性反转综合征患者SRY基因的研究
摘要
目的：探讨性反转综合征患者发病的分子遗传学机理；建立一种有效地在大片段中检测突变的方法，为临床诊治提供科学依据。
方法：应用聚合酶链反应扩增12例性反转患者SRY基因片段；PCR产物通过限制性酶切—单链构象多态性方法检测未知突变；基因测序以进一步明确突变的内容和性质；应用荧光原位杂交技术证实患者X、Y染色体结构及有无嵌合体存在。
结果：2例46XY女性和1例46XX男性患者SRY基因缺失，9例46XY女性患者PCR扩增出现609bp特异性片段；通过限制性酶切-单链构象多态性方法检测发现其中1例患者单链电泳带发生变异；基因序列分析进一步明确该例患者SRY基因HMG盒内相应于第244位核苷酸的位置存在点突变 (A→T)，导致第82位密码子由原来编码天冬酰胺Asn(AAT)变为编码酪氨酸Tyr(TAT)，发生错义突变；荧光原位杂交结果与经典细胞遗传学的染色体核型分析结果完全一致，且未发现嵌合体存在。
结论：SRY基因的研究有助于阐明性反转发病的分子遗传学机理及SRY基因在性别决定中的作用；所建立的限制性酶切—单链构象多态性方法可用于实验室检测大片段中的突变。
Objective: to explore the mechanism of molecular genetics in the patients with sex reverse syndrome; to develop a efficient method applied to screen for mutation in the long segments, which offers scientific ground for clinical diagnosis and treatment.
Methods: The SRY gene segment in the genomic DNA of twelve patients with sex reverse syndrome was amplified by polymerase-chain reaction(PCR); the PCR products were screened mutations using restriction endonuclease fingerprinting-single strand conformation polymorphism(REF-SSCP); mutations were confirmed by DNA sequencing; fluorescence in situ hybridization(FISH) was used to detect X、Y chromosome and mosaicism.
Results: SRY gene was deleted in two 46XY female patients and one 46XX male patient, 609-bp segments of SRY gene were amplified in nine 46XY female patients; using restriction endonuclease fingerprinting-Single strand Conformation polymorphism (REF-SSCP), electrophoretic bands of DNA single strand in one patienet differed from that in normal male control; by DNA sequenceing techniques, A novel missense mutation was identified within the HMG-box of SRY gene in this patient. A T is replaced by T in codon 82 at position +244, resulting in a substitution of asparagine(AAT) for tyrosine(TAT), this point mutation has not been descried earlier and be a new mutation; the results by fluorescence in situ hybridization(FISH) were consistent with kayotypes by cytogentics and chromosome mosaicism was not found.
Conclusion: the study of SRY gene have provided valuable information for the understanding of molecular mechanism of the patients with sex reversal syndrome. this finding also supports the hypothesis that SRY play an important role in normal sex determination. The present REF-SSCP method is applicable to screen mutations in long segment DNA in laboratory.
方法：应用聚合酶链反应扩增12例性反转患者SRY基因片段；PCR产物通过限制性酶切—单链构象多态性方法检测未知突变；基因测序以进一步明确突变的内容和性质；应用荧光原位杂交技术证实患者X、Y染色体结构及有无嵌合体存在。
结果：2例46XY女性和1例46XX男性患者SRY基因缺失，9例46XY女性患者PCR扩增出现609bp特异性片段；通过限制性酶切-单链构象多态性方法检测发现其中1例患者单链电泳带发生变异；基因序列分析进一步明确该例患者SRY基因HMG盒内相应于第244位核苷酸的位置存在点突变 (A→T)，导致第82位密码子由原来编码天冬酰胺Asn(AAT)变为编码酪氨酸Tyr(TAT)，发生错义突变；荧光原位杂交结果与经典细胞遗传学的染色体核型分析结果完全一致，且未发现嵌合体存在。
结论：SRY基因的研究有助于阐明性反转发病的分子遗传学机理及SRY基因在性别决定中的作用；所建立的限制性酶切—单链构象多态性方法可用于实验室检测大片段中的突变。
Objective: to explore the mechanism of molecular genetics in the patients with sex reverse syndrome; to develop a efficient method applied to screen for mutation in the long segments, which offers scientific ground for clinical diagnosis and treatment.
