视网膜母细胞瘤家系的基因突变检测和遗传咨询
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摘要
【背景】视网膜母细胞瘤(Retinoblastoma,RB)是婴幼儿最常见的眼内恶性肿瘤。其致病基因RB1位于染色体13q14,是人类发现的第一个抑癌基因。遗传性RB与非遗传性RB在临床表现、突变特征和处理方面各不相同。分子遗传学研究能够更早发现并明确RB风险,从而为进一步诊断和治疗提供依据,最终实现改善预后的目的,因此具有重要意义。RB1基因突变谱极为广泛,给检测带来困难,但分子生物学的发展已经提供了许多有效工具,联合应用能够获得良好的检出率。
【目的】收集视网膜母细胞瘤患者(家系),完成基因诊断,了解突变携带状态,并将结果应用于遗传咨询;丰富RB突变谱。
【方法】提取患者外周血和肿瘤组织(如果有)的基因组DNA,设计引物扩增RB1基因各外显子及其侧翼序列和启动子,直接测序与参考序列比对,以检测碱基更替、小片段缺失/插入等小突变。
【结果】在1例遗传性RB家系外周血中检出生殖细胞突变g.39470 G＞T,产生终止密码子,先证者肿瘤组织中检出体细胞突变g.2078 ins C,证实肿瘤发生的“twohit”模型。在1例散发双侧RB患者外周血中检出生殖细胞突变g.45844 G＞A,产生终止密码子。其中g.39470 G＞T和g.45844 G＞A均为首次报告。另外在2例散发单侧RB患者中未检出生殖细胞突变,对其中1例肿瘤组织的序列分析显示可能存在杂合性缺失现象。
【结论】直接测序法应用于视网膜母细胞瘤的基因诊断是可行的,利用较低成本即可检出大部分突变,阴性者可继续选择其他手段予以检测。基因诊断可应用于指导遗传咨询,对携带致病突变者加强随访可尽早发现肿瘤以保存视力。
Background:Retinoblastoma(RB) is the most common intraocular malignancy in childhood.The retinoblastoma gene RB1 is located on chromosome 13q14,and known as the first tumor suppressor gene identified.Hereditary form and non-hereditary form of retinoblastoma vary a lot in terms of clinical features,mutation type and management. For hereditary RB,molecular genetic study can identify the patients at risk as early as possible,thus enabling a better prognosis.An extraordinarily broad spectrum of mutations has been determined for RB1 gene,whereas development of molecular biology has provided abundant accesses to a satisfactory detection rate when used in combination.
Objective:To establish genetic diagnosis for the RB patients and families collected;to translate the genetic results to the clinical setting for genetic counseling;to update the RB1 mutation spectrum.
Method:Mutation analysis was carried out by extraction of genomic DNA from peripheral white blood cell and tumor tissue(if applicable),respectively,amplification of each exon with its flanking introns and promoter region,direct sequencing and comparison with reference sequences.
Results:In one case of hereditary RB,a germline mutation of g.39470 G＞T and a somatic mutation of g.2078 ins C were identified,validating the "two hit model".In one case of sporadic bilateral RB,a germline mutation of g.45844 G＞A was identified.The two substitutions were both novel mutations.No germline mutation was identified in another 2 sporadic unilaterally affected cases,but loss of heterozygosity was highly suspected in one of the patients.
Conclusion:Direct sequencing was feasible for genetic diagnosis of retinoblastoma,as most mutations could be identified with relatively low costs,and negative results could proceed to other genetic techniques.Genetic diagnosis could be applied for genetic counseling,and close follow-up of carriers might allow early detection of the tumor to preserve eyesight.
