Please wait a minute...
ZJUS-B
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)  2016, Vol. 17 Issue (1): 76-82    DOI: 10.1631/jzus.B1500072
Article     
Association between SMN2 methylation and disease severity in Chinese children with spinal muscular atrophy
Yan-yan Cao(),Yu-jin Qu,Sheng-xi He,Yan Li,Jin-li Bai,Yu-wei Jin,Hong Wang,Fang Song()
Department of Medical Genetics, Capital Institute of Pediatrics, Beijing 100020, China
Download: HTML     PDF(728KB)
Export: BibTeX | EndNote (RIS)      

Abstract  

The homozygous loss of the survival motor neuron 1 (SMN1) gene is the primary cause of spinal muscular atrophy (SMA), a neuromuscular degenerative disease. A genetically similar gene, SMN2, which is not functionally equivalent in all SMA patients, modifies the clinical SMA phenotypes. We analyzed the methylation levels of 4 CpG islands (CGIs) in SMN2 in 35 Chinese children with SMA by MassARRAY. We found that three CpG units located in CGI 1 (nucleotides (nt) ?871, ?735) and CGI 4 (nt +999) are significantly hypomethylated in SMA type III compared with type I or II children after receiving Bonferroni correction. In addition to the differentially methylated CpG unit of nt ?871, the methylation level of the nt ?290/?288/?285 unit was negatively correlated with the expression of SMN2 full-length transcripts (SMN2-fl). In addition, the methylation level at nt +938 was inversely proportional to the ratio of SMN2-fl and lacking exon 7 transcripts (SMN2-(7, fl/(7), and was not associated with the SMN2 transcript levels. Thus, we can conclude that SMN2 methylation may regulate the SMA disease phenotype by modulating its transcription.

Key words： CpG island      Methylation      Survival motor neuron 2 (SMN2)      Spinal muscular atrophy     
Received: 24 March 2015      Published: 01 January 2016
Fund:  Project supported by the National Natural Science Foundation of China(Nos. 81050034, 81500979);the Research Foundation of the Capital Institute of Pediatrics(No. Fangxiang-2014-01);the Beijing Talents Fund(No. 2014000021469G228)
Corresponding Authors: Yan-yan Cao,Fang Song     E-mail: yanyancao2@163.com;songf_558@263.net
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
Yan-yan Cao
Yu-jin Qu
Sheng-xi He
Yan Li
Jin-li Bai
Yu-wei Jin
Hong Wang
Fang Song
Cite this article:

Yan-yan Cao,Yu-jin Qu,Sheng-xi He,Yan Li,Jin-li Bai,Yu-wei Jin,Hong Wang,Fang Song. Association between SMN2 methylation and disease severity in Chinese children with spinal muscular atrophy. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2016, 17(1): 76-82.

URL:

http://www.zjujournals.com/xueshu/zjus-b/10.1631/jzus.B1500072     OR     http://www.zjujournals.com/xueshu/zjus-b/Y2016/V17/I1/76