Methods: The SRY gene segment in the genomic DNA of twelve patients with sex reverse syndrome was amplified by polymerase-chain reaction(PCR); the PCR products were screened mutations using restriction endonuclease fingerprinting-single strand conformation polymorphism(REF-SSCP); mutations were confirmed by DNA sequencing; fluorescence in situ hybridization(FISH) was used to detect X、Y chromosome and mosaicism.
Results: SRY gene was deleted in two 46XY female patients and one 46XX male patient, 609-bp segments of SRY gene were amplified in nine 46XY female patients; using restriction endonuclease fingerprinting-Single strand Conformation polymorphism (REF-SSCP), electrophoretic bands of DNA single strand in one patienet differed from that in normal male control; by DNA sequenceing techniques, A novel missense mutation was identified within the HMG-box of SRY gene in this patient. A T is replaced by T in codon 82 at position +244, resulting in a substitution of asparagine(AAT) for tyrosine(TAT), this point mutation has not been descried earlier and be a new mutation; the results by fluorescence in situ hybridization(FISH) were consistent with kayotypes by cytogentics and chromosome mosaicism was not found.
Conclusion: the study of SRY gene have provided valuable information for the understanding of molecular mechanism of the patients with sex reversal syndrome. this finding also supports the hypothesis that SRY play an important role in normal sex determination. The present REF-SSCP method is applicable to screen mutations in long segment DNA in laboratory.
引文
Sinclair AH, Berta P, Palmer MS, et al. A gene from the human sex-determining region ecodes a protein with homology to a conserve DNA-binding motif. Nature, 1990, 346: 240
Zhou C, Li L, Mo Y, et al. 46, XY female sex reversal patient with a novel point mutation in the coding sequence of the SRY gene. Zhong hua yi xue yi chuan xue za zhi, 2003, 20(5): 369-72
Mc Elreavey K, Vilain E, Abbas N, et al. A regular casade hypothesis for mammalion sex determination: SRY represses a negative regular of male development. Proc Natl Acad Sci USA , 1993, 90: 3368
Koopman P, Gubbay K, Vivian N, et al. Male female mice transgenic for Sry. Nature, 1990, 348: 448
Haqq CM, King CY, Donahoe PK, et al. SRY recognizes conserved DNA sites in sex-specific promoters. Proc Natl Acad Sci USA, 1993, 90(3): 1097-1101
杜晓娟, 伏瑾, 蔡玲玲等. 人SRY基因表达及表达蛋白的结合功能研究. 中华医学遗传学杂志, 1998, 15(5): 290-292
Graves JA. Interactions between SRY and SOX genes in mammalian sex determination. Bioessays, 1998, 20: 264-269
Dardis A, Saraco N, Mendilaharzu H, et al. Report of an XX male with hypospadias and pubertal gynecomastia: SRY gene negative in blood lenkocytes but SRY gene positive in testicular cell. Horm Res. 1997, 47: 85-88
刘智, 熊正刚, 肖广惠等. 七例46XY女性的SRY基因分析. 中华医学遗传学杂志, 1995, 12(5): 258-261
Imai A, Takagi A, Tamaya T, et al. A novel sex determining region on Y(SRY) missense mutation identified in a 46XY female and also in the father. Dndocr-J, 1999, 46(5): 735-9
Schaffler A, Barth N, Winkler B, et al. Identification of a new missense mutation(Gly95Glu) in a highly conserved codon within the high-mobility
group box of the Sex-determining region Y gene: report on a 46,XY female with gonadal dysgenesis and Yolk-Sac tumor. J-Clin-Endocrinol-Metab, 2000, 85(6): 2287-92
Jordan BK, Jain M, Natarjan S, et al. Familial mutation in the testis-determining gene SRY shared by an XY female and her normal father. J Clin Endocyinol Metabe. 2002, 87(7): 3428-32
