常染色体隐性遗传性多囊肾相关新基因的克隆及其特征
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摘要
背景与目的：常染色体隐性遗传性多囊肾(Autosomal recessive polycystic kidney disease，ARPKD)是婴幼儿和儿童的一种以肾脏囊性改变为特征常见遗传性疾病，据估计其发病率在新生儿中占1／6，000-55，000，死亡率约占1／20，000。每70个成人中可能就有一个带有ARPKD的突变等位基因。通过遗传连锁分析得知，该病的致病基因位于6号染色体上6P~(21.1-12)，又叫做多囊肾／多囊肝病变1基因(polycystic kidney and hepatic disease 1，PKHD_1)。接着又将该基因可能出现的范围进一步限定到1-cM，最近又将该基因限定在～840Kb的基因组序列中。其两端的界线标志分别是端粒侧的D6S1714／D6S243和着丝粒侧的D6S1024。而PKHD_1基因可能位于端粒侧一边。根据Celera数据库中的预测，PKHD_1基因可能有两个，分别是hcG1642636和hcG1646861。根据上述资料，我们着手克隆该基因。
方法与结果：我们从近端粒侧的hcG1646861开始，根据其核苷酸序列设计PCR引物，用RT-PCR的方法扩增出一段cDNA，再以此cDNA为探针，筛选人胚胎肾脏cDNA基因文库，得到PKHD_1基因的3′末端。接着用genescan软件，预测其5′端可能存在的外显子，从其中较大的外显子中设计正向引物，在已知的cDNA序列中设计反向引物，用RT-PCR的方法克隆出该基因的中间部分。最后用5′RACE克隆到该基因的5′端部分。从而得到该基因的全部序列。我们称其为PKHD1-Tentative(PKHD1-T)基因。该基因
助失大学医乡贤鹰士学夕磐文
一
全长 11．6kb，跨过～365kb基因组序列，靠近D6S1714这个端粒侧
标志。它包括61个外显子，第一个外显子为非翻译区，启始密码
子 ATG位于第二号外显子。终止密码子 TAG位于第 61号外显子。
3’端非翻译区有 1．Ikb长，并有 pOly A的信号序列和 poly A尾。
；‘该基因编码区序列长10188hp，编码一个含3396个氨基酸残
基的蛋白质，与目前的己知蛋白相比没有明显的同源性。该基因的
编码蛋自含有多个TIG功能区，这是在兔疫球蛋白家族中常见的
一种功能结构区域。分析其氨基酸序列，提示 PKHD IT蛋白可能
是一个分泌型的蛋白，而不是一个膜蛋白。
用NCBI和Celera数据库分析和我们用 Southern blot分析均
末见有重复区域，表明该基因可能是一个单考贝基因。
Northern分析显示，该基因有分子量不同的转录产物，其中最
主要的在～11 kb 处，与我们克隆的基因大小一致。在成人和人胚
胎的肾脏有很强的表达，肝脏则有较弱的表达。这种表达类型与
ARPKD 所累及的脏器是相吻合的，原位分子杂交分析表明
PKHDJ 基因主要在肾脏的集合管上皮细胞中表达，而这正是
ARPKD 病变时肾脏出现囊性变化的部位。上述这些均强烈表明
PKHD1基因是 ARPKD的致病基因。
为进一步研究 PKHD－T基因的功能。我们又用 CDNA文库筛
选、RT－PCR和 5’RACE的方法克隆了小鼠的 pkhd基因的全部编
码区序列。并用FISh方祛将该序列定位于小鼠第1号染色体A带
上，与我们从 GenBank搜索到的 pkhd基因位置一致。pkhd基因
全长12225hp，跨越～500Kb基因组DNA长度，含有67个外显子，
比人的PKHDIT基因长，与Ward等所克隆人的PKHDI—F（PKHDI
基因的另一种转录产物）相同。编码区序列长为 11 994hp，编码一
个含有 3 998个氨基酸残基的蛋白质。与人的 PKHD相比，其核
昔酸序列的同源性达76％，氨基酸序列的相似性达88％。
在我们的文章等持发表的同时，Ward 等也克隆出了 PKHD
基因的另一种转录产物一 PKHD IF。这两种 PKHD不同转录产物
3
渺兴大学磨学虏尊士学哎衫文
一
其第 1．60号外显子基本上是相同的，只是 PKHD IF第一个外显子
5’末端比PKHDI—T基因多16个碱基，两者在基因组DNA中的
位置也完全相同。但PKHD－F有67个外显子，而PKHDI－T只有
61个外显子，第 61号外显子后的 DNA序列则完全不同。
另外，PKHD—T和 PKHD—F 两 cDNA前 60个外显子中，
还有7个单核昔酸呈多态性，其中6个单核苦酸的差异不引起编码
的氨基酸改变。只有一个单核昔酸的变化引起了氨基酸的改变Y
11 24 C，但这两个氨基酸是性质相似的氨基酸，不引起蛋白质的功
能变化，属于无意义突变
Department: Forensic Department, Medical College, Shantou University
Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. The estimated incidence of ARPKD is widely variable, ranging from 1 in 6,000 to 1 in 55,000 live births. A morbidity of 1 in 20,000 was estimated by clinical geneticists. Approximately 1 out of 70 individuals is a carrier of an ARPKD mutant allele. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. Recently, the disease interval was refined even further, to an ~840-kb genomic region. According to Celera database, there are two predicted genes, hCGl642636 and hCGl646861. Because a previous report demonstrated that the candidate gene for ARPKD may reside closer to the telomeric flanking marker (D6S1714) we focused our attention on hCG1646861.
