome.

Glucokinase:

Kcnj11:

PPAR gamma:

Encodes subunit of potassium channel. Inhibition of this channel is a key step in glucose-mediated regulation of insulin secretion.

Enzyme that converts glucose to glucose-6-phosphate,

Initial step in glucose regulation of insulin secretion

Genes that cause monogenetic forms of Type 2 Diabetes have added to our understanding and treatment of typical Type 2 Diabetes as well.

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

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.

Transcription factors involved in beta cell proliferation and differentiationHnf4A, TCF1, TCF2:

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.

• 100,000s of SNPs over entire genome can be interrogated using chip arrays or barcoded beads.

• Each study compares100-1000 individuals with Type 2 Diabetes to the same number of controlsto identify statistical differences in association with SNPs.

• GWA approach is unbiased- do not need any prior knowledge of genes.

• 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).

Genomewide Association Mapping

• Can identify multiple genetic variants that each make a small contribution to risk.

• One million single nucleotide polymorphisms have been identified placed along human genome sequence to create SNP map.

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.

Starting point for GWA mapping is SNP map of human genome.

• SNPs can be easily identified by DNA sequencing or hybridization

• 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.

Mapping by SNP association

X1 2 3 4

X1 2 3 4

recombination

X1 2 3 4

X1 2 3 4

X = allele of gene X that confers susceptibility to Type 2 Diabetes

X = allele of gene x that confers no risk

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, 4but have a different sequences.

(1)

(2)

Many generationswith recombination

X1 2 3 4

X1 2 3 4

X1 2 3 4

X1 2 3 4

After many generations X is still always associated with SNPs 1 and 2 oforiginal chromosome (1) but not always with more distant SNPS 3 and 4.

SNPs 1 and 2 will be found more often in the genomesof individuals with Type 2 Diabetes than any of the other SNPs.

SNPs 1 and 2 identify a chromosomal region that may contain angenetic variant conferring susceptibility to Type 2 Diabetes.

X1 2 3 4

X1 2 3 4 1 2 3 4

1 2 3 4

GWA analysis

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 atsame 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

X1 2 3 4

Example:

1. SNP array: SNP sequences are arranged in grid. Sequence, position on grid and genomic location are known

SLC0A8:

IGFBP2:

FTO:

Summary of GWA studies as of September 2008

Total of five independent screens

7000 cases and 12,000 controls screened in initial phase

15 genetic loci identified as consistently associated with Type 2 Diabetes

Examples of candidate genes at these loci show complexity of this disease:

Loci have relatively low odds ratios

SNPs represent ~80% of genome

Zinc membrane transporter.Highly expressed in beta cells May transport zinc needed for insulin structure into insulin secretory vesicles

Regulates translation of insulin-like growth factor 2 (IGF2).IGF2 is a signaling molecule that contributes to beta cell proliferation

FTO polymorphisms are associated with obesity.Effect on Type 2 diabetes risk is thought to be through effect on obesity.

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

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