Luận án Phát hiện người mang đột biến gen Atp7b trong các thành viên gia đình bệnh nhân wilson

nts to treat soon. Because biochemical tests can only be used in diagnosis for Wilson patients who have presented manifestations; they cannot distinguish individuals who are asymptomatic, especially in the case of heterozygote carriers (Schilsky et al. 2009; 130 Wan et al. 2006; Maleki et al. 2015; Manoochehri et al. 2014). Family screening of patients with Wilson disease is the best method of prognosis, and indicator for further treatment, for those suffering from Wilson disease (Manoochehri et al. 2014). This can help to prevent serious complications of Wilson disease, and is an effective and safe treatment (Seo et al. 2016). Among 3 fetuses were prenatal diagnosis, 1 were homozygote and would be suffered from Wilson disease, the two others were carrier. This result is important in treatment soon after birth and genetic counseling pre-marriage. The discovery of the ATP7B gene has opened up a new molecular diagnostic approach in genetic pre- marriage counseling and prenatal diagnosis, and could form the basis of genetic therapy in the future (Pfeiffer et al. 2007; Schilky et al. 2009; Zhang et al. 2011). Conclusion 27 different mutations were identified, with 7 novel mutations (H251AfsX19, P868PfsX5, (R723S;H724TfsX34), V1042CfsX79, F1026Y, IVS6+3A>G, IVS20+4A>G). The most common mutation in the study was S105X and hot-spot region were exon 2, exon 8, exon 16, exon 14, intron 14, exon 18 and exon 20 should be screen firstly for Wilson patients in Vietnam. Base on identifying the characteristic types of mutations in Vietnamese Wilson patients, procedure to detect ATP7B gene mutations were divided into 4 steps in order will facilitate faster and more effective genetic diagnoses of Wilson disease in the Vietnamese population. A total of 13 affected siblings were identified by target mutation on ATP7B gene which were identified in the proband. Among them, 5 cases were asymptomatic that would be treated soon to prevent clinical feature. This study also discoved 65 carrier in their family members. Prenatal diagnosis found 1 fetus homozygote and would be suffered from Wilson disease and 2 fetuses were carriers. The findings’ highest diagnostic importance for patients and their family members is in prognosis and the prevention of morbidity and mortality. 131 8. TÀI LIỆU THAM KHẢO 1. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR (2010) A method and server for predicting damaging missense mutations. Nature methods 7(4): 248–249. 2. Aggarwal A, Chandhok G, Todorov T, Parekh S, Tilve S, Zibert A, Bhatt M, Schmidt HH (2013) Wilson disease mutation pattern with genotype- phenotype correlations from Western India: confirmation of p.C271* as a common Indian mutation and identification of 14 novel mutations. Ann Hum Genet 77(4): 299-307. 3. Ala A, Walker AP, Ashkan K, Dooley JS, Schilsky ML (2007) Wilson’s disease. Lancet 369 (9559): 397-408. 4. Anjali BS, Igor C, Zhang TH, Ross MB, Das K, Lutsenko S, Parano S, Pavone L, Evgrafov P, Smonlensqkaya IAI, Anneren’ G, Westermark K, Uqrrutia HF, Penchaszadeh KG, Sternilieb I, Scheinberg HI, Gilliam CT, Petrukhin K (1997) Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype-phenotype correlation, and functional analyses”. Am.J.Hum.Gennet 61: 317-328. 5. Aoki T (2004) Copper deficiency and the clinical practice. Trace elements (JMAL) 47(8): 365-370. 6. Arianfar F, Dastsooz H, Fardaei M et al (2015) Common polymorphism of ATP7B gene as a good marker in linkage analysis in Wilson disease patients from sounthern Iran. Journal of advanced medical sciences and applied technology (JAMSAT) 1(1): 30-34. 7. Bost M, Piguet-Lacroix G, Parant F, Wilson CM (2012) Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis. J Trace Elem Med Biol 26(2-3): 97- 101. 8. Brewer GJ, Fred K, Askari (2005) Wilson's disease: clinical management and 132 therapy. J Hepatol 42(1): 13-21. 9. Bucossi S, Squitti R et al (2012) Association of K832R and R952K SNPs of Wilson disease gene with Alzheimer’s disease. J Alzheimers Dis 29(4): 913- 919. 10. Cater MA, La Fontaine S, Mercer JF (2007) Copper binding to the N- terminal metal-binding sites or the CPC motif is not essential for copper- induced trafficking of the human Wilson protein (ATP7B). Biochem J (1): 143-153. 11. Chang IJ, Hahn SH (2017) The genetics of Wilson disease. Handb Clin Neurol 142 (3rd series): 19-34 12. Chaudhry HS, Bhimji SS (2017) Wilson Disease. Copyright © 2018, Stat Pearls Publishing LLC. 13. Chen C, Shen B, Xiao JJ, Wu R, Canning SJD, Wang XP (2015) Currently clinical views on genetics of Wilson’s disease. Chinese Med J 128(13): 1826–1830. 14. Cheng N, Wang H, Wu W, Yang R, Liu L, Han Y, Guo L, Hu J, Xu L, Zhao J, Han Y, Liu Q, Li K, Wang X, Chen W (2017) Spectrum of ATP7B mutations and genotype-phenotype correlation in large-scale Chinese patients with Wilson Disease. Clin Genet 92(1):69-79. 15. Cho YH, Lee YH et al (2011) A case of Wilson’ disease in patient with mildly elevated liver enzyme. Korean J Farm Med 32: 205-208. 16. Choi Y, Chan AP (2015) PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 31(16): 2745-2747. 17. Coffey AJ, Durkie M, Hague S, McLay K, Emmerson J, Lo C, Klaffke S, Joyce CJ, Dhawan A, Hadzic N, Mieli-Vergani G, Kirk R, Elizabeth Allen K, Nicholl D, Wong S, Griffiths W, Smithson S, Giffin N, Taha A, Connolly S, Gillett GT, Tanner S, Bonham J, Sharrack B, Palotie A, Rattray M, Dalton 133 A, Bandmann O (2013) A genetic study of Wilson's disease in the United Kingdom. Brain 136(Pt 5):1476-87. 18. Cox DW, Prat L, Walshe JM, Heathcote J, Gaffney D (2005) Twenty-four novel mutations in Wilson disease patients of predominantly European ancestry. Hum Mutat 26(3):280. 