A fragment of RT-PCR product was amplified from primers based on the predicted hCG1646861 cDNA sequence, and used as a
A Novel Gene is a Positional Candidate for Autosomal Recessive Polycystic Kidney Disease (ARPKD)
Ph.D student: Huaqi Xiong
Mentor : Prof. Xiaohu Xu
Prof. Guanqing Wu
Department: Forensic Department, Medical College, Shantou University
Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. The estimated incidence of ARPKD is widely variable, ranging from 1 in 6,000 to 1 in 55,000 live births. A morbidity of 1 in 20,000 was estimated by clinical geneticists. Approximately 1 out of 70 individuals is a carrier of an ARPKD mutant allele. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. Recently, the disease interval was refined even further, to an ~840-kb genomic region. According to Celera database, there are two predicted genes, hCGl642636 and hCGl646861. Because a previous report demonstrated that the candidate gene for ARPKD may reside closer to the telomeric flanking marker (D6S1714) we focused our attention on hCG1646861.
A fragment of RT-PCR product was amplified from primers based on the predicted hCG1646861 cDNA sequence, and used as a
Northern analyses demonstrated that the gene has discrete bands with one peak signals at -11-kb indicating PKHD1-T is likely to have multiple alternative transcripts, and PKHD1-T is highly expressed in adult and infant kidneys and weakly in the liver. This expression pattern parallels the tissue involvement observed in ARPKD. In-situ hybridization analysis further revealed that the expression of PKHD1-T in the kidney is mainly localized to the epithelial cells of collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features make it a strong positional candidate gene for ARPKD.
For more understanding the function of PKHD1-T, we identified the homology gene in mice, called pkhdl, using RT-PCR and 5'RACE. The sequence is on chromosome 1, band A2-5 by FISH method same as we find on gene bank. The pkhdl encodes a 12225 bp transcript and is composed of 67 exons spanning an ~500-kb genomic region which longer than PKHD1-T and same as PKHD1-F. The ORF of PKHD1-T is 11994 bp long and is predicted to encode a 3398-amino-acid protein. The nucleotide of pkhdl is 76% identical and amino-acid is about 88% similar to human PKHDl.
