先天性马蹄内翻足与HoxD基因传递连锁不平衡研究
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摘要
目的
先天性马蹄内翻足(congenital clubfoot，CCF)是一种常见的严重影响足功能的先天畸形。大部分患儿需要手术治疗，其发病率约为1‰，目前其病因尚不清楚。遗传流行病学研究表明，先天性马蹄内翻足的遗传方式属于多基因遗传，其发生是遗传因素与环境因素共同作用的结果。我国对CCF的病因研究仅限于免疫组织化学、诱发电位及动物模型等方面，从基因水平研究其发病机理尚未见报道。近几年，国外有关胚胎发育的基因调控研究取得了初步进展，发现转录因子Hox基因(homeobox genes)是相关基因。目前在果蝇和鼠的基因敲除实验中发现Hox基因调节并参与胚胎后肢骨骼肌肉发育，也有Hox基因与人类肢体发育异常相关的报道，但先天性马蹄内翻足与Hox基因的关系报道较少。为了研究CCF与Hox基因的相互关系，本文以HoxD基因作为CCF的候选基因，选择HoxD基因内多态微卫星标记Hox4Ep，对65个核心家系进行等位基因检测分析，对32个杂合子父母核心家系进行了传递连锁不平衡检验(transmission disequilibrium test，TDT)，以探讨Hox基因D簇与先天性马蹄内翻足的关系。
方法
1．标本来源
收集65例CCF患者，均有典型的临床表现，经X光片及手术确诊，并排除其他畸形。取患儿及父母外周静脉血3ml，EDTA抗凝，常规酚／氯仿法提取基因组DNA。
2．微卫星DNA
选取HoxD簇基因内的一个二核苷酸重复的微卫星DNA标
记Ho－c4Ep。Ho－c4Ep选自人类基因数据库（GDB入PCR引物由上
海搏亚生物技术有限公司合成。
3．基因型分析
3．INR技术扩增微卫星DNA
PCR扩增按常规方法，在p－200型扩增仪上进行，反应体
系 25…。循环条件：94七变性40秒，55℃复性40秒，72b延伸
40秒，30个循环。
3．2 电泳
取扩增产物10gi，经6％变性聚丙烯酸胺凝胶电泳分离等位
基因，电泳条件：50吐＊、3－4h。用 0．2％AgNO。染色及 3％
N82 CO。和0．5％甲醛溶液显色，双育法等位基因型判读。
4．统计学分析
建立TDT表格，采用SPSS10．0计算机软件，进行传递不平衡
检验及相关分析。
结 果
1．收集65例核心家系中，有23例父母资料不全，难以进行
基因分型者，作者将其排除。通过凝胶电泳做基因分型分析的核
心家系共42例。
2．H。X4Ep位点稳定分离出12个等位基因，其片段分布在
110一130hp 之间。
3．由于 TDT分析要求运用杂合子父母未传递给子代的等位
基因作对照，故排除纯合子父母家系10例，最后32个家系可用于
TDT分析。
4．根据电泳带型建立TDT表格。
5．微卫星DNA Ho－c4Ep等位基因 12，杂合子型父母向子代传
递等位基因12的数目为15，未传递等位基因数目为6，差异显著
（X＝4．614，P＜0·05）。
·2·
结 论
二．CCF与H。x4EP位点存在传递连锁不平衡，提示HOxD基因
可能是先天性马蹄内翻足的易感基因。
2．HOx4Ep位点可以稳定分离出12个等位基因，其片段分布
在 110 一130hp 之间。
Objective
Congenital clubfoot ( CCF) is one of the most common congenital foot malformations in pediatric orthopedics with an incidence of 0. 1 percent. Genetic epidemical investigations suggest that there be a genetic background. CCF may require a predisposing gene acting in a particular background of polygenes or environmental influences. Hie development limb is a very complicated process, which involves in a set of genes to express at different poeriods and in different regions. Recently, the studies on embryonic development suggest that as an important regulation family, homeobox (Hox) genes are closely related to the development of organisms. Hox genes are critical for limb development based on experimental manipulations of mice and chick embryos. At present, no previous reports can be found on the genetic research of association between Hox genes and CCF. Transmission disequilibrium test ( TDT) set up in 1993 by Spielman in the study of susceptive gene of insulin - dependent diabetes mellitus (IDDM), which can avoid population stratum error. The study chose the polymorphic marker Hox4Ep at segment where HoxD genes locates. The polymerases chain reaction(PCR) products were run to isolate alleles. By the transmission pattern of the alleles, a TDT table was formed and TDT test was done. The purpose of this study is to investigate the correlation between the congenital clubfoot and HoxD genes.