【目的】收集视网膜母细胞瘤患者(家系),完成基因诊断,了解突变携带状态,并将结果应用于遗传咨询;丰富RB突变谱。
【方法】提取患者外周血和肿瘤组织(如果有)的基因组DNA,设计引物扩增RB1基因各外显子及其侧翼序列和启动子,直接测序与参考序列比对,以检测碱基更替、小片段缺失/插入等小突变。
【结果】在1例遗传性RB家系外周血中检出生殖细胞突变g.39470 G＞T,产生终止密码子,先证者肿瘤组织中检出体细胞突变g.2078 ins C,证实肿瘤发生的“twohit”模型。在1例散发双侧RB患者外周血中检出生殖细胞突变g.45844 G＞A,产生终止密码子。其中g.39470 G＞T和g.45844 G＞A均为首次报告。另外在2例散发单侧RB患者中未检出生殖细胞突变,对其中1例肿瘤组织的序列分析显示可能存在杂合性缺失现象。
【结论】直接测序法应用于视网膜母细胞瘤的基因诊断是可行的,利用较低成本即可检出大部分突变,阴性者可继续选择其他手段予以检测。基因诊断可应用于指导遗传咨询,对携带致病突变者加强随访可尽早发现肿瘤以保存视力。
Background:Retinoblastoma(RB) is the most common intraocular malignancy in childhood.The retinoblastoma gene RB1 is located on chromosome 13q14,and known as the first tumor suppressor gene identified.Hereditary form and non-hereditary form of retinoblastoma vary a lot in terms of clinical features,mutation type and management. For hereditary RB,molecular genetic study can identify the patients at risk as early as possible,thus enabling a better prognosis.An extraordinarily broad spectrum of mutations has been determined for RB1 gene,whereas development of molecular biology has provided abundant accesses to a satisfactory detection rate when used in combination.
Objective:To establish genetic diagnosis for the RB patients and families collected;to translate the genetic results to the clinical setting for genetic counseling;to update the RB1 mutation spectrum.
Method:Mutation analysis was carried out by extraction of genomic DNA from peripheral white blood cell and tumor tissue(if applicable),respectively,amplification of each exon with its flanking introns and promoter region,direct sequencing and comparison with reference sequences.
Results:In one case of hereditary RB,a germline mutation of g.39470 G＞T and a somatic mutation of g.2078 ins C were identified,validating the "two hit model".In one case of sporadic bilateral RB,a germline mutation of g.45844 G＞A was identified.The two substitutions were both novel mutations.No germline mutation was identified in another 2 sporadic unilaterally affected cases,but loss of heterozygosity was highly suspected in one of the patients.
Conclusion:Direct sequencing was feasible for genetic diagnosis of retinoblastoma,as most mutations could be identified with relatively low costs,and negative results could proceed to other genetic techniques.Genetic diagnosis could be applied for genetic counseling,and close follow-up of carriers might allow early detection of the tumor to preserve eyesight.
引文
1. Chen, et al. Genetics of retinoblastoma. Int Ophthalmol Clin 1993;33(2):57-65.
2. Abramson DH. Retinoblastoma incidence in the United States. Arch Ophthalmol 1990;108:1514.
3. Seregard S, et al. Incidence of retinoblastoma from 1958 to 1998 in Northern Europe: advantages of birth cohort analysis. Ophthalmology 2004; 111:1228-32
4. Imhof SM, et al. Stage of presentation and visual outcome of patients screened for familial retinoblastoma: nationwide registration in the Netherlands. Br J Ophthalmol 2006;90:875-88.
5. Vogel F. Genetics of retinoblastoma. Hum Genet 1979;52:l-54.
6. Knudson AGMutation and Cancer:statistical study of retinoblastoma. Proc Natl Acad Sci,USA 1971;68:820-3.
7. Friend SH, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986;323:643-6.
8. Toguchida J, et al. Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene. Genomics 1993;17:535-43.
9. DeCaprio JA, et al. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 1989;58:1085-95.
10. Greger V, et al. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet 1989;83:155-8.
11. Valverde JR, et al. RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database. BMC Genetics 2005;6:53.
12. Zhu X, et al. Mechanisms of loss of heterozygosity in retinoblastoma. Cytogenet Cell Genet. 1992;59:248-52.
13. Andrade AFB, et al. A molecular study of first and second RB1 mutational hits in retinoblastoma patients. Cancer Genet and Cytogenet 2006; 167:43-4
14. Zhang K, et al. Molecular genetics of retinoblastoma. Int Ophthalmol Clinics. 1993;33:53-65.
15. Houdayer C, et al. Comprehensive Screening for Constitutional RB1 Mutations by DHPLC and QMPSF. Hum Mutat 2004;23:193-202
16. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
17. http://www.ncbi.nlm.nih.gov/snp
18. http://www.verandi.de/joomla/index.php?option=com_wrapper&Itemid=26
19. http://www.hgmd.cf.ac.uk/ac/validate.php
20. Richter S, et al. Sensitive and Efficient Detection of RB1 Gene Mutations Enhances Care for Families with Retinoblastoma Am. J. Hum. Genet 2003;72:253-69.