Type Age (year) Gender male/female Methylation (%)
CGI 1 CGI 2 CGI 3 CGI 4
?SMA I (n=12) 0.6±0.5 8/4 76.7±8.6 59.9±5.0 13.5±4.6 70.0±3.8
?SMA II (n=15) 1.3±1.1 7/8 79.6±3.9 58.6±3.5 13.1±3.1 73.4±6.3
?SMA III (n=8) 2.9±1.3 4/4 77.8±3.1 56.8±2.8 12.8±1.7 68.0±1.7
?P-value 0.000 0.558 0.453 0.236 0.916 0.032
?Adjusted P-value 0.474 0.098 0.824 0.014
Table 1 Characteristics of SMA children and SMN2 methylation according to their clinical types
Fig. 1 Methylation analysis of four SMN2 CGIs by bisulfite treatment, cloning, and sequencing Three DNA samples were isolated from the peripheral blood of the SMA I, II, and III patients. Ten independent clones for each CGI of each patient were analyzed for methylation levels. Full circles represent methylated CpGs, while empty circles correspond to unmethylation
Fig. 2 Methylation analysis of four SMN2 CGIs by MassARRAY Black triangles represent mean methylation levels of each CpG unit within each SMN2 CGI in the SMA patients. The mean methylation levels of each SMN2 CGI are indicated by a horizontal line: (a) CGI 1: 76.7%, CGI 2: 59.9%, CGI 3: 13.5%, CGI 4: 70.0%; (b) CGI 1: 79.6%, CGI 2: 58.6%, CGI 3: 13.1%, CGI 4: 73.4%; (c) CGI 1: 77.8%, CGI 2: 56.8%, CGI 3: 12.8%, CGI 4: 68.0%
Methylation unit (dinucleotide position, nt) Methylation level (%)
 P-value
SMA I (n=12) SMA II (n=15) SMA III (n=8)
?CGI 1_2 (?871) 94.1±5.3 85.7±5.6 80.3±4.4 0.000
?CGI 1_8 (?735) 93.7±5.1 86.6±7.7 82.0±4.4 0.001
?CGI 2_9 (?290/?288/?285) 54.2±5.7 50.5±5.6 48.3±3.4 0.046
?CGI 3A_7 (?81) 3.2±3.3 6.8±3.4 2.3±1.7 0.003
?CGI 3B_5 (+160) 1.3±1.1 2.2±1.2 1.1±0.9 0.046
?CGI 3B_10 (+213) 8.0±1.6 9.4±2.5 7.0±1.6 0.032
?CGI 3B_11 (+228/+237) 8.1±3.5 11.6±2.6 8.5±2.3 0.008
?CGI 4_2 (+855) 21.5±8.6 53.5±18.2 39.3±8.6 0.000*
?CGI 4_2 (+890/+894/+898/+900) 83.1±6.5 79.4±4.0 76.4±1.4 0.010*
?CGI 4_3 (+938) 69.6±4.1 73.7±9.9 63.4±5.1 0.006*
?CGI 4_6 (+988) 49.6±6.2 50.1±11.6 36.5±7.3 0.004
?CGI 4_7 (+999) 78.1±4.2 75.7±5.9 62.2±4.3 0.000
?CGI 4_10 (+1064) 83.5±7.3 81.9±10.8 69.6±6.4 0.003
Table 2 Differentially methylated units in SMN2 among SMA types
Fig. 3 Comparison of SMN2 methylation levels by SMA types Eight CpG units had different methylation frequencies in type I or II compared with type III SMA patients (* P<0.05, ** P<0.01, and *** P<0.001 significantly differ). The bar graph depicts the mean methylation levels (±SD) of each CpG unit within the respective CGIs and each CpG unit includes one to four CpGs. The CpG unit corresponding dinucleotide position is as follows: CGI 1_2: nt ?871, CGI 1_8: nt ?735, CGI 2_9:nt ?290/?288/?285, CGI 4_2: nt +890, CGI 4_3: nt +938, CGI 4_6: nt +988, CGI 4_7: nt +999, and CGI 4_10: nt +1064
Transcription nt ?871
 nt ?290/?288/?285 nt +938
r P-value r P-value r P-value
SMN2-fl ?0.550 0.007 ?0.500 0.015 ?0.383 0.071
SMN2-δ7 ?0.512 0.012 ?0.141 0.522 ?0.024 0.913
SMN2-fl+ SMN2-δ7 ?5.990 0.003 ?0.355 0.097 ?0.109 0.621
fl/Δ7 0.077 0.726 ?0.206 0.346 ?0.515 0.012
Table 3 Correlation between differentially methylated units and SMN2 transcription
[1]   Andreassi C, Angelozzi C, Tiziano FD. Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy. Eur J Hum Genet. 2004, 12(1):59-65
doi: 10.1038/sj.ejhg.5201102 pmid: 1456031614560316
[2]   Barrès R, Yan J, Egan B. Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab. 2012, 15(3):405-411. (Available from: http://dx.doi.org/10.1016/j.cmet.2012.01.001)
doi: 10.1016/j.cmet.2012.01.001 pmid: 22405075
[3]   Bernal S, Also-Rallo E, Martínez-Hernández R. Plastin 3 expression in discordant spinal muscular atrophy (SMA) siblings. Neuromuscul Disord. 2011, 21(6):413-419. (Available from: http://dx.doi.org/10.1016/j.nmd.2011.03.009)
doi: 10.1016/j.nmd.2011.03.009 pmid: 21546251
[4]   Chang JG, Hsieh-Li HM, Jong YJ. Treatment of spinal muscular atrophy by sodium butyrate. PNAS. 2001, 98(17):9808-9813. (Available from: http://dx.doi.org/10.1073/pnas.171105098)
doi: 10.1073/pnas.171105098 pmid: 11504946
[5]   Crawford TO, Paushkin SV, Kobayashi DT. Pilot Study of Biomarkers for Spinal Muscular Atrophy Trial Group. Evaluation of SMN protein transcript and copy number in the biomarkers for spinal muscular atrophy (BforSMA) clinical study. PLoS ONE. 2012, 7(4):e33572 (Available from: http://dx.doi.org/10.1371/journal.pone.0033572)