Methods
1. DNA preparations
Peripheral venous blood samples were collected in 65 cases of CCF nuclear families with one affected child. The Pediatric Orthopedics Department of the 2nd Clinical Hospital of China Medical University supplied the families. Each patient with CCF showed typical clinical manifestation and was diagnosed by X - ray and operation. Furthermore , the secondary clubfoot with other deformities was excluded. Blood samples were stored in -70 C. DNA was extracted using phenol/chloroform method.
2. PCR amplification
Information about Hox4Ep ( microsatellite marker) was obtained from: http://www. gdb. org. DNA was amplified in 25ul PCR reaction. PCR was done with an initial denaturation at 94 C for 10min; followed by 30 cycles of denaturation at 94 C for 40sec, a unealing at 55 C for 40sec, and extension at 72 C for 40 sec, with a final 7min extension at 72 C. PCR products were examined on 2% agrose gels.
3. Genotyping
First, 10ul of PCR product was denatured with 10ul of denaturing solution at 96 C for 10 minutes. Second, it was cooled rapidly and then run on 6% denatured polyamine gel. The gels were stained by AgNO3/Na2CO3 method. The results were stored in Kodak Digital system.
4. Statistical analysis
The TDT table was formed according to the genotypes of families with heterozygous parents. And transmission disequilibrium test (TDT) was performed by SPSS10.0 software.
Results
1. Genotypes of 126 members in 42 CCF families were analyzed by amplifying the fragment using PCR.
2. There were 12 alleles at this microsatellite marker with fragment length from 110bp to 130bp, each named allele 1-12.
3. Transmission disequilibrium test was performed on 32 CCF families with heterozygotes parents.
4. A TDT table was formed by the transmission pattern of the alleles.
5. For allele 12, transmission number was 15, and no transmission number was 6. There was transmission disequilibrium at the twelfth allele( x2=4.614, P<0.05).
Conclusions
1. There was transmission disequilibrium between HoxD gene and human CCF. HoxD gene may be a susceptive gene of CCF.
2. There were 12 alleles at this microsatellite marker Hox4Ep in the Chinese population.
先天性马蹄内翻足(congenital clubfoot，CCF)是一种常见的严重影响足功能的先天畸形。大部分患儿需要手术治疗，其发病率约为1‰，目前其病因尚不清楚。遗传流行病学研究表明，先天性马蹄内翻足的遗传方式属于多基因遗传，其发生是遗传因素与环境因素共同作用的结果。我国对CCF的病因研究仅限于免疫组织化学、诱发电位及动物模型等方面，从基因水平研究其发病机理尚未见报道。近几年，国外有关胚胎发育的基因调控研究取得了初步进展，发现转录因子Hox基因(homeobox genes)是相关基因。目前在果蝇和鼠的基因敲除实验中发现Hox基因调节并参与胚胎后肢骨骼肌肉发育，也有Hox基因与人类肢体发育异常相关的报道，但先天性马蹄内翻足与Hox基因的关系报道较少。为了研究CCF与Hox基因的相互关系，本文以HoxD基因作为CCF的候选基因，选择HoxD基因内多态微卫星标记Hox4Ep，对65个核心家系进行等位基因检测分析，对32个杂合子父母核心家系进行了传递连锁不平衡检验(transmission disequilibrium test，TDT)，以探讨Hox基因D簇与先天性马蹄内翻足的关系。
方法
1．标本来源
收集65例CCF患者，均有典型的临床表现，经X光片及手术确诊，并排除其他畸形。取患儿及父母外周静脉血3ml，EDTA抗凝，常规酚／氯仿法提取基因组DNA。
2．微卫星DNA
选取HoxD簇基因内的一个二核苷酸重复的微卫星DNA标
记Ho－c4Ep。Ho－c4Ep选自人类基因数据库（GDB入PCR引物由上
海搏亚生物技术有限公司合成。
3．基因型分析
3．INR技术扩增微卫星DNA
PCR扩增按常规方法，在p－200型扩增仪上进行，反应体
系 25…。循环条件：94七变性40秒，55℃复性40秒，72b延伸
40秒，30个循环。
3．2 电泳
取扩增产物10gi，经6％变性聚丙烯酸胺凝胶电泳分离等位
基因，电泳条件：50吐＊、3－4h。用 0．2％AgNO。染色及 3％
N82 CO。和0．5％甲醛溶液显色，双育法等位基因型判读。
4．统计学分析
建立TDT表格，采用SPSS10．0计算机软件，进行传递不平衡
检验及相关分析。
结 果
1．收集65例核心家系中，有23例父母资料不全，难以进行
基因分型者，作者将其排除。通过凝胶电泳做基因分型分析的核
心家系共42例。
2．H。X4Ep位点稳定分离出12个等位基因，其片段分布在
110一130hp 之间。
3．由于 TDT分析要求运用杂合子父母未传递给子代的等位
基因作对照，故排除纯合子父母家系10例，最后32个家系可用于
TDT分析。
4．根据电泳带型建立TDT表格。
5．微卫星DNA Ho－c4Ep等位基因 12，杂合子型父母向子代传
递等位基因12的数目为15，未传递等位基因数目为6，差异显著
（X＝4．614，P＜0·05）。
·2·
结 论
二．CCF与H。x4EP位点存在传递连锁不平衡，提示HOxD基因
