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In the first case, a 60-year-old man who was using continuous subcutaneous insulin infusion (CSII), developed recurrent hypoglycemia due to insulin antibodies. This is the first report of such a case using CSII. In the second case, a 70-year-old man was follow-up case who developed hypoglycemia while using human insulin. In both cases, the hypoglycemia subsided after switching to multiple daily insulin injection and/or insulin preparation. The results of Scatchard analyses of the two cases were similar to those of cases of insulin autoimmune syndrome (IAS) that improved after recovery from hypoglycemia.The clinical characteristics and Scatchard analysis data were essentially the same as those for IAS, except for the presence of insulin administration.

Aim After the hepatitis A virus (HAV) outbreak among men who have sex with men (MSM) around 2018, the importance of HAV vaccination was emphasized, especially for MSM-living with human immunodeficiency virus (MSM-LWHIV). Aimmugen® is licensed and distributed exclusively in Japan. While administration of three doses is recommended, 85% of recipients in the general population were reported to acquire seroprotection after the second dose. In this study, we evaluated the efficacy of two or three vaccine doses along with predictors associated with the response to Aimmugen® in MSM-LWHIV. Methods We retrospectively examined anti-HA-IgG titers of MSM-LWHIV vaccinated with Aimmugen® in our hospital. Patients' data were collected from medical records. Results Between January 2018 and October 2019, 141 subjects whose median age was 46 years old, were examined. All the subjects were on antiretroviral therapy (ART) and the median CD4 count was 615/μL. The acquisition rate of protectable anti-HA-IgG titers after the second and third dose was 71.1% and 98.6%, respectively. In 114 subjects whose anti-HA-IgG titers were tested after the second-dose, factors significantly associated with better response were prolonged ART duration and higher CD4 count. The titers of anti-HA-IgG after the third dose were higher in those who became seropositive after the second-dose than those who did not. Conclusions Three-dose of Aimmugen® for MSM-LWHIV was effective while two-dose was less effective compared to non-HIV-infected people. People-LWHIV with shorter duration of ART and lesser CD4 cell count achieved lower titers of anti-HA-IgG and might require an additional vaccination. This article is protected by copyright. All rights reserved.

HLA-A, -C, -B, and -DRB1 genotypes were analyzed in 178 Japanese COVID-19 patients to investigate the association of HLA with severe COVID-19. Analysis of 32 common HLA alleles at four loci revealed a significant association between HLA-DRB1*09:01 and severe COVID-19 (odds ratio [OR], 3.62; 95% CI, 1.57-8.35; p = 0.00251 [permutation p value = 0.0418]) when age, sex, and other common HLA alleles at the DRB1 locus were adjusted. The DRB1*09:01 allele was more significantly associated with risk for severe COVID-19 compared to preexisting medical conditions such as hypertension, diabetes, and cardiovascular diseases. These results indicate a potential role for HLA in predisposition to severe COVID-19.

Hepatitis B (HB) vaccines (Heptavax-II and Bimmugen) designed based on HBV genotypes A and C are mainly used for vaccination against HB in Japan. To determine whether there are differences in the genetic background associated with vaccine responsiveness, genome-wide association studies were performed on 555 Heptavax-II and 1193 Bimmugen recipients. Further HLA imputation and detailed analysis of the association with HLA genes showed that two haplotypes, DRB1*13:02-DQB1*06:04 and DRB1*04:05-DQB1*04:01, were significantly associated in comparison with high-responders (HBsAb > 100 mIU/mL) for the two HB vaccines. In particular, HLA-DRB1*13:02-DQB1*06:04 haplotype is of great interest in the sense that it could only be detected by direct analysis of the high-responders in vaccination with Heptavax-II or Bimmugen. Compared with healthy controls, DRB1*13:02-DQB1*06:04 was significantly less frequent in high-responders when vaccinated with Heptavax-II, indicating that high antibody titers were less likely to be obtained with Heptavax-II. As Bimmugen and Heptavax-II tended to have high and low vaccine responses to DRB1*13:02, 15 residues were found in the Heptavax-II-derived antigenic peptide predicted to have the most unstable HLA-peptide binding. Further functional analysis of selected hepatitis B patients with HLA haplotypes identified in this study is expected to lead to an understanding of the mechanisms underlying liver disease.

Objectives: Patients with dengue exhibit a range of symptoms from an acute febrile illness (dengue fever, DF), to dengue hemorrhagic fever (DHF), and to the most severe outcome, dengue shock syndrome (DSS). This study was performed to determine the host genetic factors responsible for dengue severity. Two single nucleotide polymorphisms (SNPs) of the interferon lambda 1 (IFNL1) gene (rs30461 and rs7247086) were analyzed for their association with dengue severity in a Thai population. Methods: This was a case–control association study involving 877 patients under the age of 15 years (DF, n = 386; DHF, n = 416; DSS, n = 75). Genotyping was performed by TaqMan real-time PCR assay. Results: It was found that the rs7247086 variant of IFNL1 was associated with DHF, but not DSS. Genotypes CT and TT and the T allele were protective against DHF (p = 0.03, odds ratio 0.62 for CT, odds ratio 0.13 for TT; and p = 0.01, odds ratio 0.54 for the T allele). The other SNP tested was not associated with DHF or DSS. Conclusions: The rs7247086 variant of IFNL1 (the T allele) was found to be protective against DHF, suggesting that IFNL1 may play a role in the pathogenesis of DHF. Keywords: Dengue, IFNL1, Genetic association, Disease severity

