1. SNP in 3’UTR of RET messenger RNA correlates protein expression and confers higher risk to Hirschsprung disease in Taiwanese
Chia Shu-ti1,2, Lee Ying-ray3, Cheng Hui-chuan2, Chen Shu-hsin3, Hsieh Hsiao-yen3, Lu Pei-jung2
1Department of Pediatric Surgery and 3Medical Research, Chia-Yi Christian Hospital, Chiayi
2Institute of Clinical Medical Research, School of Medicine, National Cheng-Kung University, Tainan

2. Natural Mouse models

3. Hirschsprung disease, associated syndromes and genetics: a review
J. Med. Genet.45;1-14(2008)

4. RET proto-oncogene Coding protein RET 10q 11.2
Size:53,283 bases ;20 exons
mRNA:5629bp
Coding protein RET
Sequence length :1114 AA.
Function :Receptor tyrosine kinase
2 Isomforms:RET9; RET51 in c-terminus
Matured (glycosylated) form:170KD
Immmatured form:150KD

5. RET signal transduction (gain of function)
Pathology international 2006;56:
MEN 2B: Human Multiple Endocrine Neoplasia

6. Mutations in RET 1.Partially impaired kinase
2.Complete loss of the RET kinase activity
3.Decreasing the expression of the RET protein with normal kinase activity.

7. Megacolon in Taiwan Mutation rate:2/1513.3% Sequencing 20 exons
Analysis of the RET Gene in Subjects with Sporadic Hirschsprung’s Disease. J Chin Med Assoc
2008; 71:

8. Megacolon in Taiwan Mutation:2/553.6% Sequencing 20 exons
Low RET mutation frequency and polymorphism analysis of the RET
and EDNRB genes in patients with Hirschsprung disease in Taiwan J Hum Genet (2005) 50:168–174

9. Un-resolving Question
RET---Mutation only? or other mechanism?

10. Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice
Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura, and Hideki Enomoto
JCI 118(5): May 2008

11. Ret<30%phenotype of megacolon
 No aganglionosis,no renal anomaly
Ret<30%
Aganglionosis, but no renal anomaly
Ret KO total aganglionosis and renal agenesis

12. Effects influencing RET expression--SNPs in RET
Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet 2008; 45:1-14

13. Low RET mutation rate in Taiwan
>15-30% RET mutation rate in western countries
10% of HSCR has trisomy 21( Down syndrome)
<5-10% RET mutation rate in Taiwan
<1% Down syndrome in our experience
Enteric nervous system development and Hirschsprung’s disease: advances in genetic and stem cell studies . Nature Rev Neuroscience 8, (2007)
Analysis of the RET Gene in Subjects with Sporadic Hirschsprung’s Disease. J Chin Med Assoc (2008) 71:
Low RET mutation frequency and polymorphism analysis of the RET and EDNRB genes in patients with Hirschsprung disease in Taiwan J Hum Genet (2005) 50:168–174

14. Specific Aims
1.To confirm that RET expression level correlates with phenotype of congenital megacolon in human surgical specimen
1-1 Sequencing the RET to divide patients into mutation- positive and mutation-negative groups and to find novel mutation or SNPs in Taiwanese
1-2 Confirm if RET expression is decreasing in all groups

15. Specific aim 1

16. Material and Methods 30 aganglionosis patients
Cell-lines:neuroblastoma( SH-SY5Y); HeLa cell
3 normal appendix, 2 infant colon

17. Specific Aim 1 Gene sequencing of RET proto-oncogene
Sequencing RET mRNA
Determine the RET protein level in patients and control group
Western-blot  Histo-Quest™
House-keeping proteins: GAPDH and PGP 9.5 (protein gene product 9.5; UCH-L1; neuron marker in mature and immature neurons and nerve fiber)
Determine the RET messenger RNA by
q-RT-PCR

18. Histo-Quest ™ for RET protein leveling
Serial section 5μm x 5 sections
Focus on the RET(+) ganglion cells
Background noice
Quantitative evaluation of myenteric ganglion cells in normal human left colon: implications for histopathological analysis. Cell Tissue Res. 2009; 336:191–201

19. RET mRNA sequencing 1 2 3 4 5 6 7 8 9 RET mRNA Total length:5629bp
RET mRNA
Total length:5629bp
Coding sequence:3216bp
3’UTR:2300bp

20. Results

21. Results No mutation in exon was found
SNP in 3’UTR has correlation with disease and protein level

22. RET Sequencing Sequencing mRNA of RET with 9 primer pairs
Nearly Full-length sequencing
Re-confirm by germline DNA sequencing
One disease associated SNP in 3’UTR RS
NCBI
control
study AA 14 36 18 AT 22 92 10 TT 9 52 2

23. New unreported SNP in 3’UTR (T→A)
1:NCBI 北京漢族healthy; non-related control (N=45)
2:台灣嘉義surgical patients without Hirschsprung disease (N=180 )
3:Our patients( study group N=30)
NCBI
Control
Study

24. SNP in Hirschsprung disease
SNP ( intro 1 CT)
SNP (3’UTR TA)

25. Increasing Risk

28. Test for agreement

29. Test for agreement

30. Increasing risk

31. Increasing risk and Trend

32. Conclusion 1
C allele in rs and T allele in rs are protective
T allele in rs and A allele in rs are predisposing
Combination of TT in rs AA in rs take highest risk

33. Clinical types and SNPs
long segment aganglionosis
short segment aganglionosis
SNPs Rs Rs

36. Conclusion 2
No evidence supports different SNP haplotypes influence the development of length of agnglionosis segment

37. Genotype to Phenotype

38. Western blot: Questionable validity and reliability in 3 different measurements

39. RET/PGP 9.5 ratio( Protein and mRNA)1 2 3 4 5 6 7 8 9 10 11 12 13 WB
0.25
0.56
2.52
1.63
1.14
0.59
2.08
1.01
1.09
0.35
0.52
0.27
0.48
Q-PCR
0.62
0.86
1.51
0.19
0.54
1.46
0.85
1.04
0.5
1.12

40. Histo-quest to quantify RET protein level

41. Different RET(+) cell density

43. Percentage of RET positive cells in Gut tissue
No. of patient

44. Conclusion 3
The count of RET positives neuronal cells varied widely from specimen to specimen

45. RET protein expression and SNPs
RET protein intensity: (fold)
ganglion cells intensity/background

46. Results

48. Conclusion 4
Although the TT in rs and AA in rs are predisposing haplotypes, RET protein levels are higher in these haplotypes.

49. Hypoventilation syndrome
Low protein desity (1.29x) but high ganglion cellularity(2.36%)

50. Discussion

51. The only one genotype to phenotype study in RET SNP
Q-RT PCR of RET mRNA in surgical specimen
49 disease and 18 normal colon
But is this reliable?
Reduced RET expression in gut tissue of individuals carrying risk alleles of Hirschsprung’s disease. Human Molecular Genetics 2010;9:

52. Thank you