Kouji H. Harada

Identification of RNF213 as a Susceptibility Gene for Moyamoya Disease and Its Possible Role in Vascular Development

Background Moyamoya disease is an idiopathic vascular disorder of intracranial arteries. Its susceptibility locus has been mapped to 17q25.3 in Japanese families, but the susceptibility gene is unknown. Methodology/Principal Findings Genome-wide linkage analysis in eight three-generation families with moyamoya disease revealed linkage to 17q25.3 (P<10-4). Fine mapping demonstrated a 1.5-Mb disease locus bounded by D17S1806 and rs2280147. We conducted exome analysis of the eight index cases in these families, with results filtered through Ng criteria. There was a variant of p.N321S in PCMTD1 and p.R4810K in RNF213 in the 1.5-Mb locus of the eight index cases. The p.N321S variant in PCMTD1 could not be confirmed by the Sanger method. Sequencing RNF213 in 42 index cases confirmed p.R4810K and revealed it to be the only unregistered variant. Genotyping 39 SNPs around RNF213 revealed a founder haplotype transmitted in 42 families. Sequencing the 260-kb region covering the founder haplotype in one index case did not show any coding variants except p.R4810K. A case-control study demonstrated strong association of p.R4810K with moyamoya disease in East Asian populations (251 cases and 707 controls) with an odds ratio of 111.8 (P = 10−119). Sequencing of RNF213 in East Asian cases revealed additional novel variants: p.D4863N, p.E4950D, p.A5021V, p.D5160E, and p.E5176G. Among Caucasian cases, variants p.N3962D, p.D4013N, p.R4062Q and p.P4608S were identified. RNF213 encodes a 591-kDa cytosolic protein that possesses two functional domains: a Walker motif and a RING finger domain. These exhibit ATPase and ubiquitin ligase activities. Although the mutant alleles (p.R4810K or p.D4013N in the RING domain) did not affect transcription levels or ubiquitination activity, knockdown of RNF213 in zebrafish caused irregular wall formation in trunk arteries and abnormal sprouting vessels. Conclusions/Significance We provide evidence suggesting, for the first time, the involvement of RNF213 in genetic susceptibility to moyamoya disease.

Perfluorooctanoate and Perfluorooctane Sulfonate Concentrations in Surface Water in Japan

Perfluorooctanoate and Perfluorooctane Sulfonate Concentrations in Surface Water in Japan: Norimitsu Saito, et al. Institute for Environmental Sciences and Public Health of Iwate Prefecture—Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) are synthetic surfactants used in Japan. An epidemiological study of workers exposed to PFOA revealed a significant increase in prostate cancer mortality. A cross‐sectional study of PFOA‐exposed workers showed that PFOA perturbs sex hormone homeostasis. We analyzed their concentrations in surface water samples collected from all over Japan by LC/MS with a solid phase extraction method. The lowest limits of detection (LOD) (ng/L) were 0.06 for PFOA and 0.04 for PFOS. The lowest limits of quantification (LOQ) (ng/L) were 0.1 for both analytes. The levels [geometric mean (GM); geometric standard deviation (GS)] (ng/L) of PFOA and PFOS in the surface waters were GM (GS): 0.97 (3.06) and 1.19 (2.44) for Hokkaido‐Tohoku (n=16); 2.84(3.56) and 3.69 (3.93) for Kanto (n=14); 2.50 (2.23) and 1.07 (2.36) for Chubu (n=17); 21.5 (2.28) and 5.73 (3.61) for Kinki (n=8); 1.51 (2.28) and 1.00 (3.42) for Chugoku (n=9); 1.93 (2.40) and 0.89 (3.09) for Kyushu‐Shikoku (n=15). The GM of PFOA in Kinki was significantly higher than in other areas (ANOVA p<0.01). Systematic searches of Yodo and Kanzaki Rivers revealed two highly contaminated sites, a public‐water‐disposal site for PFOA and an airport for PFOS. The former was estimated to release 18 kg of PFOA/d. PFOA in drinking water in Osaka city [40 (1.07) ng/L] was significantly higher than in other areas. The present study confirms that recognizable amounts of PFOA are released in the Osaka area and that people are exposed to PFOA through drinking water ingestion.

