Matt Thomas

Determining The Association Between the Thr92Ala Type 2 Deiodinase Polymorphism and

Insulin Resistance in theOld Order Amish

Impact of Diabetes

17 Million in the U.S. Have Diabetes- 6.2% of the total population

Sixth leading cause of death in 1999

Complications Total cost of diabetes was $98

billion in 1997

Prevalence of Diabetes

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

1980

1981

1982

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

Year

Mil

lion

s of

Dia

gnos

ed C

ases

Type 2 Diabetes

Also known as- late onset diabetes- non insulin dependent- adult-type diabetes

Develops later in life Comprises 90-95% of all

diabetes cases Very heterogeneous

Normal InsulinRelease and Action

Pathogenesis

Insulin secretion - Pancreatic β -cells fail to produce

adequate insulin Insulin resistance

- Cells fail to respond properly to insulin

Result: Hyperglycemia

Hyperglycemia

Short-term- Ketoacidosis

Long-term- heart disease- stroke- high blood pressure- Blindness- kidney disease- amputations

Environment + Genetics

Multifactorial disease Environmental factors

- Diet- Physical activity

Genetic factors- Twin studies- Type 2 diabetes is polygenic- Candidate gene studies

“Thrifty Gene” hypothesis

Human ancestors- Periods of feast and famine- Active lifestyles

Improved energy storage

Humans today- Abundant diets- Sedentary lifestyles

Obesity and diabetes

Thyroid Hormones Role in regulation of the

metabolic rate and energy balance- i.e. hyper and hypo-thyroidism

Stimulate the resting metabolic rate

Drives glucose transporter-4 (GLUT4) transcription- GLUT4 protein allows glucose

uptake

Type 2 Deiodinase (DIO2) DIO2 metablolizes inactive thyroid

hormone (T4) into active thyroid hormone T3

Deiodination occurs in fat and skeletal muscle

I 3I 3’

OHO R

I 5I 5’

I 3I 3’

O R

I 55’

T3(Active)

T4(Inactive)

DIO2HO

The DIO2 Gene

Chromosome position 14q24.3- Chromosome 14- Long arm- Region 24.3

Thr92Ala DIO2 Polymorphism

Polymorphism- AG transition at nucleotide 274- Threonine is substituted by Alanine

at amino acid 92 Previous studies

- Used hyperinsulinemic-euglycemic clamp

- Thr92Ala strongly associated with decreased glucose disposal

Why Study the Old Order Amish (OOA)?

OOA are a closed founder group Large families Nearly complete genealogies Homogeneous lifestyles Amish Family Diabetes Study began in

1995- Detailed phenotypic characterization

• Used OGTT• DNA samples blood

Polymerase Chain Reaction (PCR)

Genomic DNA extracted from blood samples

Master mix:- Taq polymerase, PCR buffer, MgCl2,

primers Ran samples 40 cycles in thermocycler Sense primer:

- 5’ CTCAGGGCTGGCAAAGTCAAG 3’ Antisense primer:

- 5’ CCACACTCTATTAGAGCCATTG 3’

AG transition at nucleotide 274 Transition introduces restriction

site site- Restriction enzyme: BsgI- Digest PCR product with BsgI

Restriction Fragment Length Polymorphism (RFLP) Analysis

5 ’ . . . G T G C A ( N ) 1 6 . . . 3 ’5 ’ . . . C A G T C ( N ) 1 4 . . . 5 ’5 ’ . . . G T G C A ( N ) 1 6 . . . 3 ’5 ’ . . . C A G T C ( N ) 1 4 . . . 5 ’

Thr92 homozygote = One band (256 bp)

Thr92Ala heterozygote = Three bands (256, 186 & 70 bp)

Ala92 homozygote = Two bands (186 & 70 bp)

RFLP Results

2.5% agarose gel

Sample Data Thr92 homozygote = 11 Thr92Ala heterozygote = 12 Ala92 homozygote = 22

ID Genotype500 22501 12502 11503 12504 12

Results

Screened 1269 (90%) of 1407 samples

Sample of OOA subjects was found to be in Hardy-Weinberg equilibrium (p=0.993)

Genotype Count Allele Allele Freq.11 616 1 0.69612 535 2 0.30422 118

Results (cont.)

Insulin secretion and insulin area under the curve (IAUC) are significantly lower for those having a higher prevalence of Ala92

Total cholesterol is significantly higher for those having a higher prevalence of Ala92

11 12 22 PSubjects 616 535 118 Additive Model

Insulin Secretion 9.49 9.08 8.24 0.005IAUC 678.58 628.47 545.40 0.046

Total Cholesterol 5.68 5.81 6.02 0.037

Conclusion Results conflict with previous findings

- Vermont studies found decreased glucose disposal for Ala92 polymorphism

Differences may be due to population differences- Amish have physically active lifestyles- Linkage disequilibrium

Different evaluations of insulin resistance

Acknowledgements

Dr. Francesco Celi Dr. Jordan Warnick Dr. Cynthia Klevickis