Aim/Introduction Insulin administration was found to trigger type 1 diabetes in six Japanese type 2 diabetes patients with type 1 diabetes high‐risk human leukocyte antigen class II and the class I allele of the insulin gene variable number tandem repeat genotype. The objective of the present study was to assess the contribution of non‐human leukocyte antigen single‐nucleotide polymorphisms (SNPs) to the risk of developing insulin‐triggered type 1 diabetes. Materials and Methods We genotyped 13 type 1 diabetes susceptible SNPs in six patients and compared them with those in Japanese controls (Hap Map3‐JPT). The SNPs that showed statistically significant results were further analyzed using non‐diabetic control participants and participants with type 2 diabetes at the Ehime University Hospital. Results The risk allele frequency of BACH2 rs3757247 in the six patients was significantly more frequent than that in 86 Japanese controls (P = 0.038). No significant difference in the allele frequency was observed in the other SNPs. This result was confirmed by the findings that the risk allele frequency of BACH2 in the six patients was significantly higher than that in the non‐diabetic control participants (n = 179) and type 2 diabetes with or without insulin treatment (n = 154 or n = 152; P = 0.035, 0.034 or 0.037, respectively). Despite being statistically not significant, the six patients were all homozygous for the CLEC16A rs12708716 risk allele and five were homozygous for the CLEC16A rs2903692 risk allele. Conclusions In addition to type 1 diabetes high‐risk human leukocyte antigen class II and the class I allele of the insulin gene variable number tandem repeat genotype, the possibility that the risk variants of BACH2 and CLEC16A could contribute to the development of insulin‐triggered type 1 diabetes cannot be excluded.

Background Dengue patients develop different disease severity ranging from mild (dengue fever [DF]) to severe forms (dengue hemorrhagic fever [DHF] and the fatal dengue shock syndrome [DSS]). Host genetics are considered to be one factor responsible for the severity of dengue outcomes. To identify genes associated with dengue severity that have not been studied yet, we performed genetic association analyses of interferon lambda 3 (IFNL3), CD27, and human leukocyte antigen-DPB1 (HLA-DPB1) genes in Thai dengue patients. Methods A case–control association study was performed in 877 children (age ≤ 15 years) with dengue infection (DF, n = 386; DHF, n = 416; DSS, n = 75). A candidate single nucleotide polymorphism of each of IFNL3, CD27, and HLA-DPB1 was selected to be analyzed. Genotyping was performed by TaqMan real-time PCR assay, and the association with dengue severity was examined. Results The rs9277534 variant of HLA-DPB1 was weakly associated with DHF. The genotype GG and G allele conferred protection against DHF (p = 0.04, odds ratio 0.74 for GG genotype, p = 0.03, odds ratio 0.79 for G allele). The association became borderline significant after adjusting for confounders (p = 0.05, odds ratio 0.82). No association was detected for IFNL3 or CD27. Conclusions The present study demonstrated the weak association of the rs9277534 variant of HLA-DPB1 with protection against DHF. This variant is in the 3′ untranslated region and affects HLA-DPB1 surface protein expression. Our finding suggests that HLA-DPB1 may be involved in DHF pathogenesis.

HIV-1 Nef plays an essential role in enhancing virion infectivity by antagonizing the host restriction molecule SERINC5. Because Nef is highly polymorphic due to the selective forces of host cellular immunity, we hypothesized that certain immune-escape polymorphisms may impair Nef’s ability to antagonize SERINC5 and thereby influence viral fitness in vivo. To test this hypothesis, we identified 58 Nef polymorphisms that were overrepresented in HIV-infected patients in Japan sharing the same HLA genotypes. The number of immune-associated Nef polymorphisms was inversely correlated with the plasma viral load. By breaking down the specific HLA allele-associated mutations, we found that a number of the HLA-B*51:01-associated Y120F and Q125H mutations were most significantly associated with a reduced plasma viral load. A series of biochemical experiments showed that the double mutations Y120F/Q125H, but not either single mutation, impaired Nef’s ability to antagonize SERINC5 and was associated with decreasing virion infectivity and viral replication in primary lymphocytes. In contrast, other Nef functions such as CD4, CCR5, CXCR4 and HLA class I downregulation and CD74 upregulation remained unchanged. Taken together, our results suggest that the differential ability of Nef to counteract SERINC5 by naturally occurring immune-associated mutations was associated with the plasma viral load in vivo.

