Russell-Silver综合征11p15.5区域遗传缺陷分析
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摘要
目的探讨Russell-Silver综合征(RSS)发病机制。方法采集6例男性,年龄6~8岁的临床表型疑似RSS患儿,以及2例患儿父母、5例健康男性儿童的外周血2 m L,分离单个核细胞并提取基因组DNA,应用焦磷酸测序技术进行分析,检测染色体11p15.5上印记基因控制区域(ICR)1的H19基因的甲基化水平。应用甲基化特异性多重连接探针扩增技术(MS-MLPA)对1例焦磷酸测序结果阳性且为RSS患儿的甲基化水平进行验证分析并对相应区域的基因拷贝数进行检测。结果焦磷酸测序结果显示,6例患儿在H19-差异甲基化区域(DMR)的6个Cp G位点的甲基化率为11%~29%;患儿父母及正常对照组对应位点的甲基化率为44%~59%。焦磷酸测序结果阳性的1例患儿对应的MS-MLPA结果显示,H19基因的4个位点甲基化率在10%左右,明显低于正常水平。KCNQ1OT1基因的4个位点甲基化率约为50%,在正常范围内。所测样本的基因拷贝数均在正常范围内。结论 RSS患儿的ICR1的H19-DMR存在甲基化水平异常。
Objective To explore the pathogenesis of Russell-Silver syndrome(RSS). Methods Two milliliter peripheral blood samples were collected from 6 male patients aged 6 to 8 years with suspected RSS phenotype, the parents of 2 patients and 5 healthy boys. Mononuclear cells were isolated and genomic DNA was extracted. The methylation level of the H19 imprinting control region(ICR) 1 on chromosome 11 p15.5 was detected by pyrosequencing. The methylation status and the copy number variation in the corresponding region of one RSS patient with positive results by pyrosequencing were analysed by methylation-specific multiplex-ligation-dependent probe amplification assay(MS-MLPA). Results Pyrosequencing analysis revealed that the methylation rates on the 6 Cp G targeting sites in H19 differentially methylated region(DMR) in the 6 RSS patients were about 11%~29%, which were significantly lower than those in their parents and normal controls(44%~59%). The MS-MLPA results of one patient with positive pyrosequencing showed that the methylation rates of 4 sites in H19-DMR were about 10 %, which was obviously lower than the normal level. The methylation rates of the 4 sites in KCNQ 1 OT 1 gene were about 50 %, which was in the normal range. The copy number variations from all samples detected were in the normal range. Conclusion There is methylation aberration of H19-DMR in ICR 1 in children with RSS.
Objective To explore the pathogenesis of Russell-Silver syndrome(RSS). Methods Two milliliter peripheral blood samples were collected from 6 male patients aged 6 to 8 years with suspected RSS phenotype, the parents of 2 patients and 5 healthy boys. Mononuclear cells were isolated and genomic DNA was extracted. The methylation level of the H19 imprinting control region(ICR) 1 on chromosome 11 p15.5 was detected by pyrosequencing. The methylation status and the copy number variation in the corresponding region of one RSS patient with positive results by pyrosequencing were analysed by methylation-specific multiplex-ligation-dependent probe amplification assay(MS-MLPA). Results Pyrosequencing analysis revealed that the methylation rates on the 6 Cp G targeting sites in H19 differentially methylated region(DMR) in the 6 RSS patients were about 11%~29%, which were significantly lower than those in their parents and normal controls(44%~59%). The MS-MLPA results of one patient with positive pyrosequencing showed that the methylation rates of 4 sites in H19-DMR were about 10 %, which was obviously lower than the normal level. The methylation rates of the 4 sites in KCNQ 1 OT 1 gene were about 50 %, which was in the normal range. The copy number variations from all samples detected were in the normal range. Conclusion There is methylation aberration of H19-DMR in ICR 1 in children with RSS.
引文
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[8]Bartholdi D,Krajewska-Walasek M,Ounap K,et al.Epigenetic mutations of the imprinted IGF2-H19 domain in Silver–Russell syndrome(SRS):results from a large cohort of patients with SRS and SRS-like phenotypes[J].J Med Genet,2009,46(3):192-197.
[9]Chiesa N,De Crescenzo A,Mishra K,et al.The KCNQ1OT1imprinting control region and non-coding RNA:new properties derived from the study of Beckwith-Wiedemann syndrome and Silver-Russell syndrome cases[J].Hum Mol Genet,2012,21(1):10-25.
[10]Nakashima S,Kato F,Kosho T,et al.Silver-Russell syndrome without body asymmetry in three patients with duplications of maternally derived chromosome 11p15 involving CDKN1C[J].J Hum Genet,2015,60(2):91-95.
[11]Zeschnigk M,Albrecht B,Buiting K,et al.IGF2/H19hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia[J].Eur J Hum Genet,2008,16(3):328-334.
[12]Weber M,Hellmann I,Stadler MB,et al.Distribution,silencing potential and evolutionary impact of promoter DNA methylation in the human genome[J].Nat Genet,2007,39(4):457-466.
[13]Deaton AM,Bird A.Cp G islands and the regulation of transcription[J].Genes Dev,2011,25(10):1010-1022.