可能是先天性马蹄内翻足的易感基因。
2．HOx4Ep位点可以稳定分离出12个等位基因，其片段分布
在 110 一130hp 之间。
Objective
Congenital clubfoot ( CCF) is one of the most common congenital foot malformations in pediatric orthopedics with an incidence of 0. 1 percent. Genetic epidemical investigations suggest that there be a genetic background. CCF may require a predisposing gene acting in a particular background of polygenes or environmental influences. Hie development limb is a very complicated process, which involves in a set of genes to express at different poeriods and in different regions. Recently, the studies on embryonic development suggest that as an important regulation family, homeobox (Hox) genes are closely related to the development of organisms. Hox genes are critical for limb development based on experimental manipulations of mice and chick embryos. At present, no previous reports can be found on the genetic research of association between Hox genes and CCF. Transmission disequilibrium test ( TDT) set up in 1993 by Spielman in the study of susceptive gene of insulin - dependent diabetes mellitus (IDDM), which can avoid population stratum error. The study chose the polymorphic marker Hox4Ep at segment where HoxD genes locates. The polymerases chain reaction(PCR) products were run to isolate alleles. By the transmission pattern of the alleles, a TDT table was formed and TDT test was done. The purpose of this study is to investigate the correlation between the congenital clubfoot and HoxD genes.
Methods
1. DNA preparations
Peripheral venous blood samples were collected in 65 cases of CCF nuclear families with one affected child. The Pediatric Orthopedics Department of the 2nd Clinical Hospital of China Medical University supplied the families. Each patient with CCF showed typical clinical manifestation and was diagnosed by X - ray and operation. Furthermore , the secondary clubfoot with other deformities was excluded. Blood samples were stored in -70 C. DNA was extracted using phenol/chloroform method.
2. PCR amplification
Information about Hox4Ep ( microsatellite marker) was obtained from: http://www. gdb. org. DNA was amplified in 25ul PCR reaction. PCR was done with an initial denaturation at 94 C for 10min; followed by 30 cycles of denaturation at 94 C for 40sec, a unealing at 55 C for 40sec, and extension at 72 C for 40 sec, with a final 7min extension at 72 C. PCR products were examined on 2% agrose gels.
3. Genotyping
First, 10ul of PCR product was denatured with 10ul of denaturing solution at 96 C for 10 minutes. Second, it was cooled rapidly and then run on 6% denatured polyamine gel. The gels were stained by AgNO3/Na2CO3 method. The results were stored in Kodak Digital system.
4. Statistical analysis
The TDT table was formed according to the genotypes of families with heterozygous parents. And transmission disequilibrium test (TDT) was performed by SPSS10.0 software.
Results
1. Genotypes of 126 members in 42 CCF families were analyzed by amplifying the fragment using PCR.
2. There were 12 alleles at this microsatellite marker with fragment length from 110bp to 130bp, each named allele 1-12.
3. Transmission disequilibrium test was performed on 32 CCF families with heterozygotes parents.
4. A TDT table was formed by the transmission pattern of the alleles.
5. For allele 12, transmission number was 15, and no transmission number was 6. There was transmission disequilibrium at the twelfth allele( x2=4.614, P<0.05).
