Genomic structure of the human plasma prekallikrein gene, identification of allelic variants, and analysis in end-stage renal disease.
Genomic structure of the human plasma prekallikrein gene, identification of allelic variants, and analysis in end-stage renal disease.
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DOI：
10.1006/geno.2000.6330
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发表时间：
2000-10
期刊：
影响因子：
4.4
通讯作者：
Hongrun Yu;Pamela J. Anderson;B. I. Freedman;S. S. Rich-S.;D. Bowden
中科院分区：
文献类型：
--
作者：
Hongrun Yu;Pamela J. Anderson;B. I. Freedman;S. S. Rich-S.;D. Bowden
钾化酶是丝氨酸蛋白酶，催化激肽和其他血管活性肽的释放。此前，我们曾研究过一种组织特异性(H. Yu et al., 1996； J. Am.；Soc。肾脏病杂志。7:2559-2564)和一个血浆特异性（H. Yu等，1998，高血压31:906-911）人终期肾病（ESRD）基因。在一项非洲裔美国研究人群中，4号染色体上的人血浆钾激肽基因（KLKB1，以前称为KLK3）的短序列重复多态性与ESRD相关。通过确定KLKB1的基因组结构和寻找可能与ESRD相关的等位变异，扩展了KLKB1在ESRD中的研究。通过对KLKB1 cDNA序列设计的引物对进行序列检测和PCR产物的DNA测序，鉴定出外显子跨越PCR引物。与大鼠血浆钾likrein基因和密切相关的人因子XI基因一样，人KLKB1基因含有15个外显子和14个内含子。最长的内含子F几乎有12kb长。该基因的总长度约为30 kb。通过鸟枪法克隆含有KLKB1基因的细菌人工克隆基因组片段，获得KLKB1基因5′-近端启动子区序列，然后使用外显子1特异性探针对克隆进行筛选。利用单链构象多态性技术，利用外显子和5'-近端启动子区域两侧的引物筛选ESRD患者和对照组基因组DNA中的等位基因变异。我们在5'-近端启动子和7个外显子中发现了12个等位基因变异。值得注意的是，在KLKB1 cDNA的521位存在一个常见的多态性（占种群的30%），这导致该蛋白A2结构域重链124位的天冬酰胺被丝氨酸取代。此外，在3个ESRD家族的5例患者中，观察到7外显子A716C多态性导致重链A3结构域氨基酸H189P的变化。编码序列中的第三个多态性是C699A的移位，引起氨基酸H183Q的变化。该等位基因在6个ESRD家族的8例患者中发现，但未在任何对照dna中发现。单独或联合观察到的等位基因变异与ESRD没有统计学相关性，尽管在一些情况下（例如H183Q），人群中携带这些等位基因的人数很少，限制了我们统计检测与ESRD显著相关性的能力。在内含子M和n中发现了两个新的CA/GT重复多态性标记KLK3f和KLK3g，杂合度分别为0.65和0.84。利用相对易感效应技术分析表明，KLK3f的等位基因4和8以及KLK3g的等位基因8的频率在对照组和ESRD患者之间存在显著差异。166个ESRD家族的先证数分别为0.226、0.096和0.313，而139个健康种族匹配对照的先证数分别为0.172、0.066和0.244（三种等位基因的等位基因P和总P < 0.05）。因此，尽管KLKB1编码和5'-近端启动子的多态性与非裔美国人的ESRD没有统计学意义上的相关性，但仍有证据表明4号染色体的这一部分与ESRD有关。这一观察结果表明，KLKB1内部或附近的其他序列，或附近的另一个基因，可能有助于ESRD易感性。
Kallikreins are serine proteases that catalyze the release of kinins and other vasoactive peptides. Previously, we have studied one tissue-specific (H. Yu et al., 1996, J. Am. Soc. Nephrol. 7: 2559-2564) and one plasma-specific (H. Yu et al., 1998, Hypertension 31: 906-911) human kallikrein gene in end-stage renal disease (ESRD). Short sequence repeat polymorphisms for the human plasma kallikrein gene (KLKB1; previously known as KLK3) on chromosome 4 were associated with ESRD in an African American study population. This study of KLKB1 in ESRD has been extended by determining the genomic structure of KLKB1 and searching for allelic variants that may be associated with ESRD. Exon-spanning PCR primer sets were identified by serial testing of primer pairs designed from KLKB1 cDNA sequence and DNA sequencing of PCR products. Like the rat plasma kallikrein gene and the closely related human factor XI gene, the human KLKB1 gene contains 15 exons and 14 introns. The longest intron, F, is almost 12 kb long. The total length of the gene is approximately 30 kb. Sequence of the 5'-proximal promoter region of KLKB1 was obtained by shotgun cloning of genomic fragments from a bacterial artificial clone containing the KLKB1 gene, followed by screening of the clones using exon 1-specific probes. Primers flanking the exons and 5'-proximal promoter region were used to screen for allelic variants in the genomic DNA from ESRD patients and controls using the single-strand conformation polymorphism technique. We identified 12 allelic variants in the 5'-proximal promoter and 7 exons. Of note were a common polymorphism (30% of the population) at position 521 of KLKB1 cDNA, which leads to the replacement of asparagine with a serine at position 124 in the heavy chain of the A2 domain of the protein. In addition, an A716C polymorphism in exon 7 resulting in the amino acid change H189P in the A3 domain of the heavy chain was observed in 5 patients belonging to 3 ESRD families. A third polymorphism in the coding sequence was a C699A shift that caused an amino acid change, H183Q. This allele was observed in 8 cases from 6 ESRD families but was not found in any control DNAs. Individually or combined, the allelic variants observed are not statistically associated with ESRD, though in several cases (e.g., H183Q) the small number of people in the population carrying these alleles limits our ability to statistically test for significant association with ESRD. Two new CA/GT repeat polymorphic markers, designated KLK3f and KLK3g, that have heterozygosities of 0.65 and 0.84, respectively, were identified within introns M and N. Analysis using the relative predispositional effect technique indicated that the frequencies of alleles 4 and 8 of KLK3f and allele 8 of KLK3g were significantly different between controls and ESRD cases. They accounted for 0.226, 0.096, and 0.313, respectively, in the probands of 166 ESRD families compared to 0.172, 0.066, and 0.244 in 139 healthy race-matched controls (allele P and total P < 0.05 for all three alleles). Therefore, although polymorphisms in the coding and 5'-proximal promoter of KLKB1 show no statistically significant association with ESRD in African Americans, there is still evidence for association of this part of chromosome 4 with ESRD. This observation suggests that other sequences within or near KLKB1, or another gene nearby, may contribute to ESRD susceptibility.