An in vitro splicing assay reveals the pathogenicity of a novel intronic variant in ATP6V0A4 for autosomal recessive distal renal tubular acidosis.

BACKGROUND: Autosomal recessive distal renal tubular acidosis (dRTA) is a rare hereditary disease caused by pathogenic variants in the ATP6V0A4 gene or ATP6V1B1 gene, and characterized by hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia and nephrocalcinosis. Although several intronic nucleotide variants in these genes have been detected, all of them fell in the apparent splice consensus sequence. In general, transcriptional analysis is necessary to determine the effect on function of the novel intronic variants located out of splicing consensus sequences. In recent years, functional splicing analysis using minigene construction was used to assess the pathogenicity of novel intoronic variant in various field. METHODS: We investigated a sporadic case of dRTA with a compound heterozygous mutation in the ATP6V0A4 gene, revealed by next generation sequencing. One variant was already reported as pathogenic; however, the other was a novel variant in intron 11 (c.1029 + 5G > A) falling outside of the apparent splicing consensus sequence. Expression of ATP6V0A4 was not detected in peripheral leukocytes by RT-PCR analysis. Therefore, an in vitro functional splicing study using minigene construction was conducted to analyze the splicing pattern of the novel variant. RESULTS: A minigene assay revealed that the novel intronic variant leads to a 104 bp insertion immediately following exon 11. In addition, this result was confirmed using RNA extracted from the patient's cultured leukocytes. CONCLUSION: These results proved the pathogenicity of a novel intronic variant in our patient. We concluded that the minigene assay is a useful, non-invasive method for functional splicing analysis of inherited kidney disease, even if standard transcriptional analysis could not detect abnormal mRNA.

Estimation of measles vaccine coverage needed to prevent transmission in schools.

BACKGROUND: The aim of the present study was to produce the first estimation in Japan of the basic reproduction number (R(0)) and the minimum level of vaccine coverage needed to prevent measles outbreaks (P(c)). METHODS: A questionnaire survey was conducted during two measles outbreaks among 12-15-year-old middle school students in one prefecture in spring, from the end of February to the beginning of May 2002, and a stochastic mathematical model was constructed to calculate vaccine effectiveness (VE) and the basic reproduction number (R(0)). P(c) was calculated from R(0) and VE. RESULTS: In outbreak 1 (school A), 62 (94%) of 66 patients responded to the questionnaire. Of a total of 601 students, 534 (88.9%) responded. Of these, 82.6% (441/534) had previously received measles vaccine. In outbreak 2 (school B), 20 (99%) of 21 patients responded. Of a total of 375 students, 373 (99.5%) responded. Of these, 317 (85.0%) received measles immunization. Mathematical analysis was as follows: in outbreak 1 R(0) was 7.40 (95% confidence interval [CI]: 7.36-7.44) and VE was 76.55% (95%CI: 53.24-87.54). In outbreak 2, R(0) was 18.89 (95%CI: 18.88-18.90) and VE was 98.54% (95%CI: 94.89-99.73). Consequently, P(c) was 112.97% (95%CI: 92.29-145.52) in outbreak 1 and 96.11% (95%CI: 93.81-98.53) in outbreak 2. CONCLUSION: Because of the lower VE in outbreak 1, measles virus transmission could not have been stopped even if all students received a single dose of vaccine. In outbreak 2, with higher VE, the outbreak could have been prevented by increasing the proportion of students who had been vaccinated.

Development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification.

We developed a useful method for the detection of rubella virus genome RNA by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and compared the sensitivity of RT-LAMP with that of other virological tests: reverse transcription-PCR (RT-PCR) and virus isolation. The rubella virus genome was amplified by RT-LAMP from clinical isolates obtained between 1987 and 2004 with similar sensitivities to the Takahashi vaccine strain. The detection limit of RT-LAMP was compared with that of RT-PCR using the Takahashi vaccine strain. We detected rubella virus genome material corresponding to 30 PFU/ml in a culture fluid sample by RT-LAMP within 60 min after the extraction of RNA with equal sensitivity to RT-nested PCR. The positive result rates of RT-LAMP, RT-PCR, and virus isolation were also compared using throat swabs obtained from patients who were clinically diagnosed with acute rubella virus infection in 2004 in Tochigi, Japan. Among nine patients with clinical rubella, the positive result rates were three/nine (33.3%) for virus isolation, six/nine (66.7%) for RT-PCR, and seven/nine (77.8%) for RT-LAMP. Consequently, RT-LAMP for rubella virus would be expected to be a reliable rapid diagnostic tool in the clinical setting.

Superconductivity of the Sr2Ca12Cu24O41 spin-ladder system: are the superconducting pairing and the spin-gap formation of the same origin?

Pressure-induced superconductivity in a spin-ladder cuprate Sr2Ca12Cu24O41 has not been studied on a microscopic level thus far although the superconductivity was already discovered in 1996. We have improved the high-pressure technique using a large high-quality crystal, and succeeded in studying the superconductivity using 63Cu nuclear magnetic resonance. We found that the anomalous metallic state reflecting the spin-ladder structure is realized and the superconductivity possesses an s-wave-like character in the meaning that a finite gap exists in the quasiparticle excitation: At a pressure of 3.5 GPa, we observed two excitation modes in the normal state from the relaxation rate T-11. One gives rise to an activation-type component in T-11, and the other T-linear component linking directly with the superconductivity. This gapless mode likely arises from free motion of holon-spinon bound states appearing by hole doping, and the pairing of them likely causes the superconductivity.