19. Curtis D, Durkie M, Balac (Morris) P, Sheard D, Goodeve A, Peake I, Quarrell O, Tanner S (1999) A study of Wilson disease mutations in Britain. Hum Mutat 14(4): 304-311. 20. Das SK, Ray K (2006) Wilson's disease: an update. Nat Clin Pract Neurol 2(9): 482-493. 21. De Bie P, Muller P, Wijmenga C, Klomp LW (2007) Molecular pathogenesis of Wilson and Menkes disease: correlation of mutations with molecular defects and disease phenotype. J Med Genet 44(11): 673-688. 22. de Jong A, Dondorp WJ, Frints SG, de Die-Smulders CE, de Wert GM (2011) Advances in prenatal screening: the ethical dimension. Nat Rev Genet 12(9): 657-663. 23. Deguti MM, Genschel J, Cancado EL, Barbosa ER, Bochow B, Mucenic M, Porta G, Lochs H, Carrilho FJ, Schmidt HH (2004) Wilson disease: Novel mutations in ATP7B gene and clinical correlation in Brazilian patients. Hum Mutat 23(4): 398. 24. Đỗ Thanh Hương (2016) Phân tích mối tương quan giữa đột biến gen ATP7B và kiểu hình của bệnh nhân Wilson ở Việt Nam. Luận án Tiến sĩ Y học. Trường Đại học Y Hà Nội. 25. Đỗ Thanh Hương, Nguyễn Văn Liễu, Khuất Thị Nga, Nguyễn Thị Vân Anh, Phan Tuấn Nghĩa (2010) Xác định đột biến thường gặp R778L của gen ATP7B trên người Việt Nam mắc WD. Tạp chí Nhi khoa 23(3&4): 231-235. 26. Dong Y, Zhu H (2005) Single-strand conformational polymorphism analysis: basic principles and routine practice. Methods Mol Med 108: 149-157. 134 27. Drubay D, Gautheret D, Michiels S (2018) A benchmark study of scoring methods for non-coding mutations. Bioinformatics 34(10):1635-1641. 28. Erlinger S (2016) A microbial peptide to rescue severe and fulminant Wilson disease? Clin Res Hepatol Gastroenterol 40(6): 647-649. 29. Fan Y, Yu L, Jiang Y, Xu Y, Yang R, Han Y, Cui Y, Ren M, Zhao S (2000) Identification of a mutation hotspot in exon 8 Wilson disease gene by cycle sequencing. Chin Med J (Engl) 113(2): 172-174. 30. Fatemi N (2011) Structural and Functional Studies of ATP7B, the Copper(I)- transporting P-type ATPase Implicated in Wilson Disease. A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy. Graduate Department of Biochemistry University of Toronto. 31. Fatemi N, Sarkar B (2002) Molecular mechanism of copper transport in Wilson disease. Environ Health Perspect 110 (5): 695-698. 32. Ferenci P (2004) Pathophysiology and clinical features of Wilson disease. Metab Brain Dis 19(3-4): 229-239. 33. Ferenci P (2006) Regional distribution of mutations of the ATP7B gene in patients with Wilson disease: impact on genetic testing. Hum Genet 120(2): 151-159. 34. Ferenci P, Czlonkowska A, Stremmel W, Houwen R, Rosenberg W, Schilsky M, Jansen P, Moradpour D (European Association for the study of the liver) (2012) EASL Clinical practice guidelines: Wilson ‘s disease. J Hepatol 56(3): 671-85. 35. Ferenci P, Steindl-Munda P, Vogel W, Jessner W, Gschwantler M, Stauber R, Datz C, Hackl F, Wrba F, Bauer P, Lorenz O (2005) Diagnostic value of quantitative hepatic copper determination in patients with Wilson's Disease. Clin Gastroenterol Hepatol 3(8): 811-818. 36. Figus A, Angius A, Loudianos G, Bertini C, Dessi V, Loi A, Deiana M, Lovicu M, Olla N, Sole G, et al. (1995) Molecular pathology and haplotype 135 analysis of Wilson disease in Mediterranean populations. Am J Hum Genet 57(6): 1318-13124. 37. Figus A, Lampis R, Devoto M, Ristaldi SM, Ideo A, Virgilis DS, Nurchi MA, Corrias A, Corda R, Lai EM, Tocco A, Deplano A, Solinas A, Zancan L, Lee W-H, Cao A, Pirastu M, Balestrieri A (1989) Carrier detection and early diagnosis of Wilson ‘s disease by restriction fragment length polymorphism analysis. Journal of Medical Genetics 26: 78-82. 38. Firneisz G, Lakatos PL, Szalay F, Polli C, Glant TT, Ferenci P (2002) Common mutations of ATP7B in Wilson disease patients from Hungary. Am J Med Genet 108(1): 23-28. 39. Folhoffer A, Ferenci P, Csak T, Horvath A, Hegedus D, Firneisz G, Osztovits J, Kosa JP, Willheim-Polli C, Szonyi L, Abonyi M, Lakatos PL, Szalay F (2007) Novel mutations of the ATP7B gene among 109 Hungarian patients with Wilson's disease. Eur J Gastroenterol Hepatol 19(2): 105-111. 40. Forbes JR, Cox DW (1998) Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant? Am J Hum Genet 63(6): 1663-1674. 41. Galehdari H, Tangestani R (2012) Molecular genetics diagnosis of Wilson disease: The first reported case of ATP7B gene mutation at condon 778 in Southwest Iran. Iranian Journal of Pathology 7(4): 262-266. 42. García-Villarreal L, Daniels S, Shaw SH, Cotton D, Galvin M, Geskes J, Bauer P, Sierra-Hernández A, Buckler A, Tugores A (2000) High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the Island of Gran Canaria (Canary Islands, Spain): a genetic and clinical study. Hepatology 32(6): 1329-1336. 43. Geng J, Wang J, Yao RE, Liu XQ, Fu QH (2013) Identification of one novel and nine recurrent mutations of the ATP7B gene in 11 children with Wilson 136 disease. World J Pediatr 9(2): 158-162. 44. Gromadzka G, Chabik G, Mendel T, Wiechowska A, Rudnicka M, Czlonkowska A (2010) Middle-aged heterozygous carriers of Wilson’s disease do not present with significant phenotypic deviations related to copper metabolism. Journal of Genetics 89(4): 463-7. 45. Gu YH, Kodama H, Du SL, Gu QJ, Sun HJ, Ushijima H (2003) Mutation spectrum and polymorphisms in ATP7B identified on direct sequencing of all exons in Chinese Han and Hui ethnic patients with Wilson ‘disease. Clin Genet 64(6): 479-484. 46. Gunjan D, Shalimar, Nadda N, Kedia S, Nayak B, Paul SB, Gamanagatti SR, Acharya SK (2017) Hepatocellular Carcinoma: An Unusual Complication of Longstanding Wilson Disease. J Clin Exp Hepatol 7(2): 152-154. 47. Gupta A, Aikath D, Neogi R, Datta S, Basu K, Maity B, Trivedi R, Ray J, Das KS, Gangopadhyay KP, Ray K (2005) Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotype– phenotype correlation in Indian patients. Hum Genet 118: 49–57. 