21. Horsthemke B. Genetics and Cytogenetics of Retinoblastoma. Cancer Genet Cytogenet 1992;63:1-7.
22. Kloss K, et al. Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma. Am J Med Genet 1991;39:196-200.
23. Blanquet V, et al. Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing. Hum Mol Genet 1993;2:975-9.
24. Weir-Thompson E, et al. A familial RB1 mutation detected by the HOT technique is homozygous in a second primary neoplasm. Oncogene 1991;6:2353-6.
25. Dunn JM, et al. Identification of germline and somatic mutations affecting the retinoblastoma gene. Science 1988;241:1797-800
26. Shimizu T, et al. Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis. Am J Hum Genet 1994;54:793-800.
27. 黄倩等,RB患者肿瘤及体细胞中Rb基因点突变谱及其特征和意义,中华眼底病杂志, 1995; 11 (4): 240-3.
28. Kato MV, et al. Parental origin of germ-line and somatic mutations in the retinoblastoma gene. Hum Genet 1994;94:31-8.
29. Xu K, et al. Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn.Am J Ophthalmol 2004; 137:18-23.
30. Dhanjal S, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol 2007;91:1090-1.
31. Lohmann DR, et al. Constitutional RB1 -Gene Mutations in Patients with Isolated Unilateral Retinoblastoma. Am. J. Hum. Genet 1997;61:282-94.
32. Nichols KE, et al. Sensitive multistep clinical molecular screening of 180 unrelated individuals with retinoblastoma detects 36 novel mutations in the RB1 gene. Hum Mutat 2005;25:566-74.
33. Fan BJ, et al. Molecular diagnostics of genetic eye diseases. Clin Biochem 2006;39:231-9.
34. http://www.fruitfly.org/seq_tools/splice.html
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36. Lohmann D, et al. Best Practice Guidelines for Molecular Analysis of Retinoblastoma. EMQN 2002. Available from http://www.emqn.org/emqn
37. Moll AC, et al. At what age could screening for familial retinoblastoma be stopped? A register based study 1945-98. Br Ophthalmol 2000;84:1170-2.
38. Sippel KC, et al. Frequency of Somatic and Germ-Line Mosaicism in Retinoblastoma: Implications for Genetic Counseling. Am. J. Hum. Genet 1998;62:610-9.
39. Dehainault C, et al. Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene. Nucleic Acids Res 2004;32:e139.
40. Braggio E, et al. Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" mediated SSCP analysis. J Clin Pathol 2004;57:585-90.
41. Dehainault C, et al. A deep intronic mutation in the RB1 gene leads to intronic sequence exonisation. Eur J Hum Genet 2007;15:473-7.
1 Chen,et al.Genetics of retinoblastoma.Int Ophthalmol Clin 1993;33:57-65.
2 Imhof SM,et al.Stage of presentation and visual outcome of patients screened for familial retinoblastoma:nationwide registration in the Netherlands.Br J Ophthalmol 2006;90:875-8.
3 Abramson DH.Retinoblastoma incidence in the United States.Arch Ophthalmol 1990;108:1514.
4 Seregard S,et al.Incidence of retinoblastoma from 1958 to 1998 in Northern Europe:advantages of birth cohort analysis.Ophthalmology 2004;111:1228-32
5 潘作新等,视网膜母细胞瘤116例的临床和病理分析。青岛大学医学院学报,1981年第1期。
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14 Knudson AG.Mutation and Cancer:statistical study of retinoblastoma.Proc Natl Acad Sci,USA 1971;68:820-3.
15 Friend SH,et al.A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma Nature 1986;323:643-6.
16 Toguchida J,et al.Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene.Genomics 1993;17:535-43.
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21 Greger V,et al.Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma.Hum Genet 1989;83:155-8.
22 Valverde JR,et al.RBI gene mutation up-date,a meta-analysis based on 932 reported mutations available in a searchable database.BMC Genetics 2005;6:53.
23 Zhu X,et al.Mechanisms of loss of heterozygosity in retinoblastoma.Cytogenet Cell Genet.1992;59:248-52.
24 Andrade AFB,et al.A molecular study of firrst and second RB1 mutational hits in retinoblastoma patients.Cancer Genet Cytogenet 2006;167:43-4.
25 Zhang K,et al.Molecular genetics of retinoblastoma.Int Ophthalmol Clin 1993;33:53-65.
26 http://RB1-lsdb.d-lohmann.de/index.php?option=com_wrapper&Itemid=26
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33 Cowell JK,et al.A novel mutation in the promotor region in a family with a mild form of retinoblastoma indicates the location of a new regulatory domain for the RB1 gene.Oncogene 1996;12:431-6.