The Influence of Time, Sex and Geographic Factors on Levels of Perfluorooctane Sulfonate and Perfluorooctanoate in Human Serum over the Last 25 years

The Influence of Time, Sex and Geographic Factors on Levels of Perfluorooctane Sulfonate and Perfluorooctanoate in Human Serum over the Last 25 years: Kouji Harada, et al. Department of Health and Environmental Sciences, Kyoto University Graduate School of Medicine— Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are important perfluorochemicals (PFCs) in various applications. Recently, it has been shown that these chemicals are widespread in the environment, wildlife and humans. But the kinds of factors that affect their levels in serum are unclear, and it is also not clear whether exposure to them is increasing or not. To investigate the impacts of time, geographical location and sex on the levels of these chemicals, we measured PFOS and PFOA concentrations in human sera samples collected both historically and recently in Miyagi, Akita and Kyoto Prefectures in Japan. The PFOS and PFOA levels in sera [Geometric Mean (Geometric Standard Deviation)] (µg/L) in 2003 ranged from 3.5 (2.9) in Miyagi to 28.1 (1.5) in Kyoto for PFOS and from 2.8 (1.5) to 12.4 (1.4) for PFOA. Historical samples collected from females demonstrated that PFOS and PFOA concentrations have increased by factors of 3 and 14, respectively, over the past 25 yr. There are large sex differences in PFOS and PFOA concentrations in serum at all locations. Furthermore, there are predominant regional differences for both PFOS and PFOA concentrations. In Kyoto the concentrations of PFOA in dwellers who had lived in the Kinki area for more than 2 yr were significantly higher than in people who had recently moved into the area, in both sexes. This finding suggests that there are sources of PFOA in the Kinki area that have raised the PFOA serum levels of its inhabitants. Further studies are needed to elucidate these sources in the Kinki area of Japan.

Downregulation of Securin by the variant RNF213 R4810K (rs112735431, G>A) reduces angiogenic activity of induced pluripotent stem cell-derived vascular endothelial cells from moyamoya patients.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p<0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p<0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.

Radiation dose rates now and in the future for residents neighboring restricted areas of the Fukushima Daiichi Nuclear Power Plant

Significance There is a potential risk of human exposure to radiation owing to the March 2011 Fukushima Daiichi Nuclear Power Plant accident. In this study, we evaluated radiation dose rates from deposited radiocesium in three areas neighboring the restricted and evacuation areas in Fukushima. The mean annual radiation dose rate in 2012 associated with the accident was 0.89–2.51 mSv/y. The mean dose rate estimates in 2022 are comparable with variations of the average 2 mSv/y background radiation exposure from natural radionuclides in Japan. Furthermore, the extra lifetime integrated dose after 2012 is estimated to elevate lifetime risk of cancer incidence by a factor of 1.03 to 1.05 at most, which is unlikely to be epidemiologically detectable. Radiation dose rates were evaluated in three areas neighboring a restricted area within a 20- to 50-km radius of the Fukushima Daiichi Nuclear Power Plant in August–September 2012 and projected to 2022 and 2062. Study participants wore personal dosimeters measuring external dose equivalents, almost entirely from deposited radionuclides (groundshine). External dose rate equivalents owing to the accident averaged 1.03, 2.75, and 1.66 mSv/y in the village of Kawauchi, the Tamano area of Soma, and the Haramachi area of Minamisoma, respectively. Internal dose rates estimated from dietary intake of radiocesium averaged 0.0058, 0.019, and 0.0088 mSv/y in Kawauchi, Tamano, and Haramachi, respectively. Dose rates from inhalation of resuspended radiocesium were lower than 0.001 mSv/y. In 2012, the average annual doses from radiocesium were close to the average background radiation exposure (2 mSv/y) in Japan. Accounting only for the physical decay of radiocesium, mean annual dose rates in 2022 were estimated as 0.31, 0.87, and 0.53 mSv/y in Kawauchi, Tamano, and Haramachi, respectively. The simple and conservative estimates are comparable with variations in the background dose, and unlikely to exceed the ordinary permissible dose rate (1 mSv/y) for the majority of the Fukushima population. Health risk assessment indicates that post-2012 doses will increase lifetime solid cancer, leukemia, and breast cancer incidences by 1.06%, 0.03% and 0.28% respectively, in Tamano. This assessment was derived from short-term observation with uncertainties and did not evaluate the first-year dose and radioiodine exposure. Nevertheless, this estimate provides perspective on the long-term radiation exposure levels in the three regions.