48. Han. WY (2002) Identification of novel mutations and the three most common mutations in the human ATP7B gene of Korean patients with Wilson disease. Genetics in Medicine 4, 43S–48S. 49. Hoàng Lê Phúc, Kerstin Zinober, Claudia Willheim, Peter Ferenci (2008) Tầm soát bệnh Wilson: vai trò của phân tích đột biến phân tử gien ATP7B. Tạp chí Y học TP.Hồ Chí Minh 12(4): 246-251 50. Hoàng Lê Phúc, Krsin Zinober, Claudia Willheim, Peter Ferenci (2013) Phân tích đột biến phân tử gien ATP7B ở bệnh nhân Wilson Việt Nam. Nhi khoa 6(4): 21-28. 51. Hoàng Lê Phúc, Phạm Lê An, Trần Diệp Tuấn (2015) Xác định bước đầu mối liên quan kiểu gien-kiểu hình của bệnh Wilson ờ trẻ em Việt Nam. Nhi 137 khoa 8(6): 67-76 52. Huster D, Finegold MJ, Morgan CT, Burkhead JL, Nixon R, Vanderwerf SM, Gilliam CT, Lutsenko S (2006) Consequences of copper accumulation in the livers of the Atp7b–/– (Wilson disease gene) knockout mice. Am J Pathol 168(2): 423–434. 53. Huster D, Hoppert M, Lutsenko S, et al (2003) Defective cellular localization of mutant ATP7B in Wilson’s disease patients and hepatoma cell lines. Gastroenterology 124(2): 335–345. 54. Huster D, Kühne A, Bhattacharjee A, Raines L, Jantsch V, Noe J, Schirrmeister W, Sommerer I, Sabri O, Berr F, Mössner J, Stieger B, Caca K, Lutsenko S (2012) Diverse functional properties of Wilson disease ATP7B variants. Gastroenterology 142(4): 947-956. 55. Jang JH, Lee T, Bang S, Kim YE, Cho EH (2017) Carrier frequency of Wilson's disease in the Korean population: a DNA-based approach. J Hum Genet 62(9): 815-818. 56. Jian X, Liu X (2014) In Silico prediction of deleteriousness for nonsynonymous and splice-altering single nucleotidevariants in the human genome. Methods Mol Biol 1498:191-197. 57. Kakavas VK, Plageras P, Vlachos TA, Papaioannou A, Noulas VA (2008) PCR-SSCP: A method for the molecular analysis of genetic diseases. Mol Biotechnol 38: 155-163. 58. Karki R, Pandya D, Elston RC, Ferlini C (2015) Defining "mutation" and "polymorphism" in the era of personal genomics. BMC Med Genomics 8:37. 59. Kenney SM, Cox DW (2007) Sequence variation database for the Wilson disease copper transporter, ATP7B. Hum Mutat 28(12): 1171-1177. 60. Kim GH, Yang JY, Park JY, Lee JJ, Kim JH, Yoo HW (2008) Estimation of Wilson's disease incidence and carrier frequency in the Korean population by screening ATP7B major mutations in newborn filter papers using the SYBR 138 green intercalator method based on the amplification refractory mutation system. Genet Test 12(3): 395-399. 61. Kim TJ, Kim IO, Kim WS, Cheon JE, Moon SG, Kwon JW, Seo JK, Yeon KM (2006) MR imaging of the brain in Wilson disease of childhood: findings before and after treatment with clinical correlation. AJNR Am J Neuroradiol 27(6): 1373-1378. 62. Kircher M, Witten DM, Jain P, O'Roak BJ, Cooper GM, Shendure J (2014) A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 46(3): 310-5. 63. Kucinskas L, Jeroch J, Vitkauskiene A, Sakalauskas R, Petrenkiene V, Kucinskas V, Naginiene R, Schmidt H, Kupcinskas L (2008) High frequency of the c.3207C>A (H1069Q) mutation in ATP7B gene of Lithuanian patients with hepatic presentation of Wilson's disease. World J Gastroenterol 14(38): 5876-5879. 64. Kumar P, Henikoff S, Ng P C (2009) Predicting the effects ofcoding non- synonymous variants on protein function using the SIFT algorithm. Nature Protocols 4(7): 1073–1082. 65. Kumar SS, Kurian G, Eapen CE, Roberts EA (2012). Genetics of Wilson's disease: a clinical perspective. Indian J Gastroenterol 31(6): 285-293. 66. Kusuda Y, Hamaguchi K, Mori T, Shin R, Seike M, Sakata T (2000) Novel mutations of the ATP7B gene in Japanese patients with Wilson disease”. J Hum Genet 45(2): 86-91. 67. Lalioti V, Sandoval I, Cassio D, Duclos-Vallée J-C (2010) Molecular pathology of Wilson ‘s disease: a brief. J Hepatol 52: 1151-1153. 68. Le Anh Tuan Pham, Trong Tue Nguyen, Hoang Bich Nga Le, Dat Quoc Tran, Cam Tu Ho, Thinh Huy Tran, Van Thanh Ta, The Hung Bui, Van Khanh Tran (2017) Genetic analysis of 55 northern Vietnamese patients with Wilson disease: seven novel mutations in ATP7B. Journal of Genetics 96(6): 933–939. 139 69. Lê Đình Lương (2001) Nguyên lý kĩ thuật di truyền. Nhà xuất bản Khoa học Kĩ thuật, Hà Nội. 70. Lê Đức Hinh (2006) Đặc điểm lâm sàng của bệnh nhân Wilson ở Việt Nam. Tạp chí y học Việt Nam 363: 1-9. 71. Lee BH, Kim JH, Lee SY, Jin HY, Kim KJ, Lee JJ, Park JY, Kim GH, Choi JH, Kim KM, Yoo HW (2011) Distinct clinical courses according to presenting phenotypes and their correlations to ATP7B mutations in a large Wilson's disease cohort. Liver Int 31(6): 831-839. 72. Lee HJ, Seong WJ, Hong SY, Bae JY (2015) Successful pregnancy outcome in a Korean patient with symptomatic Wilson's disease. Obstet Gynecol Sci 58(5): 409-413. 73. Leggio L, Malandrino N, Loudianos G, Abenavoli L, Lepori MB, Cappristo E, Virgiliis SD, Gasbarrini G, Addolorato G (2007) Analysis of the T1288R mutation of the Wilson disease ATP7B gene in four generations of a family: Possible genotype-phenotype correlation with hepatic onset. Dig. Dis. Sci 52: 2570-2575. 74. Lepori MB, Zappu A, Incollu S, Dessì V, Mameli E, Demelia L, Nurchi AM, Gheorghe L, Maggiore G, Sciveres M, Leuzzi V, Indolfi G, Bonafé L, Casali C, Angeli P, Barone P, Cao A, Loudianos G (2012) Mutation analysis of the ATP7B gene in a new group of Wilson's disease patients: contribution to diagnosis. J Pediatr 140(5): 499-501. 75. Li XH, Lu Y, Ling Y, Fu QC, Xu J, Zang GQ, Zhou F, De-Min Y, Han Y, Zhang DH, Gong QM, Lu ZM, Kong XF, Wang JS, Zhang XX (2011) Clinical and molecular characterization of Wilson‘s disease in China: identification 04 14 novel mutations. BMC Med Genet 12: 16. 76. Liborio S, Ivana a, Giandomenico P, Valentina G (2012) Use of the MLPA assay in the molecular diagnosis of gene copy number alternations in human genetic diseases. Int.J.Mol Sci 13: 3245-3247. 