肝豆状核变性患者ATP7B基因突变分析
摘要
目的
肝豆状核变性（Hepatolenticular Degeneration, HLD)，亦称Wilson病（Wilson disease,WD），是一种伴随铜代谢障碍的常染色体隐性遗传性疾病，全世界发病率约为1︰30000～100000。临床症状系由铜在体内蓄积所致，主要表现为肝功能异常、神经精神系统功能障碍、角膜色素环及肾功能异常等。WD的致病基因为ATP7B，定位于13q14.3区，截止目前已发现其致病基因突变400余种，突变位点几乎遍布整个基因编码区。对WD的基因突变的深入研究，有助于WD的早期诊断和治疗。本研究运用人类基因组DNA提取、PCR扩增及DNA序列分析等技术对WD的致病基因ATP7B全部21个外显子及邻近内含子区域进行突变扫描，以获得确切的基因突变类型。初步分析WD患者基因型与表型的关系，为WD分子病因学研究提供数据，有助于我省WD患者的基因诊断和遗传咨询。方法
采集12例WD患者和50名无血缘关系健康对照者的外周血，采用经典Miller蛋白酶K-氯化钠盐析法从其外周血白细胞中提取基因组DNA，应用聚合酶链反应(PCR)和DNA直接测序技术对12例WD患者进行ATP7B基因突变位点检测，同时应用限制性内切酶酶切分析的方法证实发现的突变。参阅国内外已有研究和报道，应用统计学方法，初步分析WD基因型和表型间的关系。
结果
1.本研究共检出ATP7B基因突变4个：R778L、A874V、T935M和P992L均为已有报道突变。
2. HpaII酶切反应未发现健康对照者存在R778L突变。
3.本研究共发现ATP7B基因的SNPs 5个：A406S、L456V、L770L、K832R和2866-13g>c。均已被NCBI的SNP数据库收录。
4.本研究所检出的基因变异均为点突变。
5.本研究中R778L的突变率为41.7%。
6. Arg778Leu突变和Thr935Met突变的患者与无该点突变的患者基因型与临床表型比较P>0.05，临床表现未见明显差异。
结论
1.本次研究再次证实ATP7B基因第8外显子R778L为高频突变位点，与前人研究一致，说明R778L也是山西省WD患者的热点突变。
2.临床上可用HpaII酶切反应作为WD患者初步筛选方法。
3. Arg778Leu突变和Thr935Met突变与临床表型在本研究中均未发现有相关性。
4.本研究所得结果与前人只研究我国热点突变所得结果基本一致，对WD的基因诊断是否只进行热点突变的检测即可，有待于加大样本量进行确定。
Objective
Wilson disease (WD) is an autosomal recessive genetic disease with copper metabolic barrier.The worldwide prevalence of Wilson disease is estimated to be between 1 in 30, 000 and 1 in100,000. Clinical symptoms caused by the toxic accumulation of overload copper in the body,mainly for liver dysfunction, neuropsychiatric system dysfunction, Kayser–Fleischer ring andabnormal renal function. Wilson disease is caused by mutations of ATP7B gene, located in13q14.3. To date, almost 400 variations reported as causative mutations of WD, almostthroughout the coding region. The deep research for WD gene mutations may help the earlydiagnosis and treatment of WD. In this study, we will use the DNA extraction, PCRamplification and DNA sequence analysis techniques on WD gene ATP7B all 21 exons andadjacent intron regions of mutation scanning to obtain the exact gene mutation. we preliminaryanalysis the relationship between genotype and phenotype of WD patients, provide data to themolecular etiology of the WD to help WD patients in Shanxi province with the diagnosis andgenetic counseling.
Methods
Total genomic DNA was extracted by the Miller classic proteinase K-sodium chloridesalting, from the peripheral blood leukocytes from 12 patients with WD and 50 randomly testedsubjects. Polymerase chain reaction (PCR) and direct DNA sequencing were used to detectATP7B mutations of 12 WD patients. Restriction enzyme analysis was used to confirm thepathogenicity of a mutation. The genotype and phenotype of WD patients were gathered in orderto analyse the genotype-phenotype correlations with statistical.