34 Dryja TP,et al.Molecular etiology of low-penetrance retinoblastoma in two pedigrees.Am J Hum Genet 1993;52:1122-8.
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36 杜琴等,RB1基因第24和25外显子缺失导致低外显性视网膜母细胞瘤,中华医学遗传性杂志2002:19:370-4.
37 Horsthemke B.Genetics and Cytogenetics of Retinoblastoma.Cancer Genet Cytogenet 1992;63:1-7.
38 Kloss K,et al.Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma.Am J Med Genet 1991;39:196-200.
39 McGee TL,et al.Detection of the XbaI RFLP within the retinoblastoma locus by PCR.Nucleic Acids Res 1990;18:207.
40 Alonso J,et al.A Microsatellite Fluorescent Method for Linkage Analysis in Familial Retinoblastoma and Deletion Detection at the RB1 Locus in Retinoblastoma and Osteosarcoma.Diagnostic Molecular Pathology 2001;10:9-14.
41 Blanquet V,et al.Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing.Hum Mol Genet.1993;2:975-9.
42 Weir-Thompson E,et al.A familial RB1 mutation detected by the HOT technique is homozygous in a second primary neoplasm.Oncogene.1991;6:2353-6.
43 Dunn JM,et al.Identification of germline and somatic mutations affecting the retinoblastoma gene.Science 1988;241:1797-800.
44 Hogg A,et al.Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing.Oncogene 1992;7:1445-51.
45 Shimizu T,et al.Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis.Am J Hum Genet 1994;54:793-800.
46 苏冠芳等,PCR-SSCP联合序列分析法在RB1基因突变检测中的应用,中华眼底病杂志1994;10:17-20.
47 黄倩等,RB患者肿瘤及体细胞中Rb基因点突变谱及其特征和意义,中华眼底病杂志,1995;11:240-3.
48 Lohmann DR.The Spectrum of RB1 Germ-Line Mutations in Hereditary Retinoblastoma.Am.J.Hum.Genet 1996;58:940-9.
49 Mateu E,et al.Genetics of Retinoblastoma:A Study.Cancer Genet Cytogenet 1997;95:40-50.
50 Sampieri K,et al.Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations J Hum Genet 2006;51:209-21.
51 Braggio E,et al.Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by “exon by exon” mediated SSCP analysis.J Clin Pathol 2004;57:585-90.
52 Suzanne Richter,et al.Sensitive and Efficient Detection of RB1 Gene Mutations Enhances Care for Families with Retinoblastoma.Am.J.Hum.Genet 2003;72:253-69.
53 Houdayer C,et al.Comprehensive Screening for Constitutional RB1 Mutations by DHPLC and QMPSF.Hum Mutat 2004;23:193-202.
54 Dehainault C,et al.Multiplex PCR/liquid chromatography assay for detection of gene rearrangements:application to RB1 gene.Nucleic Acids Res 2004;32:e139.
55 Sellner LN,et al.Screening for RB1 Mutations in Tumor Tissue Using Denaturing High Performance Liquid Chromatography,Multiplex Ligation-Dependent Probe Amplification,and Loss of Heterozygosity Analysis.Pediatr Dev Pathol 2006;9:31-7.
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59 Lohmann D,et al.Best Practice Guidelines for Molecular Analysis of Retinoblastoma.EMQN 2002.Available from http://www.emqn.org/emqn
60 Moll AC,et al.At what age could screening for familial retinoblastoma be stopped? A register based study 1945-98. Br Ophthalmol 2000; 84:1170-2.
61 Imhof SM, et al. Stage of presentation and visual outcome of patients screened for familial retinoblastoma: nationwide registration in the Netherlands.Br J Ophthalmol 2006;90(7):875-8.
62 Xu K, et al. Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn. Am J Ophthalmol 2004; 137:18-23.
63 Dhanjal S, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol 2007;91:1090-1.
64 Balmer A, et al. Diagnosis and current management of retinoblastoma. Oncogene 2006;25:5341-9.
65 http://www.genetests.org/servlet/access?prg=j&db=genetests&site=gt&id=8888891&fcn=c& qry=2161 &res=&key=5On4EAO89T5qx&show_flag=c
66 Swaminathan R, et al. Childhood cancers in Chennai, India, 1990-2001: incidence and survival. Int J Cancer 2008;122:2607.
67 Sippel KC, et al. Frequency of Somatic and Germ-Line Mosaicism in Retinoblastoma: Implications for Genetic Counseling. Am. J. Hum. Genet 1998;62:610-9.
