【作者】 熊华淇；

【导师】 徐小虎； 吴冠青；

【作者基本信息】 汕头大学 ， 法医病理学， 2002， 博士

【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，PKHD₁）。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PKHD₁基因可能位于端粒侧一边。根据Celera数据库中的预测，PKHD₁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PKHD₁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Y11 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，属于无意义突变更多还原

【Abstract】 Department: Forensic Department, Medical College, Shantou UniversityAutosomal 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 aA Novel Gene is a Positional Candidate for Autosomal Recessive Polycystic Kidney Disease (ARPKD)Ph.D student: Huaqi XiongMentor : Prof. Xiaohu XuProf. Guanqing WuDepartment: Forensic Department, Medical College, Shantou UniversityAutosomal 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 aNorthern 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更多还原