Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness.

BACKGROUND: Bartter syndrome (BS) is a genetic disorder accompanied by hypokalaemic metabolic alkalosis. BS with sensorineural deafness (SND, OMIM602522) is a newly identified phenotype caused by mutations in the BSND gene that encodes barttin, a beta-subunit for chloride channel ClC-Ka and ClC-Kb and classified as type IV BS. Type IV BS features the most severe phenotype entailing life-threatening neonatal volume depletion and chronic renal failure developing during infancy. A recent report described a case of BS with SND from a consanguineous family who showed homozygous mutations in the CLCNKA and CLCNKB genes. This case indicated the possibility of the occurrence of digenic inheritance in BS with SND resulting from double mutations in the CLCNKA and CLCNKB genes. SUBJECT AND RESULTS: The current report concerns a 2-year-old girl from a non-consanguineous family with BS accompanied by SND. In our case, four loss-of-function mutations, consisting of mutations in both parental alleles in both CLCNKA and CLCNKB, were identified. The paternal allele had a nonsense mutation (Q260X) in CLCNKA and a splicing site mutation (IVS17+1 g>a) in CLCNKB. The maternal allele had a large deletion mutation (about 12 kbp) extending from CLCNKA to CLCNKB. Our case provides clear evidence that loss-of-function alleles in both alleles of both CLCNKA and CLCNKB results in a phenotype indistinguishable from that of mutations in BSND (type IV BS). CONCLUSIONS: Recent advances in genetics have resulted in a better understanding of many human inherited diseases, but most of them are monogenic disorders and more complex inheritance patterns remain unresolved. Our case provides clear evidence of digenic inheritance outside the scope of Mendelian inheritance disorders.

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.

Hyperthermic effects on DNA repair of UV-irradiated Dictyostelium discoideum.

DNA repair of a lower eukaryote, Dictyostelium discoideum, has been investigated through the analysis of heat effects on cell mortality and DNA repair of UV-irradiated amoeboid cells. In a wild-type strain (NC4), an increase in temperature immediately after UV irradiation resulted in an increase in cell mortality, though similar heat treatment before UV irradiation had no such effect. Similar results were obtained in another wild-type strain, HPS83. In NC4, heat treatment after UV irradiation did not inhibit the nicking of DNA strands during excision repair processes, but did inhibit the rejoining of the DNA strand breaks. Removal of thymine-containing pyrimidine dimers from DNA molecules was also depressed by heat treatment after UV irradiation. In contrast, heat treatment before UV irradiation had no effect on any stage of the nicking process, the excision of the dimers or the rejoining. On the other hand, a radiation-sensitive mutant (TW8) defective in an incision step of the excision repair process did not show an increase in cell mortality in response to heat treatment administered either before or after UV irradiation. Though the optimum temperature for cell growth of the amoebae was 23 degrees C, the critical temperature for effective enhancement of cell killing was ca. 30 degrees C. Hence we assume that the excision repair of UV-damaged DNA is selectively sensitive to heat treatment.

An ovulation inducing agent containing clomiphene citrate causes DNA-strand breaks without SOS responses in Escherichia coli.

Effects of Clomid, an ovulation-inducing drug containing clomiphene citrate, on Escherichia coli were investigated. Radiation-sensitive mutants, uvrA and recA, were more sensitive to Clomid than the parental wild-type strain. DNA synthesis in these two strains was more depressed by Clomid than that in the wild-type strain. Clomid caused DNA-strand breaks, but few SOS responses such as mutation, induction of prophage and expression of the umuC+ gene were induced.

Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli.

A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B. Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene. Plasmid survival was also measured by transforming activity of the indicator strain. The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%). Some hot-spots in the mutations were observed by sequencing the suppressor gene. Mutagenic specificity in DNA base sequences induced by HL in E. coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells. This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells.

Mutagenicity of nifurtimox in Escherichia coli.

The effects of nifurtimox, a nitrofuran derivative, on killing and mutation induction in 3 Escherichia coli strains having different DNA-repair systems for UV lesion were studied and compared with the effects of furylfuramide. Nifurtimox induces mutations at a high frequency in both Hs3OR (uvrA-) and H/r30R (radiation-resistant), although no significant killing effect is detected with Hs30R. No significantly induced mutation frequency could be detected with NG30 (recA-), which is very sensitive to killing by nifurtimox. The characteristics of lesions of DNA induced by nifurtimox and the mechanism of mutation induction in Hs30R are discussed.

Induction of umu gene expression by cross-links and other DNA lesions.

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.

Induction of SOS responses in Escherichia coli by Panfuran-S, 3-di(hydroxymethyl)amino-6-(5-nitro-2-furylethenyl)-1,2,4-triazine.

The inducibility of SOS responses by Panfuran-S, which has been used as an antimicrobial medicine in Japan, was studied in Escherichia coli cells having different DNA-repair capacities for UV lesions. Panfuran-S induced mutations at high frequencies in uvrA and the wild-type strains, and significant killing effects of Panfuran-S were detected in DNA-repair-deficient strains, uvrA and recA. The effective prophage induction was detected in two kinds of lambda-lysogenized cells treated with Panfuran-S. The expression of the umuC+ gene was apparently induced in uvrA and the wild-type strains, but not induced in lexA and recA strains. In particular, high inducibility of the gene expression was detected in uvrA strain as compared with the wild-type strain. From these results, we conclude that Panfuran-S is a DNA-damaging agent and may induce the error-prone SOS responses.