[14]Poole RL,Leith DJ,Docherty LE,et al.BeckwithWiedemann syndrome caused by maternally inherited mutation of an OCT-binding motif in the IGF2/H19-imprinting control region,ICR1[J].Eur J Hum Genet,2012,20(2):240-243.
[15]Fuke T,Mizuno S,Nagai T,et al.Molecular and clinical studies in 138 Japanese patients with Silver-Russell syndrome[J].PLo S One,2013,8(3):e60105.
[16]Scott RH,Douglas J,Baskcomb L,et al.Methylationspecific multiplex ligation-dependent probe amplification(MS-MLPA)robustly detects and distinguishes 11p15abnormalities associated with overgrowth and growth retardation[J].J Med Genet,2008,45(2):106-113.
[17]Kannenberg K,Urban C,Binder G.Increased incidence of aberrant DNA methylation within diverse imprinted gene loci outside of IGF2/H19 in Silver-Russell syndrome[J].Clin Genet,2012,81(4):366-377.
[18]Wakeling EL,Amero SA,Alders M,et al.Epigenotypephenotype correlations in Silver-Russell syndrome[J].J Med Genet,2010,47(11):760-768.
[19]Ishida M.New developments in Silver-Russell syndrome and implications for clinical practice[J].Epigenomics,2016,8(4):563-580.
[2]Eggermann T,Buiting K,Temple IK.Clinical utility gene card for:Silver-Russell syndrome[J].Eur J Hum Genet,2011,19(3).doi:10.1038/ejhg.2010.202.
[3]Wakeling EL,Amero SA,Alders M,et al.Epigenotypephenotype correlations in Silver-Russell syndrome[J].J Med Genet,2010,47(11):760-768.
[4]Priolo M,Sparago A,MammìC,et al.MS-MLPA is a specific and sensitive technique for detecting all chromosome11p15.5 imprinting defects of BWS and SRS in a single-tube experiment[J].Eur J Hum Genet,2008,16(5):565-571.
[5]Piyasena C,Cartier J,Khulan B,et al.Dynamics of DNA methylation at IGF2 in preterm and term infants during the first year of life:an observational study[J].Lancet,2015,385(Suppl 1):S81.
[6]Hubertus J,Zitzmann F,Trippel F,et al.Selective methylation of Cp Gs at regulatory binding sites controls NNAT expression in Wilms tumors[J].PLo S One,2013,8(6):e67605.
[7]Sachwitz J,Strobl-Wildemann G,Fekete G,etal.Examinations of maternal uniparental disomy and epimutations for chromosomes 6,14,16 and 20 in SilverRussell syndrome-like phenotypes[J].BMC Med Genet,2016,17:20.
[8]Bartholdi D,Krajewska-Walasek M,Ounap K,et al.Epigenetic mutations of the imprinted IGF2-H19 domain in Silver–Russell syndrome(SRS):results from a large cohort of patients with SRS and SRS-like phenotypes[J].J Med Genet,2009,46(3):192-197.
[9]Chiesa N,De Crescenzo A,Mishra K,et al.The KCNQ1OT1imprinting control region and non-coding RNA:new properties derived from the study of Beckwith-Wiedemann syndrome and Silver-Russell syndrome cases[J].Hum Mol Genet,2012,21(1):10-25.
[10]Nakashima S,Kato F,Kosho T,et al.Silver-Russell syndrome without body asymmetry in three patients with duplications of maternally derived chromosome 11p15 involving CDKN1C[J].J Hum Genet,2015,60(2):91-95.
[11]Zeschnigk M,Albrecht B,Buiting K,et al.IGF2/H19hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia[J].Eur J Hum Genet,2008,16(3):328-334.
[12]Weber M,Hellmann I,Stadler MB,et al.Distribution,silencing potential and evolutionary impact of promoter DNA methylation in the human genome[J].Nat Genet,2007,39(4):457-466.
[13]Deaton AM,Bird A.Cp G islands and the regulation of transcription[J].Genes Dev,2011,25(10):1010-1022.
[14]Poole RL,Leith DJ,Docherty LE,et al.BeckwithWiedemann syndrome caused by maternally inherited mutation of an OCT-binding motif in the IGF2/H19-imprinting control region,ICR1[J].Eur J Hum Genet,2012,20(2):240-243.
[15]Fuke T,Mizuno S,Nagai T,et al.Molecular and clinical studies in 138 Japanese patients with Silver-Russell syndrome[J].PLo S One,2013,8(3):e60105.
[16]Scott RH,Douglas J,Baskcomb L,et al.Methylationspecific multiplex ligation-dependent probe amplification(MS-MLPA)robustly detects and distinguishes 11p15abnormalities associated with overgrowth and growth retardation[J].J Med Genet,2008,45(2):106-113.
[17]Kannenberg K,Urban C,Binder G.Increased incidence of aberrant DNA methylation within diverse imprinted gene loci outside of IGF2/H19 in Silver-Russell syndrome[J].Clin Genet,2012,81(4):366-377.
[18]Wakeling EL,Amero SA,Alders M,et al.Epigenotypephenotype correlations in Silver-Russell syndrome[J].J Med Genet,2010,47(11):760-768.
[19]Ishida M.New developments in Silver-Russell syndrome and implications for clinical practice[J].Epigenomics,2016,8(4):563-580.