140 77. Lin CW, Er TK, Tsai FJ, Liu TC, Shin PY, Chang JG (2010) Development of high-resolution melting method for the screening of Wilson disease-related ATP7B gene mutations. Clin Chim Acta 411 (17-18): 1223-12231. 78. Liu M, Jin M, Chen X, Wan B, Guo Y, Sheng M, Chen L, Zhao L, Huang D, Li Y (2017) ATP7B mutation detection and pathogenicity analysis: One atypical case of Wilson's disease with adrenocortical insufficiency. J Mol Neurosci 64(1):20-28. 79. Liu XQ, Zhang YF, Liu TT, Hsiao KJ, Zhang JM, Gu XF, Bao KR, Yu LH, Wang MX (2004) Correlation of ATP7B genotype with phenotype in Chinese patients with Wilson disease. World J Gastroenterol 10(4): 590-593. 80. Loudianos G, Dessi V, Lovicu M, Angius A, Altuntas B, Giacchino R, Marazzi M, Marcellini M, Sartorelli MR, Sturniolo GC, Kocak N, Yuce A, Akar N, Pirastu M, Cao A (1999) Mutation analysis in patients of Mediterranean descent with Wilson disease: identification of 19 novel mutations. J Med Genet 36(11): 833-836. 81. Loudianos G, Dessì V, Lovicu M, Angius A, Kanavakis E, Tzetis M, Kattamis C, Manolaki N, Vassiliki G, Karpathios T, Cao A, Pirastu M. (1998) Haplotype and mutation analysis in Greek patients with Wilson disease. Eur J Hum Genet 6(5): 487-491. 82. Ludwig J, Moyer TP, Rakela J (1994) The liver biopsy diagnosis of Wilson's disease. Methods in pathology. Am J Clin Pathol 102(4): 443-446. 83. Luoma LM, Deeb TM, Macintyre G, Cox DW (2010) Functional analysis of mutations in the ATP loop of the Wilson disease copper transporter, ATP7B. Hum Mutat 31(5): 569-577. 84. Lutsenko S, Barnes NL, Bartee MY, Dmitriev OY (2007) Function and regulation of human copper-transporting ATPase. Physiol Rev 87(3): 1011- 1146. 85. Lutsenko S, Tsivkovskii R, Cooper J Matthew, MacArthur C Brian, 141 Bachinger Hans-Peter (2002) Biochemistry of the Wilson’s Disease Protein. Handbook of copper pharmacology and toxicology. Humana Press Inc, Totowa, NJ. 86. Lutsenko S. (2014) Modifying factors and phenotypic diversity in Wilson’s disease. Ann N Y Acad Sci 1315(1): 56–63. 87. Lv W, Wei X, Guo R, Liu Q, Zheng Y, Chang JBai T, Li H, Zhang J, Song Z, Cram DS, Liang D, Wu L (2015) Non invasive prenatal testing for Wilson disease by use of circulating single-molecule amplification and resequencing technology (cSMART). Clin Chem 61(1): 172-181. 88. Mak MC, Lam C-W, Tam S (2008) Diagnostic accuracy of serum ceruloplasmin in Wilson disease: Determination of sensitivity and specificity by ROC curve among analysis among ATP7B-genotype subjects. Clinical chemistry 54(8): 1356-1362. 89. Maleki. I, Zali M.R, Abadi H.N (2013) Novel mutation of ATP7B gene in Iranian patients with Wilson‘disease. Res Mol Med 1(1): 44-47. 90. Malik A, Khawaja A, Sheikh L (2013) Wilson's disease in pregnancy: case series and review of literature.BMC Res Notes 18(6): 421. 91. Manoochehri J, Masoumi RD, Faraji H, Mohammadi S, Dastsooz H, Moradi T, Rezaei E, Sadeghi KH, Fardaei M (2014) Family screening for a novel ATP7B gene mutation, c.2335T>G, in the South of Iran. Iran J Ped Hematol Oncol 4(1): 26-31. 92. Medicin V, Rossaro L (2006) Wilson’s disease: a genetic but treatable liver disorder. Homepage:www.hcvadvocate.org. 93. Miron PM (2012) Preparation, culture, and analysis of amniotic fluid samples. Curr Protoc Hum GenetChapter 8:Unit8.4. 94. Nguyễn Thị Mai Hương, Nguyễn Thị Phương Mai, Lý Thị Thanh Hà, Ngô Diễm Ngọc, Nguyễn Phạm Anh Hoa, Nguyễn Thanh Liêm, HW.Yoo (2010) Phát hiện đột biến gen ATP7B gây bệnh Wilson. Tạp chí Nhi Khoa 3(3&4): 142 236-240. 95. Ogino S, Gulley ML, den Dunnen JT, Wilson RB (2007) Standard mutation nomenclature in molecular diagnostics: practical and educational challenges. J Mol Diagn 9(1): 1-6. 96. Okada T, Shiono Y, Hayashi H, Satoh H, Sawada T, Suzuki A, Takeda Y, Yano M, Michitaka K, Onji M, Mabuchi H (2000) Mutational analysis of ATP7B and genotype-phenotype correlation in Japanese with Wilson disease. Human Mutation 15: 454-462. 97. Panichareon B, Taweechue K, Thongnoppakhun W, Aksornworanart M, Pithukpakorn M, Yenchitsomanus PT, Limwongse C, Limjindaporn T (2011) Six novel ATP7B mutations in Thai patients with Wilson disease. Eur J Med Genet 54(2): 103-107. 98. Park S, Park JY, Kim GH, Choi JH, Kim KM, Kim JB, Yoo HW (2007) Identification of novel ATP7B gene mutation and their functional roles in Korean patients with Wilson disease. Hum Mutat 28(11): 1108-1113. 99. Patil M, Sheth AK, Krishnamurthy CA, Devarbhavi H (2013) A Review and Current Perspective on Wilson Disease. J Clin Exp Hepatol 3(4): 321–336. 100. Petrukhin K, Fischer SG, Pirastu M, Tanzi RE, Chernov I, Devoto M, Brzustowicz LM, Cayanis E, Vitale E, Russo JJ, et al (1993) Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nat Genet 5(4): 338-343. 101. Pfeiffer RF (2007) Wilson’disease. Semin Neuro 27(2): 123-132. 102. Phan Tôn Hoàng (2015) Nghiên cứu phát hiện đột biến gen ATP7B trên bệnh nhân Wilson. Luận án Tiến sĩ y học. Bộ môn Y sinh học, Trường Đại học Y Hà Nội. 103. Roberts EA, Schilsky ML (2003) A practice guideline on Wilson disease. Hepatology 37(6): 1475-1492. 104. Rogers MF, Shihab HA, Mort M, Cooper DN, Gaunt TR, Campbell C (2017) 143 FATHMM-XF: accurate prediction of pathogenic point mutations via extended features. Bioinformatics 34(3): 511-513. 105. Ronald F, Pfeiffer (2007) Wilson‘s disease. Semin Neuro 27(2): 123-132. 106. Sambrook J, Fritsch EF, Maniatis T (2001) Molecular Cloning: A laboratory manual". Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 107. Santhosh S, Shaji RV, Eapen CE, Jayanthi V, Malathi S, Chandy M, Stanley M, Selvi S, Kurian G, Chandy GM (2006) ATP7B mutations in families in a predominantly Southern Indian cohort of Wilson's disease patients. Indian journal of gastroenterology 25(6): 277-282. 108. Scheinberg IH, Sternlieb I (1965) Wilson’ disease. Annu Rev Med 16: 119- 134. 