During the preparation of this publication, the gene responsible for ARPKD, PKHDl has been identified by other group, named as PKHDl-F. In comparison with their findings, we found that PKHDl-T is essentially identical to PKHDl. However, PKH
方法与结果：我们从近端粒侧的hcG1646861开始，根据其核苷酸序列设计PCR引物，用RT-PCR的方法扩增出一段cDNA，再以此cDNA为探针，筛选人胚胎肾脏cDNA基因文库，得到PKHD_1基因的3′末端。接着用genescan软件，预测其5′端可能存在的外显子，从其中较大的外显子中设计正向引物，在已知的cDNA序列中设计反向引物，用RT-PCR的方法克隆出该基因的中间部分。最后用5′RACE克隆到该基因的5′端部分。从而得到该基因的全部序列。我们称其为PKHD1-Tentative(PKHD1-T)基因。该基因
助失大学医乡贤鹰士学夕磐文
一
全长 11．6kb，跨过～365kb基因组序列，靠近D6S1714这个端粒侧
标志。它包括61个外显子，第一个外显子为非翻译区，启始密码
子 ATG位于第二号外显子。终止密码子 TAG位于第 61号外显子。
3’端非翻译区有 1．Ikb长，并有 pOly A的信号序列和 poly A尾。
；‘该基因编码区序列长10188hp，编码一个含3396个氨基酸残
基的蛋白质，与目前的己知蛋白相比没有明显的同源性。该基因的
编码蛋自含有多个TIG功能区，这是在兔疫球蛋白家族中常见的
一种功能结构区域。分析其氨基酸序列，提示 PKHD IT蛋白可能
是一个分泌型的蛋白，而不是一个膜蛋白。
用NCBI和Celera数据库分析和我们用 Southern blot分析均
末见有重复区域，表明该基因可能是一个单考贝基因。
Northern分析显示，该基因有分子量不同的转录产物，其中最
主要的在～11 kb 处，与我们克隆的基因大小一致。在成人和人胚
胎的肾脏有很强的表达，肝脏则有较弱的表达。这种表达类型与
ARPKD 所累及的脏器是相吻合的，原位分子杂交分析表明
PKHDJ 基因主要在肾脏的集合管上皮细胞中表达，而这正是
ARPKD 病变时肾脏出现囊性变化的部位。上述这些均强烈表明
PKHD1基因是 ARPKD的致病基因。
为进一步研究 PKHD－T基因的功能。我们又用 CDNA文库筛
选、RT－PCR和 5’RACE的方法克隆了小鼠的 pkhd基因的全部编
码区序列。并用FISh方祛将该序列定位于小鼠第1号染色体A带
上，与我们从 GenBank搜索到的 pkhd基因位置一致。pkhd基因
全长12225hp，跨越～500Kb基因组DNA长度，含有67个外显子，
比人的PKHDIT基因长，与Ward等所克隆人的PKHDI—F（PKHDI
基因的另一种转录产物）相同。编码区序列长为 11 994hp，编码一
个含有 3 998个氨基酸残基的蛋白质。与人的 PKHD相比，其核
昔酸序列的同源性达76％，氨基酸序列的相似性达88％。
在我们的文章等持发表的同时，Ward 等也克隆出了 PKHD
基因的另一种转录产物一 PKHD IF。这两种 PKHD不同转录产物
3
渺兴大学磨学虏尊士学哎衫文
一
其第 1．60号外显子基本上是相同的，只是 PKHD IF第一个外显子
5’末端比PKHDI—T基因多16个碱基，两者在基因组DNA中的
位置也完全相同。但PKHD－F有67个外显子，而PKHDI－T只有
61个外显子，第 61号外显子后的 DNA序列则完全不同。
另外，PKHD—T和 PKHD—F 两 cDNA前 60个外显子中，
还有7个单核昔酸呈多态性，其中6个单核苦酸的差异不引起编码
的氨基酸改变。只有一个单核昔酸的变化引起了氨基酸的改变Y
11 24 C，但这两个氨基酸是性质相似的氨基酸，不引起蛋白质的功
能变化，属于无意义突变
Department: Forensic Department, Medical College, Shantou University
Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. The estimated incidence of ARPKD is widely variable, ranging from 1 in 6,000 to 1 in 55,000 live births. A morbidity of 1 in 20,000 was estimated by clinical geneticists. Approximately 1 out of 70 individuals is a carrier of an ARPKD mutant allele. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. Recently, the disease interval was refined even further, to an ~840-kb genomic region. According to Celera database, there are two predicted genes, hCGl642636 and hCGl646861. Because a previous report demonstrated that the candidate gene for ARPKD may reside closer to the telomeric flanking marker (D6S1714) we focused our attention on hCG1646861.