Results
1. Four mutations were detected in the ATP7B gene in this study: R778L, A874V, T935Mand P992L, all of them have been reported.
2. The newly identified mutation R778L was not found in PCR amplified products from 50randomly tested subjects by restriction enzyme analysis.
3. Eight single nucleotide polymorphisms (SNPS): A406S, L456V, L770L, K832R and2866-13 g>c were detected in this study. All of them have been found in SNP database of NCBI.
4. All gene mutations in this study were point mutation.
5. The mutation rate of R778L is 41.7% in this study.
6. R778L and T935M mutation were not related to gender, the onset age and clinicalphenotype.
ConclusionConclusions
1. R778L is the hot point mutation of ATP7B gene in ShanXi Han Patients with Wilson'disease;
2. In clinic, HpaII restriction enzyme analysis can be used as an initial screening method inpatients with WD.
3. R778L and T935M mutation were not related to gender, the onset age and clinicalphenotype.
4. The results of this study consistent of previous study results only detectied of hot spotmutation. Whether genetic diagnosis of WD only detected the hot spot mutation, would bedetermined by the sample size increased.
肝豆状核变性（Hepatolenticular Degeneration, HLD)，亦称Wilson病（Wilson disease,WD），是一种伴随铜代谢障碍的常染色体隐性遗传性疾病，全世界发病率约为1︰30000～100000。临床症状系由铜在体内蓄积所致，主要表现为肝功能异常、神经精神系统功能障碍、角膜色素环及肾功能异常等。WD的致病基因为ATP7B，定位于13q14.3区，截止目前已发现其致病基因突变400余种，突变位点几乎遍布整个基因编码区。对WD的基因突变的深入研究，有助于WD的早期诊断和治疗。本研究运用人类基因组DNA提取、PCR扩增及DNA序列分析等技术对WD的致病基因ATP7B全部21个外显子及邻近内含子区域进行突变扫描，以获得确切的基因突变类型。初步分析WD患者基因型与表型的关系，为WD分子病因学研究提供数据，有助于我省WD患者的基因诊断和遗传咨询。方法
采集12例WD患者和50名无血缘关系健康对照者的外周血，采用经典Miller蛋白酶K-氯化钠盐析法从其外周血白细胞中提取基因组DNA，应用聚合酶链反应(PCR)和DNA直接测序技术对12例WD患者进行ATP7B基因突变位点检测，同时应用限制性内切酶酶切分析的方法证实发现的突变。参阅国内外已有研究和报道，应用统计学方法，初步分析WD基因型和表型间的关系。
结果
1.本研究共检出ATP7B基因突变4个：R778L、A874V、T935M和P992L均为已有报道突变。
2. HpaII酶切反应未发现健康对照者存在R778L突变。
3.本研究共发现ATP7B基因的SNPs 5个：A406S、L456V、L770L、K832R和2866-13g>c。均已被NCBI的SNP数据库收录。
4.本研究所检出的基因变异均为点突变。
5.本研究中R778L的突变率为41.7%。
6. Arg778Leu突变和Thr935Met突变的患者与无该点突变的患者基因型与临床表型比较P>0.05，临床表现未见明显差异。
结论
1.本次研究再次证实ATP7B基因第8外显子R778L为高频突变位点，与前人研究一致，说明R778L也是山西省WD患者的热点突变。
2.临床上可用HpaII酶切反应作为WD患者初步筛选方法。
3. Arg778Leu突变和Thr935Met突变与临床表型在本研究中均未发现有相关性。
4.本研究所得结果与前人只研究我国热点突变所得结果基本一致，对WD的基因诊断是否只进行热点突变的检测即可，有待于加大样本量进行确定。
Objective
Wilson disease (WD) is an autosomal recessive genetic disease with copper metabolic barrier.The worldwide prevalence of Wilson disease is estimated to be between 1 in 30, 000 and 1 in100,000. Clinical symptoms caused by the toxic accumulation of overload copper in the body,mainly for liver dysfunction, neuropsychiatric system dysfunction, Kayser–Fleischer ring andabnormal renal function. Wilson disease is caused by mutations of ATP7B gene, located in13q14.3. To date, almost 400 variations reported as causative mutations of WD, almostthroughout the coding region. The deep research for WD gene mutations may help the earlydiagnosis and treatment of WD. In this study, we will use the DNA extraction, PCRamplification and DNA sequence analysis techniques on WD gene ATP7B all 21 exons andadjacent intron regions of mutation scanning to obtain the exact gene mutation. we preliminaryanalysis the relationship between genotype and phenotype of WD patients, provide data to themolecular etiology of the WD to help WD patients in Shanxi province with the diagnosis andgenetic counseling.
