Download Huntington`s Disease

In periodic expansion several CAGs expand at once, creating a stepwise increase in the length of the trinucleotide tract. In continuous only one triplet expand at once, causing little change in the length of the trinucleotide tract. 80
Patient 1 – Cerebellum
60
Continuous Expansion
60
Frequemcy
Frequency
Result of trinucleotide repeat expansion analysis 40
20
0
Patient 2 – Cerebellum
Continuous Expansion
40
20
0
31 33 34 38 40 41 42 43 44 45 46 47 48 49 50 51 52 53 63
31
34
37
Figures to the right show the results of trinucleotide repeat analysis in cerebellum, frontal cortex and parietal cortex in the two patients. No periodicity was found in cerebellum. Both frontal cortex and parietal cortex showed periodic expansion with great increase in number of triplets. These results was consistent for both patients and with assumptions made in the beginning of the project based on previous experiments in Huntington mice.
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Patient 1 ‐ Frontal Cortex
Periodic Expansion
Frequemcy
Frequemcy
100
50
40
41
42
43
44
46
49
51
Patient 2 ‐ Frontal Cortex
Periodic Expansion
40
20
0
0
31 38 43 47 51 55 59 63 67 71 75 82 93 116 135 168 203
36 37 38 39 40 41 42 43 44 46 47 48 49 50 51 52 53 54 55 59 61 68 70 74
Number of CAG‐triplets
Number of CAG‐triplets
Patient 1 – Parietal Cortex
80
Patient 2 ‐ Parietal Cortex
Periodic Expansion
60
Periodic Expansion
Frequency
30
Frequency
39
Number of CAG‐triplets
Number of CAG‐triplets
20
10
40
20
0
0
34
36
40
44
46
48
50
52
54
56
58
60
62
64
66
68
70
73
78
96
103
113
33 35 37 39 41 43 45 47 50 54 58 62 69 73 78
Number of CAG‐triplets
Number of CAG‐triplets
Huntington’sDisease
GeneExpressionofBaseExcisionRepairEnzymesandTrinucleotide
RepeatAnalysisinHumanBrainTissue.
Rakel Ekholt1 and Martine Berg‐Jensen1 in collaboration with Linda Møllersen2 and Arne Klungland2
1Oslo and Akershus University College of Applied Sciences, Pilestredet 35, 0166 Oslo; 2Oslo Univeritetssykehus, Rikshospitalet, Department of Microbiology, Sognsvannsveien 20, 0372 Oslo
Introduction
Huntington’s disease is a neurodegenerative disease caused by aberrant expansion of CAG‐
triplets within the huntingtin gene causing an elongated and toxic polyglutamine tract. This project was to determine if this expansion in human brain tissue was periodic or continuous. Brain areas analyzed were frontal cortex, parietal cortex and cerebellum.
Results of gene expression analysis using qPCR
Result of protein expression using western blot Figures below show the results of gene expression analysis using qPCR. The results are normalized using β‐actin (dark column) and GAPDH (light column) and In the figure below the results of the two patients are combined tissue wise for each probe. From the figure it is clear that gene expression is greatest in cerebellum.
Result of the western blot analysis using antibodies to determine protein expression is shown in figure below. Results are quantified using α‐tubuline as loading control. FEN1, OGG1, NEIL1 and Polβ show greatest expression in frontal cortex while APE1 is expressed more in cerebellum. 35
4,50
30
4,00
Pasient 1 ‐ FCx
25
3,50
20
3,00
15
2,50
Pasient 1 ‐ Cbl
2,00
Pasient 2 ‐ Cbl
β‐actin
10
GAPDH
5
Pasient 2 ‐ FCx
1,50
1,00
0
0,50
0,00
FEN1
This project would also determine gene expression of various base excision repair (BER) enzymes in frontal cortex and cerebellum. Enzymes analyzed were FEN1, NEIL1, APE1, OGG1 and Polβ. Analysis of trinucleotide repeat expansion was done with PCR and fragment analysis, for gene expression qPCR and western blot were used. Previous research in mice have shown:
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Periodic CAG‐expansion in striatum and cortex, continuous CAG‐ expansion in cerebellum.
APE1 is expressed more in cerebellum than striatum
Lack of OGG1 and NEIL1 reduces somatic instability.
Polβ has higher activity in striatum than in cerebellum.
In addition to being tissue specific gene expression can also be age‐dependent.
Below the gene expression of each enzyme is compared between the patients. For all enzymes gene expression is greatest in cerebellum of patient 2. 3,5
3
FEN1
2,5
NEIL1
2,5
2
2,5
1,5
2
1,5
1,5
1
1
0,5
0,5
1
0,5
0
0
2FCx 1FCx 2CBl 1Cbl
40
Polβ
0
2FCx 1FCx 2CBl 1Cbl
5
OGG1
4
30
3
20
2
10
1
0
0
2FCx 1FCx 2CBl 1Cbl
APE1
2
2FCx 1FCx 2CBl
1Cbl
2FCx 1FCx 2CBl 1Cbl
APE1
NEIL1
OGG1
Pol β Conclusion
₋ Results of triplet expansion in brain areas seems to be consistent with results in mouse models of Huntington disease.
₋ Little consistency between results of qPCR
and western blot analysis. Only result for APE1 was similar
₋ qPCR show expected results for FEN1, APE1 and OGG1 and using western blot ₋ Expected results were found for APE1, NEIL1 and OGG1 when using western blot.