matt thomas determining the association between the thr92ala type 2 deiodinase polymorphism and...
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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
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
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
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