Methods
Total genomic DNA was extracted by the Miller classic proteinase K-sodium chloridesalting, from the peripheral blood leukocytes from 12 patients with WD and 50 randomly testedsubjects. Polymerase chain reaction (PCR) and direct DNA sequencing were used to detectATP7B mutations of 12 WD patients. Restriction enzyme analysis was used to confirm thepathogenicity of a mutation. The genotype and phenotype of WD patients were gathered in orderto analyse the genotype-phenotype correlations with statistical.
Results
1. Four mutations were detected in the ATP7B gene in this study: R778L, A874V, T935Mand P992L, all of them have been reported.
2. The newly identified mutation R778L was not found in PCR amplified products from 50randomly tested subjects by restriction enzyme analysis.
3. Eight single nucleotide polymorphisms (SNPS): A406S, L456V, L770L, K832R and2866-13 g>c were detected in this study. All of them have been found in SNP database of NCBI.
4. All gene mutations in this study were point mutation.
5. The mutation rate of R778L is 41.7% in this study.
6. R778L and T935M mutation were not related to gender, the onset age and clinicalphenotype.
ConclusionConclusions
1. R778L is the hot point mutation of ATP7B gene in ShanXi Han Patients with Wilson'disease;
2. In clinic, HpaII restriction enzyme analysis can be used as an initial screening method inpatients with WD.
3. R778L and T935M mutation were not related to gender, the onset age and clinicalphenotype.
4. The results of this study consistent of previous study results only detectied of hot spotmutation. Whether genetic diagnosis of WD only detected the hot spot mutation, would bedetermined by the sample size increased.
引文
[1] Danks DM. Hereditary disorders of copper metabolism in Wilson’s disease andMenkes’disease. Metabolic Basis of Inherited Disease, 1993, 6: 1251-1261.
[2] Tanzi RE, Petrukhin K, Chemov I, et al. The Wilson disease gene is a copper transportingATPase with homology to the menkes disease gene. Nat Genet, 1993, 5(4): 344-350.
[3] Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative c- oppertransporting P-type ATPase similar to the Menkes gene. Nat Genet, 1993, 5(4): 327-337.
[4]杨静芳,陈彪.中国人肝豆状核变性基因突变的分布特征.中国临床康复, 2004, 8(4):690-692.
[5]杨春水,梁秀龄,闫振文，等.肝豆状核变性基因启动子区的多态和突变研究.中国实用内科杂志, 2004, 24(9): 535-537.
[6] Thomas GR, Forbes JR, Roberts EA, et al. The Wilson disease gene: spectrum of mutationsand their consequences. Nat Genet, 1995, 9(2): 210-217.
[7] Chuang LM, Wu HP, Jang MH, et al. High frequency of two mutations in codon 778 in exon8 of the ATP7B gene in Taiwanese families with Wilson disease. J Med Genet, 1996, 33(6):521-523.
[8] Okada T, Shiono Y, Hayashi H, et al. Mutational analysis of ATP7B and genotype-phenotypecorrelation in Japanese with Wilson’s disease. Hum Mutat, 2000, 15(5): 454-462.
[9]张萍,施小六,凌奇荷，等.应用分子遗传学方法进行Wilson病的症状前诊断.中国医师杂志. 2002. 4(9): 924-926.
