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J Zhejiang Univ (Med Sci)  2019, Vol. 48 Issue (4): 373-377    DOI: 10.3785/j.issn.1008-9292.2019.08.04
    
Genetic study of a family of neuronal ceroid lipofuscinosis caused by a heterozygous mutation of CLN6 gene
LOU Tie1,2(),HUANG Yingzhi1,DONG Minyue1,*()
1. Key Laboratory of Reproductive Genetics, Ministry of Education, Department of Reproductive Genetics, Women's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
2. Department of Gynecology and Obstetrics, Jianggan District People's Hospital, Hangzhou 310021, China
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Abstract  

Objective: To analyze the genetic cause of a family with autosomal recessive neuronal ceroid lipofuscinoses (NCL). Methods: The proband was screened for mutations within the coding region of the candidate genes through high-throughput targeted sequencing. Potential causative mutations were verified by PCR and Sanger sequencing in the proband and his parents. RT-PCR and TA clone sequencing were performed to investigate whether the mRNAs were abnormally spliced. Results: The sequencing results revealed compound heterozygous mutations of CLN6:c.486+2T>C and c.486+4A>T, which were respectively inherited from his parents. RT-PCR and TA cloning sequencing suggested that the mRNAs were abnormally spliced in two forms due to both mutations. Conclusions: The compound heterozygous mutations of CLN6:c.486+2T>C and c.486+4A>T are possibly the genetic causes of the NCL family. Detection of the novel mutation has extended mutation spectrum of CLN6.



Key wordsNeuronal ceroid-lipofuscinoses/genetics      Etiology      Genes      Mutation      Spliceosomes      Polymerase chain reaction     
Received: 28 March 2019      Published: 30 October 2019
CLC:  R394.3  
Corresponding Authors: DONG Minyue     E-mail: tiel-2004@163.com;dongmy@zju.edu.cn
Cite this article:

LOU Tie,HUANG Yingzhi,DONG Minyue. Genetic study of a family of neuronal ceroid lipofuscinosis caused by a heterozygous mutation of CLN6 gene. J Zhejiang Univ (Med Sci), 2019, 48(4): 373-377.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2019.08.04     OR     http://www.zjujournals.com/med/Y2019/V48/I4/373


CLN6基因复合杂合突变导致神经元蜡样脂褐质沉积症一家系遗传学研究

目的: 探讨一个常染色体隐性遗传神经元蜡样脂褐质沉积症(NCL)家系的遗传学原因。方法: 应用目标区捕获高通量靶向测序对先证者进行候选基因突变筛查,并通过PCR测序在先证者及其父母中对突变位点进行验证;RT-PCR及TA克隆测序考察上述两种突变是否影响剪接。结果: 测序结果显示先证者存在CLN6:c.486+2T>C和c.486+4A>T复合杂合突变,分别来自父母双方。RT-PCR及TA克隆测序提示两种突变均可导致两种异常剪接。结论: CLN6:c.486+2T>C和c.486+4A>T复合杂合突变很可能为该NCL家系患者的遗传学病因,新突变基因丰富了CLN6基因突变谱。


