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Pathological analysis of CINCA syndrome using iPS cells

Recently, researcher Takayuki Tanaka, a member of the research group led by CiRA Deputy Director Professor Tatsutoshi Nakahata and Associate Professor Megumu Saito, prepared iPS cells(2) from patients with chronic infantile neurologic cutaneous and articular (CINCA) syndrome(1) who displayed somatic mosaicism(3) and used them to undertake a detailed analysis of the characteristics of the disease. Cells with a gene mutation were found to be closely linked to the onset of the disease.

iiPS cells are capable in vitro of unlimited proliferation and differentiation into any of the various cell types that make up the human body. Using iPS cells generated from patient cells, disease characteristics can be reproduced at cell level, which opens the prospect of using these cells for research into disease etiology and drug discovery. One useful characteristic of iPS cells is that a cell line can be generated through proliferation from a single cell. Previously, when obtaining blood cells from CINCA syndrome patients with somatic mosaicism, it was not possible to separate blood cells with the gene NLRP3 mutation characteristic of the syndrome from those without the mutation, which made it impossible to analyze and compare their different functions. The present study, however, took advantage of this special characteristic of iPS cells which allows a cell line to be generated from a single cell, making it possible for iPS cells prepared from patient skin cells to be divided into mutant and non-mutant cell lines for separate analysis.

In the study, iPS cells were prepared from two CINCA syndrome patients carrying the NLRP3 mutation in some of their body cells, a condition known as somatic mosaicism, and these were used to generate an iPS cell line with the NLRP3 mutation and a normal iPS cell line without the mutation. When each of these cell lines was induced to differentiate into a type of blood cell known as a macrophage, no difference was observed in the characteristic macrophage morphology or behavior (phagocytizing ability). However, when a comparison was made of IL-1beta cytokine(4) production,  a characteristic which is the key to CINCA syndrome, abnormally elevated levels of cytokine production were found only in those macrophages with the gene mutation. This confirmed that the close association with patient disease onset is limited to cells with NLRP3 gene mutation. Moreover, when the same experiment was repeated but this time with the addition to the cell culture of a chemical compound predicted on the basis of previous research reports to have an inhibitory action on IL-1beta production, IL-1beta production was inhibited in cells with the NLRP3 gene mutation.
The present study was conducted as a joint project by Kyoto University Department of Pediatrics, Chiba University Department of Dermatology, Kyoto University Infection and Immunity Group, Kyoto University Institute for Integrated Cell-Material Sciences, and the University of Tokyo Cancer Genomics Project. The research findings were published at the end of June in the online version of the U.S. scientific journal Blood.
Title of paper
"Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery"

Takayuki Tanaka, Kazutoshi Takahashi, Mayu Yamane, Shota Tomida, Saori Nakamura, Koichi Oshima, Akira Niwa, Ryuta Nishikomori, Naotomo Kambe, Hideki Hara, Masao Mitsuyama, Nobuhiro Morone, John E. Heuser, Takuya Yamamoto, Akira Watanabe, Aiko Sato-Otsubo, Seishi Ogawa, Isao Asaka, Toshio Heike, Shinya Yamanaka, Tatsutoshi Nakahata, and Megumu K. Saito

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1) Chronic infantile neurologic cutaneous and articular (CINCA) syndrome
One of the auto-inflammatory syndromes whose onset is associated with NLRP3 gene mutation, this is a chronic inflammatory disease with immediate postnatal onset characterized by three main symptoms: dermal eruption, central nervous lesions, and articular symptoms. Since 2000, IL-1beta overproduction has been pinpointed as the main cause and anti-IL-1 therapy has been introduced with marked results.
2) iPS cell
Induced pluripotent stem cell. Created by inserting a reprogramming factor into skin cells or other somatic cells. Pluripotent stem cells are capable of unlimited proliferation and can differentiate into the cells of any body tissue.
3) Somatic mosaicism
A condition in which, due to a gene mutation occurring in the process of individual development from the fertilized egg, cell populations of differing genotypes are co-present in a mosaic pattern among the cells of a single individual. Among CINCA syndrome patients, those with NLRP3 mutations in all somatic cells and those with somatic mosaicism leading to NLRP3 mutation in only 10-30% of cells show symptoms of largely similar severity. It was therefore not known, in patients with somatic mosaicism, whether only cells with NLRP3 mutation are involved in disease onset or whether all cells exert a pathological action due to some cause regardless of whether the mutation is present.

4) Cytokine
A protein secreted by various cells that exerts action on the behavior of specific cells. IL-1beta is a cytokine of the inflammatory type which acts on surrounding blood cells and sets them on the path to inflammation.

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