[10]吴志英,王柠,林珉婷，等.肝豆状核变性病基因第1、2和21号外显子突变的检测分析.中华医学杂志. 2001. 81(2): 105-106.
[11]吴志英,王柠,慕容慎行,等.肝豆状核变性基因第3-20号外显子突变及多态的DNA测序研究.中华神经科杂志, 1998, 31(6): 23-25.
[12] Caca K, Ferenci P, Kühn HJ, et al. High prevalence of the H1069Q mutation in East Gemanpatients with Wilson disease: rapid detection of mutation by limited sequencing andphenotype-genotype analysis. J Hepatol, 2001, 35(5): 575-581.
[13] Figus A, Angius A, Loudianos G, et al. Molecular pathology and haplotype analysis ofWilson disease in Mediterranean populations. Am J Hum Genet,1995, 57(6): 1318-1324.
[14] Iorio R, D’Ambrosi M, Mazzarella G, et al. Early occurrence of hypertransaminasemia in a13-month-old child with Wilson disease. J Pediatr Gastroenterol Nutr, 2003, 36(5):637-638.
[15] Beyersdorff A, Findeisen A. Morbus Wilson: Case report of a two-year old child as firstmanifestation. Scand J Gastroenterol, 2006, 41(4): 496-497.
16] Mak CM, Tam S, Fan ST, et al. Wilson’s disease: a patient undiagnosed for 18 years. HongKong Med J, 2006, 12(2): 154-158.
17] Lo Curto AG, Marchi A, Grasso M, et al. Early diagnosis of Wilson Disease in asix-year-old child. J Pediatr, 2006, 148(1):141.
18] Gollan JL, Gollan TJ. Wilson disease in 1998: genetic, diagnostic and therapeutic aspects. JHepatol, 1998, 28(S1): S28–S36.
19] Schilsky ML. Diagnosis and treatment of Wilson’s disease. Pediatr Transplant, 2002, 6(1):15–19.
20] Caprai S, Loudianos G, Massei F, et al. Direct diagnosis of Wilson disease by moleculargenetics. J Pediatr, 2006, 148(1): 138-140.
21] Figus A, Lampis R, Devoto M, et al. Carrier detection and early diagnosis of Wilson'sdisease by restriction fragment length polymorphism analysis. J Med Genet, 1989, 26(2):78-82.
22] Saito T. An assessment of efficiency in potential screening for Wilson's disease. JEpidemiol Community Health, 1981, 35(4): 274-280.
23] Bowcock AM, Farrer LA, Cavalli-Sforza LL, et al. Mapping the Wilson disease locus to acluster of linked polymorphic markers on chromosome 13. Am J Hum Genet, 1987, 41(1):27-35.
24]刘晓青,张建明,顾学范,等. Wilson病ATP7B基因Arg778Leu突变与临床表型之相关性探讨.中华儿科杂志. 1999. 37(6): 359-361.
25]吴志英,王柠,林珉婷,等. Wilson病基因全长外显子的突变检测和分析.中华神经科杂志, 2001, 34(3): 152-155.
26]赵鹏,张本恕.天津地区汉族肝豆状核变性患者基因突变热区的检测.天津医科大学学报, 2005, 11(2): 213-215.
27]罗开忠,杨旭,童德军，等.湖南地区肝豆状核变性基因突变热区的序列检测与分析.中华肝脏病杂志, 2006, 14(12): 920-923.
28]鲍远程,吴鹏,余元勋，等.肝豆状核变性患者ATP7B基因8、12号外显子突变的DNA分析.中风与神经疾病杂志, 2008,25(3):336-340.
29] Houwen RH, Juyn J, Hoogenraad TU, et al. H714Q mutation in Wilson disease is associatedwith late, neurological presentation. J Med Genet, 1995, 32(6): 480-482.
30] Gromadzka G, Schmidt HH, Genschel J, et al. Frameshift and nonsense mutations in thegene for ATPase7B are associated with severe impairment of copper metabolism and withan early clinical manifestation of Wilson's disease. Clin Genet, 2005, 68(6): 524-532.