109. Schilsky LM (2009) Wilson disease: Current status and the future. Biochimie 91: 1278-1281. 110. Schwarz JM, R¨odelsperger C, Schuelke M, Seelow D (2010) MutationTaster evaluates disease-causing potential of sequence alterations. Nature Methods 7(8):575–576. 111. Seo KJ (2016) Wilson disease: an update (Article in Korean). Korean J Hepatol 12(3): 333-363. 112. Shihab HA1, Gough J, Cooper DN, Stenson PD, Barker GL, Edwards KJ, Day IN, Gaunt TR (2013) Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models. Hum Mutat 34(1):57-65. 113. Shiono Y, Wakusawa S, Hayashi H, Takikawa T et al (2001) Iron accumulation in the liver of male patients with Wilson's disease. Am J Gastroenterol 96(11): 3147-3151. 114. Squitti R, Siotto M, Bucossi S, Polimanti R (2014) In silico investigation of the ATP7B gene: insights from functional prediction of non-synonymous substitution to protein structure. Biometals 27: 53-64. 144 115. Stephen W. Mercer, Jianbin Wang, and Richard Burke (2016) In Vivo modeling of the pathogenic effect of copper transporter mutations that cause Menkes and Wilson diseases, motor neuropathy, and susceptibility to Alzheimer's Disease. J Biol Chem 292(10): 4113-4122. 116. Stuppia L, Antonucci I, Palka G, Gatta V (2012) Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci 13(3): 3245-3276. 117. Sullivan AC, Chopdar A, Shun-Shin AG (2002) Dense Kayser-Fleischer ring in asymptomatic Wilson's disease (hepatolenticular degeneration). Br J Ophthalmol 86(1): 114. 118. Sundaresan S, Eapen CE, Shaji RV, Chandy M, Kurian G, Chandy G (2007) Screening for mutations in ATP7B gene using conformation-sensitive gel electrophoresis in a family with Wilson's disease. Med Sci Monit 13(3): CS38-40. 119. Suporn Treepongkaruna, Paneeya Paneey Pienvichit, Pornpimon Phuapradit, Porawee Kodcharin, Duangrurdee Wattanasirichaigoo (2010) Mutations of ATP7B gene in two Thai siblings with Wilson disease. Asia biomedicine 4(1): 163-169. 120. Tạ Thành Văn (2010) PCR và một số kỹ thuật y sinh học phân tử. Nxb Y học, Hà Nội. 121. Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgacid aminrd-Pedersen B (1986) Randomised controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet 1(8493): 1287-1293. 122. Tanzi RE, Petrukhin K, Chernov I, Pellequer JL, Wasco W, Ross B, Romano DM, Parano E, Pavone L, Brzustowicz LM, et al. (1993) The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat Genet 5(4): 344-350. 123. Thái Duy Thành (2005) Nghiên cứu một số đặc điểm lâm sàng và cận lâm 145 sàng của WD tại khoa Thần kinh Bệnh viện Bạch Mai từ năm 2002 đến 2005. Luận văn bác sĩ nội trú bệnh viện, Trường Đại học Y Hà Nội. 124. The Human Gene Mutation Database 125. Theodora M, Antsaklis A, Antsaklis P, Blanas K, Daskalakis G, Sindos M, et al (2015) Fetal loss following second trimester amniocentesis. Who is at greater risk? How to counsel pregnant women? J Matern Fetal Neonatal Med: 29(4): 590-5. 126. Thomas GR, Forbes JR, Roberts EA, Walshe JM, Cox DW (1995) The Wilson disease gene: spectrum of mutations and their consequences. Nat Genet 9(2): 210-217. 127. Thomas GR, Roberts EA, Walshe JM, Cox DW (1995) Haplotypes and mutations in Wilson disease. Am J Hum Genet 56 (6): 1315-1319. 128. Tongsong T, Wanapirak C, Sirivatanapa P, Piyamongkol W, Sirichotiyakul S, Yampochai A (1998) Amniocentesis-related fetal loss: a cohort study. Obstet Gynecol 92(1):64-7. 129. Tsai CH, Tsai FJ, Wu JY, Chang JG, Lee CC, Lin SP, Yang CF, Jong YJ, Lo MC(1998) Mutation analysis of Wilson disease in Taiwan and description of six new mutations. Hum mut 12(6): 370-376. 130. Vassiliki L, Ignacio S, Doris C, Jean-Charles DV (2010) Molecular pathology of Wilson‘s disease: a brief. J Hepatol 53: 1151-1153. 131. Vrabelova S, Letocha O, Borsky M, Kozak L (2005) Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease. Mol Genet Metab 86(1-2): 277-185. 132. Wan L, Tsai CH, Hsu CM, Huang CC, Yang CC, Liao CC, Wu CC, Hsu YA, Lee CC, Liu SC, Lin WD, Tsai FJ (2010) Mutation analysis and characterization of alternative splice variants of the Wilson disease gene ATP7B. Hepatology 52(5): 1662-1670. 133. Wan L, Tsai CH, Tsai Y, Hsu CM, Lee CC, Tsai FJ (2006) Mutation analysis of Taiwanese Wilson disease patients. Biochem Biophys Res Commun 146 345(2): 734-738. 134. Wang HL, Huang QY, Shang X, Su XQ, Xiong F, Yu YQ, Lin PH, Wei SZ, Hong FM, Xu MX (2011) Mutation analysis of 73 southern Chinese Wilson’s disease patients: identification of 10 novel mutations and its clinical correlation. J Hum Genet 59(9): 660-665. 135. Weiss HK (2016) Wilson Disease, Synonym: Hepatolenticular Degeneration.GeneReviews® [Internet], https://www.ncbi.nlm.nih.gov/books/NBK1512/#wilson.Summary.Last Update: July 29, 2016. 136. Weitzman E, Pappo O, Weiss P, Frydman M, Haviv-Yadid Y, Ben Ari Z (2014) Late onset fulminant Wilson's disease: a case report and review of the literature. World J Gastroenterol 20(46): 17656-17660. 137. Wu Z, Wang N, Murong S, Lin M (2000) Identification and analysis of mutations of the Wilson disease gene in Chinese population. Chin Med J (Engl) 113(1): 40-43. 138. Yamaguchi Y, Heiny ME, Gitlin JD (1993) Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem Biophys Res Commun 197(1): 271-277. 139. Yeo G, Burge CB (2004) Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11(2-3):377- 94. 140. Zali N, Mohebbi RS, Esteghamat S, Chiani M, Haghighi MM, Hosseini-Asl MKS, Derakhshan F, Mohammad-Alizadeh HA, Malek-Hosseini AS, Zali RM (2011) Prevalence of ATP7B Gene Mutations in Iranian Patients With Wilson Disease. Hepat Mon 11(11): 890-894. 141. Zhang DF, Teng JF (2016) Direct sequencing of mutations in the copper- transporting P-type adenosine triphosphate (ATP7B) gene for diagnosis and pathogenesis of Wilson's disease. Genet Mol Res 23; 15(3). 147 142. Zhang Y, WuZi Y (2011) Wilson’disease in Asia. Neurology Asia 1(2): 103- 109. 