Murphy EC, Zhurkin VB, Cornilescu G, et al. Structural basis for SRY-dependent 46XY sex reversal: modulation of DNA bending by a naturally occurring point mutation. J-Mol-Biol, 2001, 312(3): 481-99
Assumpcao JG, Benedetti CE, Maciel-Guerra AT, et al. Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46XY pure gondal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes. J Mol Med, 2002, 80(12): 782-90
任鲁风, 徐悦凡, 扬宇. 限制酶切指纹-单链构象多态性——一种有效的大片段中突变的检测方法. 中华医学遗传学杂志, 2002, 10(5): 416-419
Liu Q, sommer SS. Restriction endonuclease fingerprinting(REF): a sensitive method for screening mutations in long,contiguous segments of DNA. Biotechniques, 1995, 18: 470-477
Calvari V, Bertini V, De-Grandi A, et al. A new subcroscopic deletion that refines the 9p region for sex reversal. Genomics, 2000, 65(3): 203-12
Chung YP, Hwa HL, Tseng LH, et al. Prenatal diagnosis of monosomy 10q25 associated with single umbilical artery and sex reversal: report of a case. Prenat Diagn, 1998, 18(1): 73-7
Batanian JR, Grange DK, Fleming R, et al. Two unbalanced translocations involving a common 6p25 region in two XY female patients. Clin-genet, 2001, 59(1): 52-7
Goodfellow PN, Camerion G.. DAX-1, an “antitestis” gene. EXS, 2001, 91: 57-69
Preiss S, Argentaro A, Clayton A, et al. Compound effects of point
mutation causing Campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding and transcriptional activation. J-Biol-Chem, 2001, 276(30): 27864-72
Margarit E, Soler A, Carrrio A, et al. Molecular, cytogenetic, and clinical characterization of six XX males including one prenatal diagnosis. J Med Genet, 1998, 35(9): 727-30
Kusz K, Kotecki M, Wojda A, et al. Incomplete masculinisation of XX subjects carrying the SRY gene on an inactive X chromosome. J-Med-Genet , 1999, 36(6): 452-624.
Koopman P, Gubbay J, Vivian N, et al. Male development of chromosoma-
lly female mice transgenic or Sry. Nature, 1991, 351(6322): 117-121
Hossain A, Grady F, Saunders≠, et al. The human sex-determining gene SRY is a direct target of WT1. J-Bio-Che, 2001, 276(20): 16817-16823
Modan-Moses D, Litmanovitch T, Rienstein S, et al. True hermaphroditism with ambiguous genitalia due to a complicated mosaic karyotype: clinical features, cytogenetic findings, and literature review. Am J Med Genet, 2003, 116A(3): 300-3
Ortenberleg J, Cddoux C, Craver R, et al. SRY gene expression in the ovotestes of XX true hermaphrodites. J-Uro, 2002, 167: 1828-1831
Margarit E, Coll MD, Oliva R, et al. SRY gene transferred to the long arm of the X chromosome in a Y-positive XX true hermaphrodite. Am J Med Genet, 2000, 90(1): 25-8
Zhou C, Li L, Mo Y, et al. 46, XY female sex reversal patient with a novel point mutation in the coding sequence of the SRY gene. Zhong hua yi xue yi chuan xue za zhi, 2003, 20(5): 369-72
Mc Elreavey K, Vilain E, Abbas N, et al. A regular casade hypothesis for mammalion sex determination: SRY represses a negative regular of male development. Proc Natl Acad Sci USA , 1993, 90: 3368
Koopman P, Gubbay K, Vivian N, et al. Male female mice transgenic for Sry. Nature, 1990, 348: 448
Haqq CM, King CY, Donahoe PK, et al. SRY recognizes conserved DNA sites in sex-specific promoters. Proc Natl Acad Sci USA, 1993, 90(3): 1097-1101
杜晓娟, 伏瑾, 蔡玲玲等. 人SRY基因表达及表达蛋白的结合功能研究. 中华医学遗传学杂志, 1998, 15(5): 290-292
Graves JA. Interactions between SRY and SOX genes in mammalian sex determination. Bioessays, 1998, 20: 264-269