31] Tarnacka B, Gromadzka G, Rodo M, et al. Frequency of His1069Gln and Gly1267 Lysmutations in Polish Wilson’s disease population. Eur J Neurol, 2000, 7(5): 495-498.
32]杨斌,胡纪原,程楠，等.中国人Wilson病ATP7B基因Thr935Met突变与临床表型的关系研究.中国优生与遗传杂志, 2002, 10(4): 12-13.
33]刘晓青,张雅芬,刘孜孜,等.肝豆状核变性基因类型与临床表型关系研究.中华儿科杂志, 2003, 41(1): 35-38.
34]赵鹏,张本恕.肝豆状核变性ATP7B基因Arg778Leu突变型与临床表现的相关性研究.脑与神经疾病杂志, 2007, 15(2): 104-105.
[1] Danks DM. Hereditary disorders of copper metabolism in Wilson’s disease andMenkes’disease. Metabolic Basis of Inherited Disease, 1993, 6: 1251-1261.
[2]苏京锁,李保全,胡天强,等.肝豆状核变性脑部MRI的评估价值.中国临床康复, 2003,7(13): 1976-1977.
[3] Tanzi RE, Petrukhin K, Chemov I, et al. The Wilson disease gene is a copper transportingATPase with homology to the menkes disease gene. Nat Genet, 1993, 5(4): 344-350.
[4]诸福堂主编.实用儿科学,第七版.人民卫生出版社, 2000, 2056-2060.
[5] Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative coppertransporting P-type ATPase similar to the Menkes gene. Nat Genet, 1993, 5(4): 327-336.
[6]杨静芳,陈彪.中国人肝豆状核变性基因突变的分布特征.中国临床康复, 2004, 8(4):690-692.
[7]杨春水,梁秀龄,闫振文，等.肝豆状核变性基因启动子区的多态和突变研究.中国实用内科杂志, 2004, 24(9): 535-537.
[8] Thomas GR, Forbes JR, Roberts EA, et al. The Wilson disease gene: spectrum of mutationsand their consequences. Nat Genet, 1995, 9(2): 210-217.
[9] Loudianos G, Dessi V, Lovicu M, et al. Molecular characterization of Wilson disease in theSardinian population-evidence of a founder effect. Hum Mutat, 1999, 14(4): 294-303.
[10] Carcia-Villarreal L, Daniels S, Shaw SH, et al. High prevalence of the very rare Wilsondisease gene mutation Leu708Pro in the island of Gran Ganaria (Canary Islands, Spain): agenetic and clinical study. Hepatology, 2000, 32(6): 1329-1336.
[11] Chuang LM, Wu HP, Jang MH, et al. High frequency of two mutation in codon 778 in exon8 of the ATP7B gene in Taiwanese families with Wilson disease. J Med Genet, 1996, 33(6):521-523.
[12] Nanji MS, Nguyen VT, Kawasoe JH, et al. Haplotype and mutation analysis in Japanesepatients with Wilson disease. Am J Hum Genet, 1997, 60(6): 1423-1429.
[13] Okada T, Shiono Y, Hayashi H, et al. Mutational analysis of ATP7B andgenotype-phenotype correlation in Japanese with Wilson disease. Hum Mutat, 2000, 15(5):454-462.
[14] Wu ZY, Wang N, Lin MT, et al. Mutation analysis and the correlation between genotypeand phenotype of Arg778Len mutation in Chinese patients with Wilson disease. ArchNeurol, 2001, 58(6): 971-976.
[15]赵鹏,张本恕.天津地区汉族肝豆状核变性患者基因突变热区的检测.天津医科大学学报, 2005, 11(2): 213-215.
[16]罗开忠,杨旭,童德军，等.湖南地区肝豆状核变性基因突变热区的序列检测与分析.中华肝脏病杂志, 2006, 14(12): 920-923.