143. Zong YN, Kong XD (2015) Analysis and application of ATP7B gene mutations in 35 patients with hepatolenticular degeneration. Genet. Mol. Res.14(4): 18764-18770. 1 9. PHỤ LỤC Phụ lục 01 Danh sách bệnh nhân, kết quả tách DNA tổng sổ, kết quả phân tích đột biến gen của 78 bệnh nhân và các thành viên trong 55 phả hệ Số thứ tự Mã số bệnh nhân Họ và tên bệnh nhân/thành viên gia đình Đột biến Năm sinh Lượng DNA (ng/µl) Độ tinh sạch (A260/280) Bệnh nhân Phả hệ Alen 1 Alen 2 1 WBW100301 Phạm Thị H P992L L1371P 1995 34 1,87 2 WBW100501 Trần Thị Bích Ng S105X N1270S 1997 42 2,0 3 WBW100502 Nguyễn Ngọc D S105X T850I 1999 46 1,9 4 1 WBW100601 Tô Thanh V S105X S105X 2000 69 1,9 Bố S105X - 67 1,9 Mẹ S105X - 56 1,9 Anh trai S205X - 44 1,8 Em trai - - 66 1,85 Em gái - - 46 1,95 5 2 WBW100602 Nguyễn Tuyết L R778L L1371P 2001 45 1,8 Bố R778L - 65 1,9 2 Mẹ L1371P - 35 1,9 Anh trai 44 2,0 Anh trai 48 2,0 Em trai R778L - 2006 54 1,9 6 3 WBW100604 Phùng Thu Tr S105X H251AfsX19 2000 36 1,8 Bố S105X - 1972 48 1,9 Mẹ H251AfsX19 - 1972 66 1,85 Em gái - - 2008 55 1,95 7 4 WBW100605 Trần Ngọc Th S105X S105X 1997 38 1,9 Bố S105X - 56 1,8 Mẹ S105X - 34 1,9 WBW160401 Trần Ngọc H S105X S105X 2009 42 1,9 8 5 WBW100901 Trần Thị H S105X S105X 2000 35 1,9 Bố S105X - 45 2,0 Mẹ S105X - 46 2,0 Em gái S105X - 23 2,8 WBW151002B Trần Anh M S105X S105X 2013 28 1,9 Em họ S105X - 75 1,9 9 6 WBW110301 Trịnh Quốc C S105X R778L 2006 49 2,0 Bố S105X - 35 1,9 Mẹ R778L - 33 1,8 3 Anh trai R778L - 78 2,0 10 7 WBW160709 Hoàng Mạnh L I1148T P1245S 2000 54 1,9 Bố P1245S - 56 1,9 Mẹ I1148T - 34 1,8 Chị gái - - 59 1,96 11 WBW110903 Phạm Văn Th S105X E1173K 1998 56 1,85 12 8 WBW12401 Cao Ngọc A P1052L P1273Q 2003 41 1,85 Bố P1052L - 45 1,9 Mẹ P1273Q - 29 1,95 Em gái P1052L - 36 1,9 13 9 WBW120402 Bùi Thị Lệ Qu S105X V176SfsX28 2002 59 1,9 Bố V176SfsX28 - 44 2,0 Mẹ S105X - 32 1,8 Anh trai S105X - 48 1,8 14 10 WBW120501 Lê Quang Tr V176SfsX28 P1273Q 2001 46 1,9 Bố V176SfsX28 - 45 1,8 Mẹ P1273Q - 37 1,9 WBW120501B Lê Tiến Ng V176SfsX28 P1273Q 2003 43 1,9 Bác gái bên bố V176SfsX28 - 34 1,8 Chị họ V176SfsX28 - 47 2,0 Anh họ V176SfsX28 - 42 1,9 4 Bác trai bên bố V176SfsX28 - 35 1,9 Chị họ V176SfsX28 - 53 1,9 Anh họ - - 56 1,8 Chị họ - - 54 1,8 Bác gái bên bố V176SfsX28 41 1,8 Chị họ - - 38 1,8 Chị họ V176SfsX28 - 41 2,0 Anh họ - - 37 1,9 Chị họ - - 28 2,0 Bác gái bên mẹ - - 56 2,0 Chị họ - - 41 1,9 Chị họ - - 39 1,9 Chị họ - - 51 1,8 Chị họ - - 42 1,8 15 11 WBW151001 Phạm Đức M V176SfsX28 I1148T 2005 47 1,9 Bố V176SfsX28 - 43 1,8 Mẹ I1148T - 46 2,0 Em gái I1148T - 2009 38 1,9 16 12 WBW120604 Dương Đình L V176SfsX28 M769HfsX26 35 2,0 Bố M769HfsX26 - 22 2,0 Mẹ V176SfsX28 - 45 1,9 5 Chị gái - - 32 1,9 Chị gái V176SfsX28 - 56 1,9 Chị gái - - 24 1,9 Em gái - - 47 1,8 17 13 WBW120701 Đinh Tiến Đ S105X S105X 23 1,9 Bố S105X - 30 1,8 Mẹ S105X - 42 1,8 Em gái - - 58 1,9 18 14 WBW130501 Nguyễn Hữu B I1148T I1148T 1987 45 1,9 Mẹ I1148T - 29 2,0 Chị gái I1148T - 67 1,8 19 15 WBW131101 Vũ Thanh H T850I L1371P 1997 45 2,0 Mẹ L1371P - 47 1,9 Em gái T850I - 89 2,0 20 16 WBW140801 Nguyễn Minh S F1026Y IVS12-2A>G 1996 43 1,8 Mẹ F1026Y - 47 1,9 Em trai khác cha F1026Y - 62 2,0 21 17 WBW141101 Dương Mạnh D S105X S105X 36 2,0 Bố S105X - 54 2,0 Mẹ S105X - 67 1,9 Em trai S105X - 55 1,9 6 22 18 WBW160101 Đinh Gia H V176SfsX28 IVS6+3A>G 2007 43 1,8 Bố V176SfsX28 - 66 1,8 Mẹ IVS6+3A>G - 45 1,9 Em trai IVS6+3A>G - 54 1,8 23 19 WBW160201 Nguyễn Lê S S105X I1148T 2009 31 1,9 Bố I1148T - 32 1,9 Mẹ S105X - 46 1,87 24 WBW160301 Triệu Xuân H D1027H IVS14-2A>G 2008 38 1,8 25 20 PWBW160402S Hoàng Thị H T850I IVS14-2A>G 1992 42 1,9 WBW160402 Hoàng Hữu Nh T850I IVS14-2A>G 2005 55 2,0 Bố IVS14-2A>G - 30 2,0 Mẹ T850I - 55 1,9 Con trai H T850I - 2015 44 1,9 26 21 WBW160403 Vũ Thị Ch E1173K E1173K 2000 45 1,9 Bố E1173K - 53 1,87 Mẹ E1173K - 35 1,90 Chị gái-S1 E1173K - 54 1,91 Chị gái-S2 - - 34 2,0 Chị gái-S3 E1173K - 45 2,0 Em gái-S5 E1173K - 42 1,97 27 22 PWBW160501S Nguyễn Thị Thu H S105X S105X 2000 41 1,82 7 Bố S105X - 45 1,86 Mẹ S105X - 51 1,86 Em gái S105X - 45 1,92 28 23 WBW160503 Nguyễn Như Th S105X IVS20+4A>G 2003 44 1,91 Bố S105X - 1982 38 1,90 Mẹ IVS20+4A>G - 1983 43 1,87 Chị gái S105X - 2006 51 2,0 Em gái S105X - 2008 40 1,93 Em gái S105X - 2009 33 1,86 Em trai IVS20+4A>G - 2016 56 1,80 Chú họ - - 1985 29 24 WBW160504 Đỗ Hoàng M S105X I1148T 2005 40 1,90 Bố I1148T - 41 1,95 Mẹ S105X - 24 1,85 Chị gái I1148T - 51 1,95 Chị gái I1148T - 77 1,91 30 25 WBW160506 Lê Bình A S105X IVS14-2A>G 2004 42 1,90 Bố S105X - 39 1,80 Mẹ IVS14-2A>G - 30 2,0 Em trai IVS14-2A>G - 2009 41 1,95 31 WBW110701 Lê Thị Huyền Tr K1010T - 2006 42 1,83 8 32 26 WBW160507 Nguyễn Thị Huyền T V1042CfsX79 IVS14-2A>G 2002 40 1,90 Bố IVS14-2A>G - 29 1,95 Mẹ V1042CfsX79 - 39 1,90 33 WBW160509 Phạm Thị Lan A S105X G1281D 2003 48 1,82 34 27 WBW160601 Nguyễn Duy H D765G D765G 2000 57 1,90 Bố D765G - 35 2,0 Mẹ D765G - 57 1,90 Chị gái D765G - 39 2,0 Chị gái D765G - 55 2,0 35 28 WBW160602 Chu Ngọc M V176SfsX28 R778L 2007 35 1,85 Bố R778L - 37 1,87 Mẹ V176SfsX28 - 48 1,85 Chị gái - - 2003 43 1,92 36 29 WBW160604 Nguyễn Thị Ph S105X S105X 2003 35 1,90 Bố S105X - 1965 64 1,84 Mẹ S105X - 1969 64 1,90 Chị gái-S1 - - 1990 45 1,91 Chị gái-S2 S105X - 1993 35 1,99 Chị gái-S3 S105X 1996 57 1,89 Chị gái-S4 - - 1998 62 1,95 WBW160604B6 Nguyễn Tuấn A S105X S105X 2007 61 1,90 9 37 30 WBW160605 Vũ Thị L S105X I1148T 45 1,91 Bố I1148T - 34 1,85 Mẹ S105X - 26 1,90 Chị gái I1148T - 1995 54 1,95 Em trai I1148T - 2004 66 1,9 38 31 WBW160606 Phạm Thị Ngọc Ph I1148T P1273Q 2007 57 1,95 Bố I1148T - 1977 63 1,9 Mẹ P1273Q - 1983 56 2,0 Chị gái P1273Q - 2003 26 1,9 Em gái - - 2012 46 2,0 Em trai I1148T - 2016 42 1,87 39 32 WBW160608 Lê Văn A IVS14-2A>G IVS14-2A>G 2006 54 1,91 WBW170604S3 Lê Thị Anh Đ IVS14-2A>G IVS14-2A>G 2003 54 2,0 Bố IVS14-2A>G - 1966 75 1,85 