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

Natural Mouse models

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

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

RET signal transduction (gain of function) Pathology international 2006;56:164-172 MEN 2B: Human Multiple Endocrine Neoplasia

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.

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; 71:406-410

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

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

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):1890-1898 May 2008

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

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

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, 466-480 (2007) Analysis of the RET Gene in Subjects with Sporadic Hirschsprung’s Disease. J Chin Med Assoc (2008) 71:406-410 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

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

Specific aim 1

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

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

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

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

Results

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

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 2742240 NCBI control study AA 14 36 18 AT 22 92 10 TT 9 52 2

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

SNP in Hirschsprung disease SNP2743357 ( intro 1 CT) SNP2742240 (3’UTR TA)

Increasing Risk

Test for agreement

Test for agreement

Increasing risk

Increasing risk and Trend

Conclusion 1 C allele in rs2743357 and T allele in rs2742240 are protective T allele in rs2742240 and A allele in rs2742240 are predisposing Combination of TT in rs2743357 +AA in rs2742240 take highest risk

Clinical types and SNPs long segment aganglionosis short segment aganglionosis SNPs Rs2743357 Rs2742240

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

Genotype to Phenotype

Western blot: Questionable validity and reliability in 3 different measurements

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

Histo-quest to quantify RET protein level

Different RET(+) cell density

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

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

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

Results

Conclusion 4 Although the TT in rs2742357 and AA in rs2742240 are predisposing haplotypes, RET protein levels are higher in these haplotypes.

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

Discussion

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;9:1461-1467

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