Dietary Intake of Radiocesium in Adult Residents in Fukushima Prefecture and Neighboring Regions after the Fukushima Nuclear Power Plant Accident: 24-h Food-Duplicate Survey in December 2011

Since the nuclear power plant accident in Fukushima in March 2011, the Japanese government has conducted screening and removal of contaminated foods from the market that exceed provisional regulation limits for radionuclides. This study aimed to provide an urgent estimate of the dietary exposure of adult residents recruited from three areas in Japan to cesium 134 ((134)Cs), cesium 137 ((137)Cs), and, for comparison, natural potassium 40 ((40)K) on December 4, 2011. Fifty-three sets of 24-h food-duplicate samples were collected in Fukushima Prefecture and neighboring regions. The (134)Cs, (137)Cs, and (40)K levels in the samples were measured using a germanium detector. Items in the food-duplicate samples were recorded and analyzed for radiocesium intake. Radiocesium was detected in 25 of 26 samples from Fukushima. The median dietary intake of radiocesium was 4.0 Bq/day (range <0.26-17 Bq/day). The estimated annual dose from radiocesium was calculated assuming that the daily intake of radiocesium was constant throughout the year. The median estimated dose level was 23 μSv/year (range <2.6-99 μSv/year). The estimated dose level of radiocesium was significantly higher in Fukushima than in the Kanto region and western Japan. Stepwise multiple linear regression analyses demonstrated that the intake of fruits and mushrooms produced in Fukushima were significant factors for the dietary intake of (137)Cs in the 26 participants from Fukushima. The average radioactivity (±SD) of locally produced persimmons and apples (n = 16) were 23 ± 28 and 30 ± 35 Bq/kg for (134)Cs and (137)Cs, respectively. The preliminary estimated dietary dose levels among Fukushima residents were much lower than the maximum permissible dose 1 mSv/year, based on new Japanese standard limits for radiocesium in foods (100 Bq/kg for general foods). In future studies, the exposure estimates should be refined by probability sampling to eliminate biases.

Tubulointerstitial damage as the major pathological lesion in endemic chronic kidney disease among farmers in North Central Province of Sri Lanka

Chronic kidney disease of uncertain etiology (CKDu) in North Central Province of Sri Lanka has become a key public health concern in the agricultural sector due to the dramatic rise in its prevalence and mortality among young farmers. Although cadmium has been suspected as a causative pathogen, there have been controversies. To date, the pathological characteristics of the disease have not been reported. Histopathological observations of 64 renal biopsies obtained at Anuradhapura General Hospital from October 2008 to July 2009 were scored according to Banff 97 Working Classification of Renal Allograft pathology. The correlations between the histological observations and clinical parameters were statistically analyzed. Interstitial fibrosis and tubular atrophy with or without nonspecific interstitial mononuclear cell infiltration was the dominant histopathological observation. Glomerular sclerosis, glomerular collapse, and features of vascular pathology such as fibrous intimal thickening and arteriolar hyalinosis were also common. Although hypertension was identified as one of the common clinical features among the cases, it did not influence the histopathological lesions in all the cases. This study concludes that tubulointerstitial damage is the major pathological lesion in CKDu. Exposure(s) to an environmental pathogen(s) should be systematically investigated to elucidate such tubulointerstitial damage in CKDu.

