Katsuyoshi Ikeda

Genotypic and phenotypic spectrum of CADASIL in Japan: the experience at a referral center in Kumamoto University from 1997 to 2014.

This study elucidates the genotypic and phenotypic spectrum and histopathological findings related to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in Japan. For this single-center retrospective observational study, we enrolled 215 patients who were clinically suspected of having CADASIL and were examined at Kumamoto University from 1997 to 2014, and we diagnosed CADASIL in 70 patients. We found 19 different NOTCH3 mutations in the patients, with the NOTCH3 Arg133Cys mutation being found most frequently. We also found the Arg75Pro mutation, a cysteine-sparing NOTCH3 mutation. CADASIL patients with this Arg75Pro mutation were frequently found throughout Japan, and fewer patients with the Arg75Pro mutation showed MRI hyperintensity in the anterior temporal pole compared with patients with other NOTCH3 mutations. Significantly more CADASIL patients with the NOTCH3 Arg133Cys mutation had hyperintensity in the external capsule compared with CADASIL patients with the other mutations not including the NOTCH3 Arg75Pro mutation. We also showed postmortem pathological findings of the first Japanese CADASIL case with the NOTCH3 Arg133Cys mutation, and histopathological findings of fresh frozen skin biopsy specimens of CADASIL patients. In conclusions, the spectrum of NOTCH3 mutations in Japanese CADASIL patients may be partially explained by founder effects. Genotype–phenotype correlations may exist in CADASIL, which should be considered so as to make an accurate diagnosis of CADASIL in each population. Fresh frozen skin biopsy specimens may aid detection of Notch3 deposits on vascular walls for an improved diagnosis of CADASIL.

Differences in reaction specificity toward lipoprotein X and abnormal LDL among 6 homogeneous assays for LDL-cholesterol.

BACKGROUND We investigated the reaction specificity toward cholesterol in lipoprotein X (Lp-X) and abnormal LDL among 6 homogeneous assays for low-density lipoprotein cholesterol (LDL-C) based on different measurement principles. METHODS The homogeneous LDL-C assays used were based on the liquid selective detergent, selective solubilization, elimination, enzyme-selective protection, calixarene complex, and phosphate complex inhibition methods. The fraction with a density of 1.006-1.063 kg/l was isolated from cholestatic sera, and the reactivity of cholesterol in the lipoprotein fractions by gel filtration for each homogeneous LDL-C assay was determined. RESULTS The liquid selective detergent and elimination methods showed increased cholesterol reactivity in the Lp-X fraction in a concentration-dependent manner, while the selective solubilization and phosphate complex inhibition methods were less reactive toward Lp-X cholesterol. Meanwhile, the homogeneous LDL-C assays showed decreased reactivity against cholesterol in abnormal LDL, with increased ratios of phospholipids and triglycerides against cholesterol. CONCLUSION The homogeneous LDL-C assays showed differential reactivity toward Lp-X and abnormal LDL. Our findings enable accurate interpretation of the LDL-C values in these homogeneous assays, and suggest that these methods should be improved to distinguish between normal LDL and abnormal LDL or Lp-X.

The Influence of Pre-analytical Factors on the Analysis of Circulating MicroRNA

Background: MicroRNAs (miRNA) are expected as useful biomarkers for various diseases. We studied the pre-analytical factors causing variation in the analysis of miRNA. Material and Methods: Blood samples were collected from 25 healthy subjects. Plasma and serum were obtained from the same samples. The levels of miR-451, -16, -126, and -223 were analyzed using RT-qPCR. Cel-miR-39 was added as a spiked-in control in each sample. Results: With the exception of miR-451, the levels of the miRNAs in plasma were higher than in serum. After high-speed centrifugation, the levels of miRNAs were almost equal between plasma and serum except for miR-451. Membrane filtration with 0.45 µm pore size reduced the levels of plasma miRNAs. The coagulation accelerators for serum processing did not affect the analysis of miRNA. The use of fraction containing particles of > 0.45 µm in size showed the inhibitory effect on the analysis of plasma miR-451. The RNase inhibitor was effective for protecting against the degradation of miRNAs. Conclusion: Plasma contains factors modifying miRNA profiles. The immediate processing of plasma with membrane filtration and RNase inhibitor may be a relevant method for achieving the stable analysis of miRNA

Sources of variation of transthyretin in healthy subjects in East and Southeast Asia: Clinical and experimental evidence for the effect of alcohol on transthyretin metabolism

BACKGROUND Although transthyretin (TTR), a negative acute phase protein, is recognized as one of the nutrition assessment proteins, factors affecting serum TTR concentrations than nutritional state in healthy subjects have not been well understood. We investigated the sources of variation of serum TTR concentrations in healthy subjects. METHODS Serum samples of 3511 healthy volunteers (2055 of Japanese subjects and 1456 of other East and Southeast Asian subjects) were collected. We measured serum TTR concentrations in addition to routine blood examinations in each sample, and assessed the relationship between each serum TTR concentrations and the clinical indices such as age, sex, body mass index (BMI), smoking, and level of daily alcohol consumption. We also investigated the direct alcohol effect of TTR expression by assessing TTR mRNA and protein levels in vivo and in vitro experiments. RESULTS Mean TTR concentrations of males were prominently higher than those of females. Multiple regression analysis revealed that serum TTR concentrations increased with age, BMI, and the level of daily alcohol consumption after adjustment for a slight regional difference. Moreover, TTR expression was up-regulated by alcohol treatment through hepatocyte nuclear factor 4α (HNF-4α) in vitro and in vivo experiments. CONCLUSIONS Sex, aging, BMI, and the level of daily alcohol consumption may be the factors affecting serum TTR concentrations in healthy subjects.