[17]吴志英,王柠,林珉婷,等. Wilson病基因全长外显子的突变检测和分析.中华神经科杂志, 2001, 34(3): 152-155.
[18] Caca K, Ferenci P, Kühn HJ, et al. High prevalence of the H1069Q mutation in East Gemanpatients with Wilson disease: rapid detection of mutation by limited sequencing andphenotype-genotype analysis. J Hepatol, 2001, 35(5): 575-581.
[19]梁秀龄.肝豆状核变性诊断与治疗中需要注意的问题.中国现代神经疾病杂志, 2007,7(1): 20-28.
[2] Tanzi RE, Petrukhin K, Chemov I, et al. The Wilson disease gene is a copper transportingATPase with homology to the menkes disease gene. Nat Genet, 1993, 5(4): 344-350.
[3] Bull PC, Thomas GR, Rommens JM, et al. The Wilson disease gene is a putative c- oppertransporting P-type ATPase similar to the Menkes gene. Nat Genet, 1993, 5(4): 327-337.
[4]杨静芳,陈彪.中国人肝豆状核变性基因突变的分布特征.中国临床康复, 2004, 8(4):690-692.
[5]杨春水,梁秀龄,闫振文，等.肝豆状核变性基因启动子区的多态和突变研究.中国实用内科杂志, 2004, 24(9): 535-537.
[6] Thomas GR, Forbes JR, Roberts EA, et al. The Wilson disease gene: spectrum of mutationsand their consequences. Nat Genet, 1995, 9(2): 210-217.
[7] Chuang LM, Wu HP, Jang MH, et al. High frequency of two mutations in codon 778 in exon8 of the ATP7B gene in Taiwanese families with Wilson disease. J Med Genet, 1996, 33(6):521-523.
[8] Okada T, Shiono Y, Hayashi H, et al. Mutational analysis of ATP7B and genotype-phenotypecorrelation in Japanese with Wilson’s disease. Hum Mutat, 2000, 15(5): 454-462.
[9]张萍,施小六,凌奇荷，等.应用分子遗传学方法进行Wilson病的症状前诊断.中国医师杂志. 2002. 4(9): 924-926.
[10]吴志英,王柠,林珉婷，等.肝豆状核变性病基因第1、2和21号外显子突变的检测分析.中华医学杂志. 2001. 81(2): 105-106.
[11]吴志英,王柠,慕容慎行,等.肝豆状核变性基因第3-20号外显子突变及多态的DNA测序研究.中华神经科杂志, 1998, 31(6): 23-25.
[12] Caca K, Ferenci P, Kühn HJ, et al. High prevalence of the H1069Q mutation in East Gemanpatients with Wilson disease: rapid detection of mutation by limited sequencing andphenotype-genotype analysis. J Hepatol, 2001, 35(5): 575-581.
[13] Figus A, Angius A, Loudianos G, et al. Molecular pathology and haplotype analysis ofWilson disease in Mediterranean populations. Am J Hum Genet,1995, 57(6): 1318-1324.
[14] Iorio R, D’Ambrosi M, Mazzarella G, et al. Early occurrence of hypertransaminasemia in a13-month-old child with Wilson disease. J Pediatr Gastroenterol Nutr, 2003, 36(5):637-638.
[15] Beyersdorff A, Findeisen A. Morbus Wilson: Case report of a two-year old child as firstmanifestation. Scand J Gastroenterol, 2006, 41(4): 496-497.
16] Mak CM, Tam S, Fan ST, et al. Wilson’s disease: a patient undiagnosed for 18 years. HongKong Med J, 2006, 12(2): 154-158.
17] Lo Curto AG, Marchi A, Grasso M, et al. Early diagnosis of Wilson Disease in asix-year-old child. J Pediatr, 2006, 148(1):141.
18] Gollan JL, Gollan TJ. Wilson disease in 1998: genetic, diagnostic and therapeutic aspects. JHepatol, 1998, 28(S1): S28–S36.
19] Schilsky ML. Diagnosis and treatment of Wilson’s disease. Pediatr Transplant, 2002, 6(1):15–19.
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