关键词: 神经元蜡样质脂褐质沉积病/遗传学,  病因学,  基因,  突变,  剪接体,  聚合酶链反应 
引物 引物序列(5′-3′) 片段长度
(bp)*
*此为标准参考序列长度,实际cDNA片段大小可随剪接异常而变化.
CLN6-4F GTGACATTCCAGCTGGAAGCGT 467
CLN6-4R GAACACTTGAGCATCCTAGCTTG
CLN6-mrE3-F CTTCCACATGGCCTACAACGTC 300
CLN6-mrE6-R GCAGTAAAGCAGCCGCTGAAGT
CLN6-mrE3_E4-F TTCTCTTGCTCAAGCTCATCGAG 300
CLN6-mrE5_E6-R GGATGTACCACATGCAGTGACC
Tab 1 Primer sequence
Fig 1 Sanger sequencing of the CLN6 gene in proband and parents
Fig 2 cDNA TA clone sequencing of the proband
[1]   BEST H L , NEVERMAN N J , WICKY H E et al. Characterisation of early changes in ovine CLN5 and CLN6 Batten disease neural cultures for the rapid screening of therapeutics[J]. Neurobiol Dis, 2017, 100:62- 74
doi: 10.1016/j.nbd.2017.01.001
[2]   GUERREIRO R , BRAS J T , VIEIRA M et al. CLN6 disease caused by the same mutation originating in Pakistan has varying pathology[J]. Eur J Paediatr Neurol, 2013, 17 (6): 657- 660
doi: 10.1016/j.ejpn.2013.04.011
[3]   HALTIA M , GOEBEL H H . The neuronal ceroid-lipofuscinoses:a historical introduction[J]. Biochim Biophys Acta, 2013, 1832 (11): 1795- 1800
doi: 10.1016/j.bbadis.2012.08.012
[4]   CHIN J J , BEHNAM B , DAVIDS M et al. Novel mutations in CLN6 cause late-infantile neuronal ceroid lipofuscinosis without visual impairment in two unrelated patients[J]. Mol Genet Metab, 2019, 126 (2): 188- 195
doi: 10.1016/j.ymgme.2018.12.001
[5]   SUN G , YAO F , TIAN Z et al. A first CLN6 variant case of late infantile neuronal ceroid lipofuscinosis caused by a homozygous mutation in a boy from China:a case report[J]. BMC Med Genet, 2018, 19 (1): 177
doi: 10.1186/s12881-018-0690-x
[6]   SATO R , INUI T , ENDO W et al. First Japanese variant of late infantile neuronal ceroid lipofuscinosis caused by novel CLN6 mutations[J]. Brain Dev, 2016, 38 (9): 852- 856
doi: 10.1016/j.braindev.2016.04.007
[7]   LEE H G , YOON B , KIM Y O et al. CLN6 mutation in a patient with progressive myoclonus epilepsy[J]. J Korean Child Neurol Soc, 2018, 26:123- 127
doi: 10.26815/jkcns.2018.26.2.123
[8]   任守臣, 高宝勤, 王雅洁 et al. 神经元蜡样脂褐质沉积病五例的临床表现、基因与超微病理特点[J]. 中华医学杂志, 2016, 96 (43): 3504- 3507
doi: 10.3760/cma.j.issn.0376-2491.2016.43.013
[9]   TEIXEIRA C A , ESPINOLA J , HUO L et al. Novel mutations in the CLN6 gene causing a variant late infantile neuronal ceroid lipofuscinosis[J]. Hum Mutat, 2003, 21 (5): 502- 508
doi: 10.1002/humu.10207
[10]   MAQUAT L E . Nonsense-mediated mRNA decay and human disease:Genome guardian and executor[J]. FASEB J, 2018, 32 (1 supplement): 99- 91
[11]   NASIF S , CONTU L , MVHLEMANN O . Beyond quality control:The role of nonsense-mediated mRNA decay (NMD) in regulating gene expression[J]. Semin Cell Dev Biol, 2018, 75:78- 87
doi: 10.1016/j.semcdb.2017.08.053
[12]   BROGNA S , WEN J . Nonsense-mediated mRNA decay (NMD) mechanisms[J]. Nat Struct Mol Biol, 2009, 16 (2): 107- 113
[13]   柴宝峰, 王美, 石文鑫 et al. 无义mRNA降解途径的机制与进化[J]. 山西大学学报(自然科学版), 2017, 40 (3): 639- 644
[14]   胡建燃, 李平 . NMD机制及其对人类遗传病的治疗意义[J]. 生命科学研究, 2016, 20 (6): 535- 541
[15]   VIPRAKASIT V, CHINCHANG W. Identification of hemoglobin YALA; a novel β thalassemia mutation due to thymidine deletion of codon 42(-T) causing β0 thalassemia and its interaction with hemoglobin E[C]. Australia: Proceedings of the Annual Scientific Meetings of the HAA Sydney, 2011.
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