Conclusions
1. There was transmission disequilibrium between HoxD gene and human CCF. HoxD gene may be a susceptive gene of CCF.
2. There were 12 alleles at this microsatellite marker Hox4Ep in the Chinese population.
引文
1. Dietz F. The genetics of idiopathic clubfoot. Clin Orthop. 2002, 401: 39-48.
2. Lochmiller C, Johnston D, Scott A, et al. Genetic epidemiology study of idiopathic talipes equinovarus. Am J Med Genet, 1998, 79: 90-96.
3. Honein MA, Paulozzi LJ, Moore CA. Family history, maternal smoking, and clubfoot: an indication of a gene - environment interaction. Am J Epidemiol. 2000, 152: 658-665.
4. Andrade M, Barnholtz JS, Amos CI, et al. Segregation analysis of idiopathic talipes equinovarus in a texan population. Am J Med Genet, 1998, 79: 97-102.
5. Chesney D, Barker S, Miedzybrodzka Z, et al. Epidemiology and genetic theories in the etiology of congenital talipes equinovarus. Bull Hosp Jt Dis, 1999,58:59-64.
6. Spielman RS, MeGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin- dependent diabetes mellitus (IDDM). Am J Hum Genet, 1993, 52: 506-516.
7. Suemori H, Noguchi S. HoxC cluster genes are dispensable for overall body plan of mouse embryonic development. Dev Biol, 2000, 220: 333-342.
8. Spitz F, Gonzalez F, Peichel C, et al. large scale transgenic and cluster deletion analysis of the HoxD complex separate an ancestral regulatory module from evolutionary innovations. Genes Dev, 2001, 15: 2209-2214.
9. Muragaki Y, Mundlos S, Upton J, et al. Altered growth and
branching patterns in synpolydactyly caused by mutations in HoxD13. Science, 1996, 272:548-551.
10. Mortlock DP, Innis JW. Mutation of HoxA13 in hand - foot - genital syndrome. Nat Genet, 1997, 15: 179-180.
11. Del CM, Jones MC, Veraksa AN, el at. Monodactylous limbs and abnormal genitalia are associated with hemizygosity for the human 2q31 region that includes the HoxD cluster. Am J Hum Genet. 1999, 65:104-110.
12. Mark M, Rijli FM, Chambon P. Homeobox genes in embryogenesis and pathogenesis. Pediatr Res, 1997, 42: 421-429.
13. Goodman FR, Scambler PJ. Human Hox gene mutations. Clin Genet, 2001, 59:1-11.
14. Meyer A. Hox gene variation and evolution. Nature, 1998, 391: 225-228.
15. Suemori H, Noguchi S. HoxC cluster genes are dispensable for overall body plan of mouse embryonic development. Der Biol, 2000, 220:333-342.
16. Horan GS, Kovacs EN, Behringer RR, et al. Mutations in paralogous Hox genes result in overlapping homeotic transformations of the axial skeleton: evidence for unique and redundant function. Der Biol, 1995, 169: 359-372.
17. Davis AP, Witte DP, Hsieh - Li HM, et al. Absence of radius and ulna in mice lacking HoxA- 11 and HoxD- 11. Nature, 1995, 375: 791-795.
18. Zakany J, Fromental RC, Warot X, et al. Regulation of nnmher and size of digits by posterior Hox genes: a dose- dependant mechanism with potential evolutionary imiplications. Proc Natl Acad Sci USA, 1997, 94: 13695-13700.
19. Charite J, Graaff W, Shen S, et al. Ectopie expression of HoxB - 8 causes duplication of the ZPA in the Forelimb and homeric transformation of axial structures. Cell, 1994, 78: 589-601.
20. Johnson RL, Tabin CJ. Molecular models for vertebrate limb development. Cell, 1997, 90: 979-990.
21. Carroll NC. Clubfoot: what have we learned m the last quarter century? J Pediatr Orthop, 1997, 17: 1-2.
22. Favier B, Rijli FM, Ramain FC, et al. Functional cooperation between the non- paralogous genes HoxA- 10 and HoxD -11 in the developing forelimb and axial skeleton. Development, 1996, 122: 449-460.
23. Delgado BE, Santos AI, Martos FLA. Retinotie acid -induced clubfoot- like deformity: pathoanatomy in rat fetuses. J Pediatr Orthop B, 1999, 8: 12-18.
24. Khoa ND, Hasunuma T, Kobata T, et al. Expression of murine HoxD9 during embryonie joint patterning and in human T lymphotropic virus type Ⅰ tax transgenic mice with arthropathy resembling rheumatoid arthritis. Arthritis Rheum, 1999, 42: 686-696.