Dardis A, Saraco N, Mendilaharzu H, et al. Report of an XX male with hypospadias and pubertal gynecomastia: SRY gene negative in blood lenkocytes but SRY gene positive in testicular cell. Horm Res. 1997, 47: 85-88
刘智, 熊正刚, 肖广惠等. 七例46XY女性的SRY基因分析. 中华医学遗传学杂志, 1995, 12(5): 258-261
Imai A, Takagi A, Tamaya T, et al. A novel sex determining region on Y(SRY) missense mutation identified in a 46XY female and also in the father. Dndocr-J, 1999, 46(5): 735-9
Schaffler A, Barth N, Winkler B, et al. Identification of a new missense mutation(Gly95Glu) in a highly conserved codon within the high-mobility
group box of the Sex-determining region Y gene: report on a 46,XY female with gonadal dysgenesis and Yolk-Sac tumor. J-Clin-Endocrinol-Metab, 2000, 85(6): 2287-92
Jordan BK, Jain M, Natarjan S, et al. Familial mutation in the testis-determining gene SRY shared by an XY female and her normal father. J Clin Endocyinol Metabe. 2002, 87(7): 3428-32
Murphy EC, Zhurkin VB, Cornilescu G, et al. Structural basis for SRY-dependent 46XY sex reversal: modulation of DNA bending by a naturally occurring point mutation. J-Mol-Biol, 2001, 312(3): 481-99
Assumpcao JG, Benedetti CE, Maciel-Guerra AT, et al. Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46XY pure gondal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes. J Mol Med, 2002, 80(12): 782-90
任鲁风, 徐悦凡, 扬宇. 限制酶切指纹-单链构象多态性——一种有效的大片段中突变的检测方法. 中华医学遗传学杂志, 2002, 10(5): 416-419
Liu Q, sommer SS. Restriction endonuclease fingerprinting(REF): a sensitive method for screening mutations in long,contiguous segments of DNA. Biotechniques, 1995, 18: 470-477
Calvari V, Bertini V, De-Grandi A, et al. A new subcroscopic deletion that refines the 9p region for sex reversal. Genomics, 2000, 65(3): 203-12
Chung YP, Hwa HL, Tseng LH, et al. Prenatal diagnosis of monosomy 10q25 associated with single umbilical artery and sex reversal: report of a case. Prenat Diagn, 1998, 18(1): 73-7
Batanian JR, Grange DK, Fleming R, et al. Two unbalanced translocations involving a common 6p25 region in two XY female patients. Clin-genet, 2001, 59(1): 52-7
Goodfellow PN, Camerion G.. DAX-1, an “antitestis” gene. EXS, 2001, 91: 57-69
Preiss S, Argentaro A, Clayton A, et al. Compound effects of point
mutation causing Campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding and transcriptional activation. J-Biol-Chem, 2001, 276(30): 27864-72
Margarit E, Soler A, Carrrio A, et al. Molecular, cytogenetic, and clinical characterization of six XX males including one prenatal diagnosis. J Med Genet, 1998, 35(9): 727-30
Kusz K, Kotecki M, Wojda A, et al. Incomplete masculinisation of XX subjects carrying the SRY gene on an inactive X chromosome. J-Med-Genet , 1999, 36(6): 452-624.
Koopman P, Gubbay J, Vivian N, et al. Male development of chromosoma-
lly female mice transgenic or Sry. Nature, 1991, 351(6322): 117-121
Hossain A, Grady F, Saunders≠, et al. The human sex-determining gene SRY is a direct target of WT1. J-Bio-Che, 2001, 276(20): 16817-16823
Modan-Moses D, Litmanovitch T, Rienstein S, et al. True hermaphroditism with ambiguous genitalia due to a complicated mosaic karyotype: clinical features, cytogenetic findings, and literature review. Am J Med Genet, 2003, 116A(3): 300-3
Ortenberleg J, Cddoux C, Craver R, et al. SRY gene expression in the ovotestes of XX true hermaphrodites. J-Uro, 2002, 167: 1828-1831
Margarit E, Coll MD, Oliva R, et al. SRY gene transferred to the long arm of the X chromosome in a Y-positive XX true hermaphrodite. Am J Med Genet, 2000, 90(1): 25-8