A fragment of RT-PCR product was amplified from primers based on the predicted hCG1646861 cDNA sequence, and used as a
A Novel Gene is a Positional Candidate for Autosomal Recessive Polycystic Kidney Disease (ARPKD)
Ph.D student: Huaqi Xiong
Mentor : Prof. Xiaohu Xu
Prof. Guanqing Wu
Department: Forensic Department, Medical College, Shantou University
Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. The estimated incidence of ARPKD is widely variable, ranging from 1 in 6,000 to 1 in 55,000 live births. A morbidity of 1 in 20,000 was estimated by clinical geneticists. Approximately 1 out of 70 individuals is a carrier of an ARPKD mutant allele. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. Recently, the disease interval was refined even further, to an ~840-kb genomic region. According to Celera database, there are two predicted genes, hCGl642636 and hCGl646861. Because a previous report demonstrated that the candidate gene for ARPKD may reside closer to the telomeric flanking marker (D6S1714) we focused our attention on hCG1646861.
A fragment of RT-PCR product was amplified from primers based on the predicted hCG1646861 cDNA sequence, and used as a
Northern analyses demonstrated that the gene has discrete bands with one peak signals at -11-kb indicating PKHD1-T is likely to have multiple alternative transcripts, and PKHD1-T is highly expressed in adult and infant kidneys and weakly in the liver. This expression pattern parallels the tissue involvement observed in ARPKD. In-situ hybridization analysis further revealed that the expression of PKHD1-T in the kidney is mainly localized to the epithelial cells of collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features make it a strong positional candidate gene for ARPKD.
For more understanding the function of PKHD1-T, we identified the homology gene in mice, called pkhdl, using RT-PCR and 5'RACE. The sequence is on chromosome 1, band A2-5 by FISH method same as we find on gene bank. The pkhdl encodes a 12225 bp transcript and is composed of 67 exons spanning an ~500-kb genomic region which longer than PKHD1-T and same as PKHD1-F. The ORF of PKHD1-T is 11994 bp long and is predicted to encode a 3398-amino-acid protein. The nucleotide of pkhdl is 76% identical and amino-acid is about 88% similar to human PKHDl.