Mẹ IVS14-2A>G - 1972 56 1,95 Chị gái - - 1990 33 1,90 Chị gái IVS14-2A>G - 1994 55 1,85 40 33 WBW160609 Nguyễn Như H S105X c.2169del13 2003 43 1,90 Bố S105X - 1978 53 1,95 Mẹ c.2169del13 - 1979 32 1,92 Chị gái S105X - 2000 35 1,91 10 Em trai c.2169del13 - 2010 63 1,9 41 34 WBW160610S2 Kiều Thị Thu Tr P992L P992L 2001 32 1,92 WBW160610 Kiều Văn Qu P992L P992L 2003 53 1,95 Bố P992L - 1967 35 1,9 Mẹ P992L - 1975 54 1,9 Chị gái P992L - 1995 28 1,8 42 35 WBW160704 Nguyễn Tiến S S105X S105X 2010 55 2,0 Bố S105X - 1977 62 1,9 Mẹ S105X - 1980 54 1,95 Chị gái S105X - 2001 56 2,0 Chị gái - - 2003 33 2,0 Em trai khác cha - - 2008 43 1,98 43 WBW160706 Pham Anh T - - 2003 44 2,0 44 WBW160707 Bùi Văn Đ P992L D1027H 2005 40 1,85 45 36 WBW170808 Hoàng Khánh L S105X S105X 2000 53 1,93 Bố S105X - 1971 34 1,88 Mẹ S105X - 1977 53 1,90 Em trai N N 2002 77 1,95 Cậu S105X N 1982 50 1,85 46 37 WBW160710 Hoàng Duy H S105X S105X 2008 45 1,92 Bố S105X - 1978 75 2,0 11 Mẹ S105X - 1978 56 1,90 Anh trai S105X - 2004 45 2,0 47 38 WBW160713 Nguyễn Anh T S105X I1148T 2004 58 1,97 Bố I1148T - 1966 67 1,85 Mẹ S105X - 1973 54 1,96 Chị gái I1148T - 1994 60 1,9 48 39 WBW160714 Hoàng Thị M S105X L902P 2003 45 1,9 Bố S105X - 1974 22 1,8 Mẹ L902P - 1976 43 2,0 Anh trai S105X - 1996 61 1,87 59 WBW160719 Nguyễn Anh V S105X P1245S 1993 42 1,92 50 40 WBW160801 Phạm Quang Đ V176SfsX28 P1052L 2009 66 1,90 Bố V176SfsX28 - 1971 65 1,85 Mẹ P1052L - 54 1,92 Anh trai V176SfsX28 1995 44 1,88 51 41 WBW160802 Hoàng Tùng K S105X S105X 2005 65 1,89 Bố S105X - 1979 64 2,0 Mẹ S105X - 1980 43 2,0 Anh trai S105X - 2001 56 1,83 Anh trai S105X - 45 2,0 WBW160802S2 Hoàng Thanh X S105X S105X 2008 65 1,89 12 52 42 WBW160805 Phạm Thị Hải Y S105X E1173K 2004 33 1,91 Bố E1173K -- 1978 56 1,94 Mẹ S105X - 34 2,0 Em trai S105X - 2011 45 1,82 53 WBW160901 Tòng Thị B L1371P L1371P 2004 33 1,84 54 43 WBW160903 Bùi Quang Đ S105X R778L 2003 54 1,98 Bố R778L - 45 1,81 Mẹ S105X - 1969 32 1,90 Anh trai R778L - 58 1,93 dì S105X - 54 1,98 55 44 WBW120601 Hà Mạnh T S105X I1148T 1994 43 2,0 WBW161001B Hà Minh Qu S105X I1148T 2009 63 1,91 Bố S105X - 34 1,90 Mẹ I1148T - 26 2,0 Anh trai 1991 36 1,91 Chị gái 1990 78 1,95 56 45 WBW160904 Lê Sỹ Qu L902P P1273Q 2012 64 1,85 WBW170304B Lê Sỹ Đức T L902P P1273Q 2016 43 1,99 Bố L902P - 1985 34 2,0 Mẹ P1273Q - 1988 50 1,98 57 WBW161101 Nguyễn Thị Hương Th IVS14-2A>G IVS14-2A>G 2002 46 1,87 13 58 PWBW161101 Nguyễn Thị Th IVS14-2A>G IVS14-2A>G 1999 47 1,90 59 PWBW161103 Đặng Thị Kim Ng S105X I1148T 2009 57 2,0 60 WBW161103 Đỗ Xuân H S105X S105X 2001 49 1,89 61 WBW170102 Nguyễn Thị Đức Đ R778L D1027H 2005 56 1,95 62 46 WBW161202 Nguyễn Thị Thu Th K1010T L1371P 2003 66 2,0 PWBW17050B Nguyễn Văn Gi K1010T L1371P 47 1,95 Mẹ L1371P - 42 1,83 63 47 WBW170301 Nguyễn Thị Phương M P1273Q L1371P 2002 51 1,97 Bố P1273Q - 56 1,91 64 48 WBW170303 Nguyễn Đức Yến Nh R778L R778L 2006 68 1,98 WBW170305 Nguyễn Đức Yến L R778L R778L 2011 45 2,0 Bố R778L - 1974 56 2,0 Mẹ R778L - 1980 47 1,90 Chị gái R778L - 2001 58 1,83 Em trai út R778L - 2013 49 1,99 65 PWBW170306F Nguyễn Minh T IVS6+3A>G M769HfsX26 64 1,93 Con trai IVS6+3A>G - 66 49 WBW170605 Phạm Tiến Qu L1371P L1371P 2007 54 2,0 Bố L1371P - 68 1,81 Mẹ L1371P - 43 1,87 67 WBW170703 Dương Văn L S105X T850I 2006 36 1,84 14 68 50 WBW170702 Đoàn Xuân Tr M769HfsX26 T850I 2006 64 1,93 Bố T850I - 66 2,0 Mẹ M769HfsX26 - 36 1,99 Em trai M769HfsX26 - 37 1,80 69 51 WBW170704 Đoàn Văn C S105X P868PfsX5 2007 45 1,91 Bố S105X - 67 1,87 Mẹ P868PfsX5 - 58 2,0 Em gái S105X - 48 1,9 70 52 WBW170802 Nguyễn Đức M S105X T850I 2006 22 2,0 Bố T850I - 35 2,0 Mẹ S105X - 56 1,92 Anh trai - - 29 1,89 71 53 WBW170807 Bùi Thế H T850I D1027H 38 1,96 Mẹ T850I - 28 2,0 Em trai D1027H - 48 1,88 72 54 WBW170806 Vũ Thùy L T850I D1027H 2003 30 2,0 WBW170805 Vũ Nguyễn Hà D T850I D1027H 2005 43 1,98 73 WBW160701 Triệu Minh Qu - - 34 1,86 74 WBW170901 Nguyễn Thị Ngọc A S105X S105X 2007 65 2,0 75 WBW171005 Phạm Quang T S105X S105X 2003 45 1,93 76 WBW171101 Phạm Đức A S105X S105X 2003 64 1,92 15 77 55 WBW171104 Nguyễn Minh Kh S105X S105X 2003 39 1,88 Bố S105X - 1980 45 1,99 Mẹ S105X - 1982 38 1,87 Anh trai S105X - 2002 48 1,91 Em gái ? ? 2007 78 WBW171105 Lương Thị Thu H S105X V176SfsX28 2005 67 1,93 Ghi chú: (-): Không bị đột biến; đổ màu ghi: đã mất; chữ nghiêng: anh/chị/em ruột của bệnh nhân bị bệnh Wilson; chữ đậm, nghiêng: Bệnh nhân Wilson chưa có biểu hiện lâm sàng. 16 Phụ lục 02 Danh sách và kiểu gen của 78bệnh nhân Wilson thuộc 78 gia đình STT MSBN Họ và tên Kiểu gen (alen/alen) Năm Sinh Địa chỉ 1 WBW100301 Phạm Thị H P992L/L1371P 1995 Hải Dương 2 WBW100501 Trần Thi Bích Ng S105X/N1270S 1997 Nam Định 3 WBW100502 Nguyễn Ngọc D S105X/T850I 1999 Thái Bình 4 WBW100601 Tô Thanh V S105X/S105X 2000 Hải Dương 5 WBW100602 Nguyễn Tuyết L R778L/L1371P 2001 Hải Dương 6 WBW100604 Phùng Thu Tr S105X/H251AfsX19 2000 Hải Phòng 7 WBW100605 Trần Ngọc Th S105X/S105X 1997 Hà Nội 8 WBW100901 Trần Thị H S105X/S105X 2000 Hải Dương 9 WBW110301 Trịnh Quốc C S105X/R778L 2006 Hải Dương 10 WBW160709 Hoàng Mạnh L I1148T/P1245S 2000 Ninh Bình 11 WBW110903 Phạm Văn Th S105X/E1173K 1998 Hưng Yên 12 WBW120401 Cao Ngọc A P1052L/P1273Q 2003 Hưng Yên 13 WBW120402 Bùi Thị Lệ Qu S105X/V176SfsX28 2002 Quảng Ninh 14 WBW120501 Lê Quang Tr V176SfsX28/P1273Q 2001 Hưng Yên 15 WBW151001 Phạm Đức M V176SfsX28/I1148T 2005 Quảng Ninh 16 WBW120604 Dương Đình L V176SfsX28/M769HfsX26 2000 Thái Nguyên 17 17 WBW120701 Đinh Tiến Đ S105X/S105X 2002 Thái Bình 18 WBW130501 Nguyễn Hữu B I1148T/I1148T 1987 Hà Nội 19 WBW131101 Vũ Thanh H T850I/L1371P 1997 Hải Phòng 20 WBW140801 Nguyễn Minh S F1026Y/IVS12-2A>G 1996 Hà Nội 21 WBW141101 Dương Mạnh D S105X/S105X 1997 Hà Nam 22 WBW160101 Đinh Gia H V176SfsX28/IVS6+3A>G 2007 Hà Nội 23 WBW160201 Nguyễn Lê S S105X/I1148T 2009 Hà Nội 24 WBW160301 Triệu Xuân H D1027H/IVS14-2A>G 2008 