Odd-numbered perfluorocarboxylates predominate over perfluorooctanoic acid in serum samples from Japan, Korea and Vietnam

Perfluorooctanoic acid (PFOA) has recently attracted attention as a potential health risk following environmental contamination. However, information detailing exposure to perfluorinated carboxylic acids (PFCAs) other than PFOA is limited. We measured the concentrations of PFCAs (from perfluorohexanoic acid to perfluorotetradecanoic acid) in serum samples obtained from patients in Japan (Sendai, Takayama, Kyoto and Osaka) between 2002 and 2009, Korea (Busan and Seoul) between 1994 and 2008 and Vietnam (Hanoi) in 2007/2008. Total PFCA levels (geometric mean) were increased from 8.9 ng mL(-1) to 10.3 ng mL(-1) in Japan; from 7.0 ng mL(-1) to 9.2 ng mL(-1) in Korea; and were estimated at 4.7 ng mL(-1) in Vietnam. PFCAs of greater length than PFOA were significantly increased in Sendai, Takayama and Kyoto, Japan, and levels of long-chain PFCAs exceeded PFOA levels in serum. Among these PFCAs, perfluoroundecanoic acid (PFUnDA) was the predominant component (28.5%), followed by perfluorononanoic acid (PFNA 17.5%), perfluorodecanoic acid (PFDA 7.9%), perfluorotridecanoic acid (PFTrDA 6.1%) and perfluorododecanoic acid (PFDoDA 1.8%). Odd-numbered PFCAs (PFNA, PFUnDA and PFTrDA) were also observed in Korea and Vietnam and their presence increased significantly in Korea between 1994 and 2007/2008. The proportion of long-chain PFCAs in serum was relatively high compared to reports in Western countries. Further investigations into the sources and exposure routes are needed to predict the future trajectory of these serum PFCA levels.

Dietary Exposure to Short-Chain Chlorinated Paraffins Has Increased in Beijing, China

Short-chain chlorinated paraffins (SCCPs) persist in the environment and bioaccumulate in biota and are under review by the Stockholm Convention on persistent organic pollutants. SCCP levels were measured semiquantitatively in pooled 24 h food composite samples from Chinese (n = 10), Korean (n = 10), and Japanese (n = 40) adults in the 1990 s and 2007-2009. In Japan, SCCPs were detected in 14 of 20 pooled samples in the 1990 s and 13 of 20 pooled samples in 2009. Between these two time points, the geometric mean (GM) of the dietary intake of total SCCPs per body weight was comparable in Japan (54 ng kg-bw(-1) day(-1) in the 1990 s and 54 ng kg-bw(-1) day(-1) in the 2000s). In Beijing, SCCP levels were elevated by 2 orders of magnitude from 1993 to 2009 (GM: 620 ng kg-bw(-1) day(-1) in 2009). The 95th percentile estimate of the dietary intake was 1200 ng kg-bw(-1) day(-1) (>1% of tolerable daily intake). In Seoul, no samples in 1994 contained detectable SCCP levels and only one sample in 2007 showed trace levels of SCCPs. Preliminary evidence on the significant increase in SCCP exposure in Beijing in 2009 warrants urgent investigations to refine dietary intake estimates by targeting food types and source identification.

Environmental and biological monitoring of persistent fluorinated compounds in Japan and their toxicities

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) comprise a class of per- and poly-fluorinated compounds that have been detected in the environment as well as in humans. The aim of this review is to summarize several monitoring studies in Japan and characterize the toxicokinetics of these compounds. We found that the levels of contamination by these compounds had unique patterns in Japan. The levels of PFOA in serum from inhabitants of the Kansai region were higher than those of other regions. The PFOA levels in air and water samples from the Kansai region were also relatively high. The estimated intakes from these routes partly explain the differences in the serum levels. The toxicokinetics of these compounds have been investigated. Serum samples from male participants had significantly higher geometric means for PFOS and PFOA compared to samples from female participants. This sex-related difference was partly simulated by menstrual blood loss. There are large interspecies differences in the excretion pathways of these compounds. The serum clearances of PFOA via urine were 300–1,000-fold lower in humans than in Wistar rats and Japanese macaques. On the other hand, the biliary excretion of these compounds was comparable in rats and humans, and the long half-lives in humans may be attributable to the low levels of urinary excretion and high biliary reabsorption rates. These findings suggest that qualitative differences in the excretion routes exist between humans and other species. For risk assessment of these compounds, further information regarding sources of exposure and their toxicokinetics is needed.