During the preparation of this publication, the gene responsible for ARPKD, PKHDl has been identified by other group, named as PKHDl-F. In comparison with their findings, we found that PKHDl-T is essentially identical to PKHDl. However, PKH
引文
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33． Scott DA, Drury S, Sundstrom RA, Bishop J, Swiderski RE, Carmi R, Ramesh A, Elbedour K, Srikumari Srisailapathy CR, Keats BJ, Sheffield VC, Smith RJ (2000) . Refining the DFNB7-DFNB11 deafness locus using intragenic polymorphisms in a novel gene, TMEM2． Gene 246: 265-274．
34． Upadhya P, Churchill G, Birkenmeier EH, Barker JE, Frankel WN (1999) . Genetic modifiers of polycystic kidney disease in intersubspecific KAT2J mutants. Genomics 58: 129-137．
35． Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly V, Cunningham VK, Bacallao B, Ishibashi M, Milliner DS, Torres VE, Harris PC (2002) . The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nature Genetics 30: 259-269．
36． Wu G., D'Agati V., Cai Y., Markowitz G., Park J. H., Reynolds D. M., Maeda Y., Le T. C., Hou H., Jr., Kucherlapati R., Edelmann W., and Somlo S. (1998) . Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell 93: 177-88．
37． Wu GQ, Markowitz GS, Li L, D'Agati VD, Factor SM, Geng L, Tibara S, Tuchman J, Cai Y, Park JH, van Adelsberg J, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S (2000) . Cardiac defects and renal failure in mice with targeted mutations in Pkd2． Nature Genetics 24: 75-78．
38． Xiao W.Oefner PJ(2001) .Denaturing high-performance liquid chromatography:a review.Hum Mutat. 17:429-474．
39． Zerres K., Mucher G., Bachner L., Deschennes G., Eggermann T., Kaariainen H., Knapp M., Lennert T., Misselwitz J., von Muhlendahl K. E., and et al. (1994) . Mapping of the gene for
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41． Zerres K., Rudnik-Schoneborn S., Deget F., Holtkamp U., Brodehl J., Geisert J., and Scharer K. (1996) . Autosomal recessive polycystic kidney disease in 115 children: clinical presentation, course and influence of gender. Arbeitsgemeinschaft fur Padiatrische, Nephrologie. Acta Paediatr 85: 437-45．
42． Zerres K., Rudnik-Schoneborn S., Steinkamm C., Becker J., and Mucher G. (1998b). Autosomal recessive polycystic kidney disease. J Mol Med 76: 303-9．
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32． Sanzen T., Harada K., Yasoshima M., Kawamura Y., Ishibashi M., and Nakanuma Y. (2001) . Polycystic kidney rat is a novel animal model of Caroli's disease associated with congenital hepatic fibrosis. Am J Pathol 158: 1605-12．
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36． Wu G., D'Agati V., Cai Y., Markowitz G., Park J. H., Reynolds D. M., Maeda Y., Le T. C., Hou H., Jr., Kucherlapati R., Edelmann W., and Somlo S. (1998) . Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell 93: 177-88．
37． Wu GQ, Markowitz GS, Li L, D'Agati VD, Factor SM, Geng L, Tibara S, Tuchman J, Cai Y, Park JH, van Adelsberg J, Hou H Jr, Kucherlapati R, Edelmann W, Somlo S (2000) . Cardiac defects and renal failure in mice with targeted mutations in Pkd2． Nature Genetics 24: 75-78．
38． Xiao W.Oefner PJ(2001) .Denaturing high-performance liquid chromatography:a review.Hum Mutat. 17:429-474．
39． Zerres K., Mucher G., Bachner L., Deschennes G., Eggermann T., Kaariainen H., Knapp M., Lennert T., Misselwitz J., von Muhlendahl K. E., and et al. (1994) . Mapping of the gene for
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40． Zerres K., Mucher G., Becker J., Steinkamm C., Rudnik-Schoneborn S., Heikkila P., Rapola J., Salonen R., Germino G. G., Onuchic L., Somlo S., Avner E. D., Harman L. A., Stockwin J. M., and Guay-Woodford L. M. (1998a). Prenatal diagnosis of autosomal recessive polycystic kidney disease (ARPKD): molecular genetics, clinical experience, and fetal morphology. Am J Med Genet 76: 137-44．
41． Zerres K., Rudnik-Schoneborn S., Deget F., Holtkamp U., Brodehl J., Geisert J., and Scharer K. (1996) . Autosomal recessive polycystic kidney disease in 115 children: clinical presentation, course and influence of gender. Arbeitsgemeinschaft fur Padiatrische, Nephrologie. Acta Paediatr 85: 437-45．
42． Zerres K., Rudnik-Schoneborn S., Steinkamm C., Becker J., and Mucher G. (1998b). Autosomal recessive polycystic kidney disease. J Mol Med 76: 303-9．