Phú Thọ 25 WBW160402 Hoàng Thị H T850I/IVS14-2A>G 2005 Thanh Hóa 26 WBW160403 Vũ Thị Ch E1173K/E1173K 2000 Nam Định 27 PWBW160501S Nguyễn Thị Thu H S105X/S105X 2000 Hưng Yên 28 WBW160503 Nguyễn Như Th S105X/IVS20+4A>G 2003 Vĩnh Phúc 29 WBW160504 Đỗ Hoàng M S105X/I1148T 2005 Ninh Bình 30 WBW160506 Lê Bình A S105X/IVS14-2A>G 2004 Hải Dương 31 WBW110701 Lê Thị Huyền Tr K1010T/- 2006 Hưng Yên 32 WBW160507 Nguyễn Thị Huyền Tr V1042CfsX79/IVS14-2A>G 2002 Hà Nội 33 WBW160509 Phạm Thị Lan A S105X/G1281D 2003 Ninh Bình 34 WBW160601 Nguyễn Duy H D765G/D765G 2000 Vĩnh Phúc 35 WBW160602 Chu Ngọc M V176SfsX28/R778L 2007 Hưng Yên 36 WBW160604 Nguyễn Thị Ph S105X/S105X 2003 Thái Bình 18 37 WBW160605 Vũ Thị L S105X/I1148T 1998 Nam Định 38 WBW160606 Phạm Thị Ngọc Ph I1148T/P1273Q 2007 Hải Dương 39 WBW160608 Lê Văn A IVS14-2A>G/IVS14-2A>G 2006 Hải Dương 40 WBW160609 Nguyễn Như H S105X/c.2169del13 2003 Hà Nội 41 WBW160610S2 Kiều Thị Thu Tr P992L/P992L 2001 Hà Nội 42 WBW160704 Nguyễn Tiến S S105X/S105X 2010 Hà Nội 43 WBW160706 Phạm Anh T -/- 2003 Hà Nội 44 WBW160707 Bùi Văn Đ P992L/D1027H 2005 Hải Dương 45 WBW170808 Hoàng Khánh L S105X/S105X 2000 Hải Phòng 46 WBW160710 Hoàng Duy H S105X/S105X 2008 Ninh Bình 47 WBW160713 Nguyễn Anh T S105X/I1148T 2004 Sơn La 48 WBW160714 Hoàng Thị M S105X/L902P 2003 Thái Bình 49 WBW160719 Nguyễn Anh V S105X/P1245S 1993 Hà Nội 50 WBW160801 Phạm Quang Đ V176SfsX28/P1052L 2009 Hà Nội 51 WBW160802 Hoàng Tùng K S105X/S105X 2008 Hà Nội 52 WBW160805 Phạm Thị Hải Y S105X/E1173K 2004 Tuyên Quang 53 WBW160901 Tòng Thị B L1371P/L1371P 2004 Sơn La 54 WBW160903 Bùi Quang Đ S105X/R778L 2003 Quảng Ninh 55 WBW120601 Hà Mạnh T S105X/I1148T 1994 Hưng Yên 56 WBW160904 Lê Sỹ Qu L902P/P1273Q 2012 Quảng Ninh 19 57 WBW161101 Nguyễn Thị Hương Th IVS14-2A>G/IVS14-2A>G 2002 Hải Dương 58 PWBW161101 Nguyễn Thị Th IVS14-2A>G/IVS14-2A>G 1999 Nam Định 59 PWBW161103 Đặng Thị Kim Ng S105X/I1148T 2009 Nghệ An 60 WBW161103 Đỗ Xuân H S105X/S105X 2001 Hà Nam 61 WBW170102 Nguyễn Thị Đức Đ R778L/D1027H 2005 Thanh Hóa 62 WBW161202 Nguyễn Thị Thu Th K1010T/L1371P 2003 Phú Thọ 63 WBW170301 Nguyễn Thị Phương M P1273Q/L1371P 2002 Thanh Hóa 64 WBW170303 Nguyễn Đức Yến Nh R778L/R778L 2006 Hà Nội 65 PWBW170306F Nguyễn Minh T IVS6+3A>G/M769HfsX26 1990 Nam Định 66 WBW170605 Phạm Tiến Qu L1371P/L1371P 2007 Hà Nam 67 WBW170703 Dương Văn L S105X/T850I 2006 Hưng Yên 68 WBW170702 Đoàn Xuân Tr M769HfsX26/T850I 2006 Ninh Bình 69 WBW170704 Đoàn Văn C S105X/P868PfsX5 2007 Quảng Ninh 70 WBW170802 Nguyễn Đức M S105X/T850I 2006 Hà Nội 71 WBW170807 Bùi Thế H T850I/D1027H 1990 Hà Nội 72 WBW170806 Vũ Thùy L T850I/D1027H 2005 Hà Nội 73 WBW160701 Triệu Minh Qu -/- 2003 Hà Nội 74 WBW170901 Nguyễn Thị Ngọc A S105X/S105X 2007 Vĩnh Phúc 75 WBW171005 Phạm Quang T S105X/S105X 2003 Hải Dương 76 WBW171101 Phạm Đức A S105X/S105X 2003 Vĩnh Phúc 20 77 WBW171104 Nguyễn Minh Kh S105X/S105X 2003 Vĩnh Phúc 78 WBW171105 Lương Thị Thu H S105X/V176SfsX28 2005 Yên Bái Ghi chú: (-) Không phát hiện đột biến; chữ đậm: Đột biến mới. Hình ảnh đi Nhận xét: Kết quả của gen ATP7B đã được khu băng đặc hiệu, sáng, sắc nét và kích thư 21 Phụ lục 03 ện di sản phẩm PCR 21 exon của gen ATP7B điện di sản phẩm PCR trên hình cho thấy, toàn b ếch đại thành công, mỗi exon được th ớc tương ứng với tính toán lý thuy ộ 21 exon ể hiện bằng 1 ết. Trình tự đột bi Bệnh nhân mã số WBW160503 có 2 đ S105X trên exon 2 làm thay đ ATP7B thành nulceotid A khi thành bộ ba TAG mã hóa IVS20+4A>G trên intron 20 làm 4 nucleotid kể từ exon 20) thành 22 Phụ lục 04 ến gen của bệnh nhân có đột biến mới IVS20+4A>G ột biến dị hợp tử: (1) đột bi ổi nucleotid C tại vị trí c.314 trên c ến cho bộ ba TCG mã hóa Serine (S) th acid amin kết thúc (X); (2) đột bi thay đổi nucleotid A tại vị trí c.4124+4 (xuôi chi nucleotid G. ến dị hợp tử DNA của gen ứ 105 chuyển ến dị hợp tử ều 23 Phụ lục 05 Bảng mã di truyền và viết tắt các acid amin Phả hệ và trình tự đột bi bệnh Wilson chưa có bi 1. Gia đình bệnh nhân Phả hệ Nam mang gen b Nam bị bệnh ? Chưa được phân tích gen/ chưa có biểu hiệ Nhận xét: Phả hệ có m năm 2007; bệnh nhân mã s của bệnh, em trai út của b lâm sàng. Bố, mẹ bệnh nhân là ngư đột biến. 24 Phụ lục 06 ến gen của các phả hệ được phát hiện sớ ểu hiện lâm sàng mã số WBW120601 của gia đình bệnh nhân mã số WBW120601 ệnh Nữ mang gen bệnh Nữ đã mất n bệnh Bệnh nhân đã biểu hiệ Ca sàng lọc chưa có bi sàng ột người con gái mắc bệnh Wilson (III ố WBW120601(III.3) cũng bị bệnh và đ ệnh nhân (III4) (mã số WBW161001B) chưa có bi ời mang gen bệnh, chị gái lớn (III m người mắc n triệu chứng ểu hiện lâm .2) đã tử vong ã có biểu hiện ểu hiện .1) không có Trình tự gen c Nhận xét: bố bệnh nhân (II.2) bị đột biến dị hợp t biến dị hợp tử kép S105X và I1148T. 25 ủa gia đình bệnh nhân mã số WBW120601 (II.1) bị đột biến dị hợp tử I1148T, m ử S105X. Cả 2 anh em trai bệnh nhân (III.2) b ẹ bệnh nhân ị đột biến đột 2. Gia đình bệnh nhân Phả hệ Nam mang gen b Nam bị bệnh ? Chưa được phân tích gen/ chưa có biểu hiện b 26 mã số WBW160610 của gia đình bệnh nhân mã số WBW16061 ệnh Nữ mang gen bệnh Nữ đã mất ệnh Bệnh nhân đã biểu hiện tri Ca sàng lọc chưa có bi sàng 0 ệu chứng ểu hiện lâm Trình tự gen 27 của gia đình bệnh nhân mã số WBW16 0610 3. Gia đình bệnh nhân mã s Phả hệ c Nam mang gen b Nam bị bệnh ? Chưa được phân tích gen/ chưa có biểu hiện b 28 ố PWBW160402S ủa gia đình bệnh nhân mã số PWBW160402S ệnh Nữ mang gen bệnh Nữ đã mất ệnh Bệnh nhân đã biểu hiện tri Ca sàng lọc chưa có biể sàng ệu chứng u hiện lâm Trình tự đột biế 29 n gen của gia đình bệnh nhân mã số PWBW160 402S Kết quả sàng lọc đ 30 Phụ lục 07 ột biến đích cho các thành viên trong gia đ nhân mã số WBW120501 ình bệnh 31 32 Phụ lục 08 Kết quả tách DNA tổng số của 3 mẫu dịch ối sau nuôi cấy Mã số Hàm lượng DNA (ng/µl) Độ tinh sạch (OD260/280nm) AFW160501 78 1,87 AFW151105 145 1,90 AFW150804 137 1,85