doi: 10.1371/journal.pone.0033572
[6]   Hahnen E, Eyüpoglu IY, Brichta L. In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy. J Neurochem. 2006, 98(1):193-202. (Available from: http://dx.doi.org/10.1111/j.1471-4159.2006.03868.x)
doi: 10.1111/j.1471-4159.2006.03868.x
[7]   Hauke J, Riessland M, Lunke S. Survival motor neuron gene 2 silencing by DNA methylation correlates with spinal muscular atrophy disease severity and can be bypassed by histone deacetylase inhibition. Hum Mol Genet. 2009, 18(2):304-317. (Available from: http://dx.doi.org/10.1093/hmg/ddn357)
doi: 10.1093/hmg/ddn357 pmid: 2638778
[8]   Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 2012, 13(7):484-492. (Available from: http://dx.doi.org/10.1038/nrg3230)
doi: 10.1038/nrg3230 pmid: 22641018
[9]   Maunakea AK, Chepelev I, Cui K. Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition. Cell Res. 2013, 23(11):1256-1269. (Available from: http://dx.doi.org/10.1038/cr.2013.110)
doi: 10.1038/cr.2013.110 pmid: 23938295
[10]   Murrell A, Rakyan VK, Beck S. From genome to epigenome. Hum Mol Genet. 2005, 14(Suppl. 1):R3-R10. (Available from: http://dx.doi.org/10.1093/hmg/ddi110)
doi: 10.1093/hmg/ddi110
[11]   Prior TW, Krainer AR, Hua Y. A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet. 2009, 85(3):408-413. (Available from: http://dx.doi.org/10.1016/j.ajhg.2009.08.002)
doi: 10.1016/j.ajhg.2009.08.002 pmid: 19716110162
[12]   Qu YJ, Du J, Li EZ. Subtle mutations in the SMN1 gene in Chinese patients with SMA: p.Arg288Met mutation causing SMN1 transcript exclusion of exon7. BMC Med Genet. 2012, 13 86
doi: 10.1186/1471-2350-13-86 pmid: 3523059
[13]   Schumacher A, Petronis A. Epigenetics of complex diseases: from general theory to laboratory experiments. Curr Top Microbiol Immunol. 2006, 310 81-115
doi: 10.1007/3-540-31181-5_6 pmid: 16909908
[14]   Tiziano FD, Pinto AM, Fiori S. SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR. Eur J Hum Genet. 2010, 18(1):52-58. (Available from: http://dx.doi.org/10.1038/ejhg.2009.116)
doi: 10.1038/ejhg.2009.116 pmid: 2987170
[1] Meng-wen Zhang, Kenji Fujiwara, Xu Che, Shu Zheng, Lei Zheng. DNA methylation in the tumor microenvironment[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2017, 18(5): 365-372.
[2] Xiao-min Hua, Juan Wang, Dong-meng Qian, Jing-yi Song, Hao Chen, Xiu-li Zhu, Rui Zhou, Yu-dan Zhao, Xiu-zhi Zhou, Ling Li, Li Zhang, Xu-xia Song, Bin Wang. DNA methylation level of promoter region of activating transcription factor 5 in glioma[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2015, 16(9): 757-762.
[3] Jie-hong Zhao, Ji-shun Zhang, Yi Wang, Ren-gang Wang, Chun Wu, Long-jiang Fan, Xue-liang Ren. DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2011, 12(11): 935-942.
[4] Jian-zhong Di, Xiao-dong Han, Wen-ye Gu, Yu Wang, Qi Zheng, Pin Zhang, Hui-min Wu, Zhong-zheng Zhu. Association of hypomethylation of LINE-1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2011, 12(10): 805-811.
[5] Yan-qin Huang, Ying Yuan, Wei-ting Ge, Han-guang Hu, Su-zhan Zhang, Shu Zheng. Comparative features of colorectal and gastric cancers with microsatellite instability in Chinese patients[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2010, 11(9): 647-653.
[6] Hong-xing Li, Lei Xiao, Cheng Wang, Jia-li Gao, Yong-gong Zhai. Epigenetic regulation of adipocyte differentiation and adipogenesis[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2010, 11(10): 784-791.
[7] Yu-hua LIANG, Xiao-ling CHEN, Zhong-sheng YU, Chun-yue CHEN, Sheng BI, Lian-gen MAO, Bo-lin ZHOU, Xian-ning ZHANG. Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2009, 10(1): 29-34.
[8] RUAN Wen-jing, LIN Jie, XU En-ping, XU Fang-ying, MA Yu, DENG Hong, HUANG Qiong, LV Bing-jian, HU Hu, CUI Jing, DI Mei-juan, DONG Jian-kang, LAI Mao-de. IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2006, 7(11): 929-932.
[9] CHEN Chuan-bo, LI Tao. A hybrid neural network system for prediction and recognition of promoter regions in human genome*[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2005, 6(5): 17-.