常染色体優性多発性嚢胞腎患者の分子遺伝学的検討

常染色体優性多発性嚢胞腎(ADPKD)患者家系の連鎖解析に用いられてきたマーカーは、本来は、PKD1遺伝子のポジショナルクローニングの際にできるだけ候補領域を狭めることを目的として開発されたものである。したがって、ヘテロ接合体性が低い等の問題点があり、実際の臨床の場での連鎖解析には不十分であった。本研究では、2塩基繰り返し配列を含むいわゆるマイクロサテライトマーカー5種(D16S521、D16S3024、UTR_05049_L33243、D16S3027、D16S423)を新たに検討し、これらと従来用いられて来たマーカーであるKG8を比較した。遺伝子型の同定には、ゲノムDNAを蛍光ラベルしたプライマーを用いてPCR法で増幅し、自動DNAフラグメント解析装置で解析した。健常人50名の100遺伝子型を用いた検討では、D16S3024、D16S3027およびD16S423はへテロ接合体性が0.8以上と高く、従来のマーカーに比べ有用性が高いと考えられた。実際に、ADPKD家系を用いた検討で、D16S3024およびD16S423は連鎖解析に有用であった。また、蛍光プライマーを用いて自動DNAフラグメント解析装置で解析する方法は従来の方法に比較して簡便に施行でき、大量のサンプルの処理に適した方法であると考えられた。本研究により、実際の臨床に供し得る常染色体優性多発性嚢胞腎(ADPKD)の高感度・高出力連鎖解析法を確立しえたと考える。今後、本法はADPKDの遺伝子型_表現型関係解析、変異解析、非PKD1非PKD2家系のマッピングおよび原因遺伝子のクローニングなどに際して有力な手段となり得ると考えられる。The need for an efficient linkage analysis strategy for autosomal dominant poycystic kidney disease (ADPKD) is rather increasing after the cloning of major responsible gene, PKD2, with large and complex gene structure. In general, markers for linkage study should have high heterozygosity, proximity to the disease locus, and productivity. To meet these requirement, we applied the high-throughput genotyping strategy using microsatellite markers to analyze linkage to PKD1. From the marker detabase of Cooperative Human Linkage Center, one intragenic (UTR.05049_L33243), two distal (D16S521, D16S3024), and two proximal (D16S3027, D16S423) markers were chosen for this evaluation. Genomic fragments including dinucleotide repeats were amprified with fluorescent-primers, and the sizes of the fragments were evaluated using ABI Prism 377 and GeneScan software. Heterozygosity of each marker was calculated based on the 100 genotypes obtained from 50 normal Japanese population. UTR_05049_L33243, as well as another intragenic polymorphic marker, KG8, had low heterozygosity (0.45 and 0.36, respectively). Three of the four neighboring markers, D16S3024, D16S3027, D16S423, had high heterozygosity (>0.80). In addition, three Japanese families inheriting ADPKD were analyzed for linkage to PKD1. LOD score tables were made using FASTLINK software. Two families were positively linked to PKD1, whereas the other one was unlinked. We conclude that high-throuput genotyping using D16S3 024, D16S3027, and D16S423 is very useful in the linkage analysis of ADPKD.研究課題/領域番号:09671158, 研究期間(年度):1997-1998出典：「常染色体優性多発性嚢胞腎患者の分子遺伝学的検討」研究成果報告書　課題番号09671158 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Tissue Gene Expression of Renin-Angiotensin System in Human Type 2 Diabetic Nephropathy

OBJECTIVE—Recent studies have proved that blockade of the renin-angiotensin system (RAS) retards the progression of diabetic nephropathy, whereas hyporeninemia is known as a typical state in diabetic subjects. The purpose of this study is to determine whether expression levels of RAS differ between nondiabetic and diabetic renal tissues with accurate quantitative method. RESEARCH DESIGN AND METHODS—Subjects were 66 nondiabetic and 8 diabetic patients with biopsy-proven renal diseases. The eight diabetic subjects suffered from type 2 diabetes with overt proteinuria. Renal histology revealed typical diffuse or nodular lesions with linear IgG deposit on immunofluorescent staining and thickened basement membrane on electronic microscopy. Total RNA from a small part of the renal cortical biopsy specimens was reverse-transcribed, and the resultant cDNA was amplified for new major components of RAS (i.e., renin, renin receptor, angiotensinogen, ACE, ACE2, angiotensin II type 1 receptor, and angiotensin II type 2 receptor) and measured. RESULTS—Among these components, a significant upregulation was observed in the ACE gene in diabetic renal tissue. CONCLUSIONS—The results suggest that renal tissue RAS might be activated in the respect that ACE gene expression is upregulated in spite of a tendency to low renin expression in type 2 diabetic nephropathy

A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.Publisher\u27s version/PDF may be used after 12 months embarg

金沢大学の学生定期健康診断における尿検査の実態

研究業

Hereditary angioedema complicated with chronic renal failure: report of sibling cases.

Hereditary angioedema (HAE) is known as a deficiency state of C1 inhibitor (C1 INH), an important protease inhibitor protein involved in the complement system. As with other components of the classical pathway of the complement system, a state of its deficiency often causes clinical immunoregulatory disorders. A 45-yr-old brother and a 63-yr-old sister with HAE both developed chronic renal failure, probably due to chronic glomerulonephritis, and required regular hemodialysis. This is, to our knowledge, the first report of sibling cases of HAE associated with chronic renal failure