68 Kivela T, et al. Retinoblastoma Associated with Chromosomal 13q14 Deletion Mosaicism. Ophthalmology 2003; 110:1983-8.
69 Chakraborty S, et al. Identification of genes associated with tumorigenesis of retinoblastoma by microarray analysis. Genomics 2007;90:344-53.
70 Cohen Y, et al. Hypermethylation of CpG island loci of multiple tumor suppressor genes in retinoblastoma. Exp Eye Res 2008;86:201-6.
71 Lohmann DR, et al. Constitutional RB1-Gene Mutations in Patients with Isolated Unilateral Retinoblastoma. Am. J. Hum. Genet 1997;61:282-94.
72 Nichols KE, et al. Sensitive multistep clinical molecular screening of 180 unrelated individuals with retinoblastoma detects 36 novel mutations in the RB1 gene. Hum Mutat 2005;25:566-74.
73 Fan BJ, et al. Molecular diagnostics of genetic eye diseases. Clin Biochem 2006;39:231 -9.
2. Abramson DH. Retinoblastoma incidence in the United States. Arch Ophthalmol 1990;108:1514.
3. Seregard S, et al. Incidence of retinoblastoma from 1958 to 1998 in Northern Europe: advantages of birth cohort analysis. Ophthalmology 2004; 111:1228-32
4. Imhof SM, et al. Stage of presentation and visual outcome of patients screened for familial retinoblastoma: nationwide registration in the Netherlands. Br J Ophthalmol 2006;90:875-88.
5. Vogel F. Genetics of retinoblastoma. Hum Genet 1979;52:l-54.
6. Knudson AGMutation and Cancer:statistical study of retinoblastoma. Proc Natl Acad Sci,USA 1971;68:820-3.
7. Friend SH, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986;323:643-6.
8. Toguchida J, et al. Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene. Genomics 1993;17:535-43.
9. DeCaprio JA, et al. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 1989;58:1085-95.
10. Greger V, et al. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet 1989;83:155-8.
11. Valverde JR, et al. RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database. BMC Genetics 2005;6:53.
12. Zhu X, et al. Mechanisms of loss of heterozygosity in retinoblastoma. Cytogenet Cell Genet. 1992;59:248-52.
13. Andrade AFB, et al. A molecular study of first and second RB1 mutational hits in retinoblastoma patients. Cancer Genet and Cytogenet 2006; 167:43-4
14. Zhang K, et al. Molecular genetics of retinoblastoma. Int Ophthalmol Clinics. 1993;33:53-65.
15. Houdayer C, et al. Comprehensive Screening for Constitutional RB1 Mutations by DHPLC and QMPSF. Hum Mutat 2004;23:193-202
16. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi
17. http://www.ncbi.nlm.nih.gov/snp
18. http://www.verandi.de/joomla/index.php?option=com_wrapper&Itemid=26
19. http://www.hgmd.cf.ac.uk/ac/validate.php
20. Richter S, et al. Sensitive and Efficient Detection of RB1 Gene Mutations Enhances Care for Families with Retinoblastoma Am. J. Hum. Genet 2003;72:253-69.
21. Horsthemke B. Genetics and Cytogenetics of Retinoblastoma. Cancer Genet Cytogenet 1992;63:1-7.
22. Kloss K, et al. Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma. Am J Med Genet 1991;39:196-200.
23. Blanquet V, et al. Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing. Hum Mol Genet 1993;2:975-9.
24. Weir-Thompson E, et al. A familial RB1 mutation detected by the HOT technique is homozygous in a second primary neoplasm. Oncogene 1991;6:2353-6.
25. Dunn JM, et al. Identification of germline and somatic mutations affecting the retinoblastoma gene. Science 1988;241:1797-800
26. Shimizu T, et al. Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis. Am J Hum Genet 1994;54:793-800.
27. 黄倩等,RB患者肿瘤及体细胞中Rb基因点突变谱及其特征和意义,中华眼底病杂志, 1995; 11 (4): 240-3.
28. Kato MV, et al. Parental origin of germ-line and somatic mutations in the retinoblastoma gene. Hum Genet 1994;94:31-8.
29. Xu K, et al. Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn.Am J Ophthalmol 2004; 137:18-23.
30. Dhanjal S, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol 2007;91:1090-1.
31. Lohmann DR, et al. Constitutional RB1 -Gene Mutations in Patients with Isolated Unilateral Retinoblastoma. Am. J. Hum. Genet 1997;61:282-94.
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