2. Lochmiller C, Johnston D, Scott A, et al. Genetic epidemiology study of idiopathic talipes equinovarus. Am J Med Genet, 1998, 79: 90-96.
3. Honein MA, Paulozzi LJ, Moore CA. Family history, maternal smoking, and clubfoot: an indication of a gene - environment interaction. Am J Epidemiol. 2000, 152: 658-665.
4. Andrade M, Barnholtz JS, Amos CI, et al. Segregation analysis of idiopathic talipes equinovarus in a texan population. Am J Med Genet, 1998, 79: 97-102.
5. Chesney D, Barker S, Miedzybrodzka Z, et al. Epidemiology and genetic theories in the etiology of congenital talipes equinovarus. Bull Hosp Jt Dis, 1999,58:59-64.
6. Spielman RS, MeGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin- dependent diabetes mellitus (IDDM). Am J Hum Genet, 1993, 52: 506-516.
7. Suemori H, Noguchi S. HoxC cluster genes are dispensable for overall body plan of mouse embryonic development. Dev Biol, 2000, 220: 333-342.
8. Spitz F, Gonzalez F, Peichel C, et al. large scale transgenic and cluster deletion analysis of the HoxD complex separate an ancestral regulatory module from evolutionary innovations. Genes Dev, 2001, 15: 2209-2214.
9. Muragaki Y, Mundlos S, Upton J, et al. Altered growth and
branching patterns in synpolydactyly caused by mutations in HoxD13. Science, 1996, 272:548-551.
10. Mortlock DP, Innis JW. Mutation of HoxA13 in hand - foot - genital syndrome. Nat Genet, 1997, 15: 179-180.
11. Del CM, Jones MC, Veraksa AN, el at. Monodactylous limbs and abnormal genitalia are associated with hemizygosity for the human 2q31 region that includes the HoxD cluster. Am J Hum Genet. 1999, 65:104-110.
12. Mark M, Rijli FM, Chambon P. Homeobox genes in embryogenesis and pathogenesis. Pediatr Res, 1997, 42: 421-429.
13. Goodman FR, Scambler PJ. Human Hox gene mutations. Clin Genet, 2001, 59:1-11.
14. Meyer A. Hox gene variation and evolution. Nature, 1998, 391: 225-228.
15. Suemori H, Noguchi S. HoxC cluster genes are dispensable for overall body plan of mouse embryonic development. Der Biol, 2000, 220:333-342.
16. Horan GS, Kovacs EN, Behringer RR, et al. Mutations in paralogous Hox genes result in overlapping homeotic transformations of the axial skeleton: evidence for unique and redundant function. Der Biol, 1995, 169: 359-372.
17. Davis AP, Witte DP, Hsieh - Li HM, et al. Absence of radius and ulna in mice lacking HoxA- 11 and HoxD- 11. Nature, 1995, 375: 791-795.
18. Zakany J, Fromental RC, Warot X, et al. Regulation of nnmher and size of digits by posterior Hox genes: a dose- dependant mechanism with potential evolutionary imiplications. Proc Natl Acad Sci USA, 1997, 94: 13695-13700.
19. Charite J, Graaff W, Shen S, et al. Ectopie expression of HoxB - 8 causes duplication of the ZPA in the Forelimb and homeric transformation of axial structures. Cell, 1994, 78: 589-601.
20. Johnson RL, Tabin CJ. Molecular models for vertebrate limb development. Cell, 1997, 90: 979-990.
21. Carroll NC. Clubfoot: what have we learned m the last quarter century? J Pediatr Orthop, 1997, 17: 1-2.
22. Favier B, Rijli FM, Ramain FC, et al. Functional cooperation between the non- paralogous genes HoxA- 10 and HoxD -11 in the developing forelimb and axial skeleton. Development, 1996, 122: 449-460.
23. Delgado BE, Santos AI, Martos FLA. Retinotie acid -induced clubfoot- like deformity: pathoanatomy in rat fetuses. J Pediatr Orthop B, 1999, 8: 12-18.
24. Khoa ND, Hasunuma T, Kobata T, et al. Expression of murine HoxD9 during embryonie joint patterning and in human T lymphotropic virus type Ⅰ tax transgenic mice with arthropathy resembling rheumatoid arthritis. Arthritis Rheum, 1999, 42: 686-696.