There is an urgent need to develop an automated malaria diagnostic system that can easily and rapidly detect malaria parasites and determine the proportion of malaria-infected erythrocytes in the clinical blood samples. In this study, we developed a quantitative, mobile, and fully automated malaria diagnostic system equipped with an on-disc SiO2 nanofiber filter and blue-ray devices. The filter removes the leukocytes and platelets from the blood samples, which interfere with the accurate detection of malaria by the blue-ray devices. We confirmed that the filter, which can be operated automatically by centrifugal force due to the rotation of the disc, achieved a high removal rate of leukocytes (99.7%) and platelets (90.2%) in just 30 s. The automated system exhibited a higher sensitivity (100%) and specificity (92.8%) for detecting Plasmodium falciparum from the blood of 274 asymptomatic individuals in Kenya when compared to the common rapid diagnosis test (sensitivity = 98.1% and specificity = 54.8%). This indicated that this system can be a potential alternative to conventional methods used at local health facilities, which lack basic infrastructure.

Racemic RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol; trade name: Rhododenol [RD]), which is used in topical skin-lightening cosmetics, was unexpectedly reported in Japan to induce leukoderma or vitiligo called RD-induced leukoderma (RIL) after repeated application. To our knowledge, no studies have investigated chemical-induced vitiligo pathogenesis on a genome-wide scale. Here, we conducted a genome-wide association study (GWAS) for 147 cases and 112 controls. CDH13, encoding a glycosylphosphatidylinositol-anchored protein called T-cadherin (T-cad), was identified as the strongest RIL susceptibility gene. RD sensitivity was remarkably increased by T-cad knockdown in cultured normal human melanocytes. Furthermore, we confirmed tyrosinase upregulation and downregulation of the anti-apoptotic molecules (BCL-2 and BCL-XL), suggesting that T-cad is associated with RD via tyrosinase or apoptotic pathway regulation. Finally, monobenzyl ether of hydroquinone sensitivity also tended to increase with T-cad knockdown, suggesting that the T-cad could be a candidate susceptibility gene for RIL and other chemical-induced vitiligo forms. This is the first GWAS for chemical-induced vitiligo, and it could be a useful model for studying the disease's genetic aspects.

Approximately 5-10% of individuals who are vaccinated with a hepatitis B (HB) vaccine designed based on the hepatitis B virus (HBV) genotype C fail to acquire protective levels of antibodies. Here, host genetic factors behind low immune response to this HB vaccine were investigated by a genome-wide association study (GWAS) and Human Leukocyte Antigen (HLA) association tests. The GWAS and HLA association tests were carried out using a total of 1,193 Japanese individuals including 107 low responders, 351 intermediate responders, and 735 high responders. Classical HLA class II alleles were statistically imputed using the genome-wide SNP typing data. The GWAS identified independent associations of HLA-DRB1-DQB1, HLA-DPB1 and BTNL2 genes with immune response to a HB vaccine designed based on the HBV genotype C. Five HLA-DRB1-DQB1 haplotypes and two DPB1 alleles showed significant associations with response to the HB vaccine in a comparison of three groups of 1,193 HB vaccinated individuals. When frequencies of DRB1-DQB1 haplotypes and DPB1 alleles were compared between low immune responders and HBV patients, significant associations were identified for three DRB1-DQB1 haplotypes, and no association was identified for any of the DPB1 alleles. In contrast, no association was identified for DRB1-DQB1 haplotypes and DPB1 alleles in a comparison between high immune responders and healthy individuals. Conclusion: The findings in this study clearly show the importance of HLA-DR-DQ (i.e., recognition of a vaccine related HB surface antigen (HBsAg) by specific DR-DQ haplotypes) and BTNL2 molecules (i.e., high immune response to HB vaccine) for response to a HB vaccine designed based on the HBV genotype C. (Hepatology 2018).

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014-2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage-specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

Objective: To know whether the effect of interferon-induced protein with tetratricopeptide repeats (IFIT) 1 polymorphism influences the susceptibility of cerebral malaria outcome. Methods: Case-control association study was performed among 314 Thai patients (110 with cerebral malaria and 204 with uncomplicated malaria) infected with Plasmodium falciparum. Genotyping for five tag-single nucleotide polymorphisms of IFIT1 was performed by endpoint genotyping. Results: Genotype frequencies of all tag-SNPs (single nucleotide polymorphisms) showed no association with malaria outcome. However, C allele of rs11203109 was associated with the protection from cerebral malaria (OR=0.62, 95% CI=0.38-0.99, P=0.048). Two single nucleotide polymorphisms (rs5786868 and rs57941432) were in linkage disequilibrium with rs11203109. Conclusions: This suggests that our associated single nucleotide polymorphism (rs11203109) might be a genetic marker of cerebral malaria progression in the Thai population.

We previously reported that single nucleotide polymorphism (SNP)-420 CG (rs1862513) in the promoter region of RETN was associated with type 2 diabetes. Plasma resistin was tightly correlated with SNP-420 genotypes. SNP-420 is a CpG-SNP affecting the sequence of cytosine-phosphate-guanine dinucleotides.To examine whether methylation at SNP-420 affects plasma resistin, we analyzed plasma resistin and methylation at RETN SNP-420.Genomic DNA was extracted from peripheral white blood cells in 2078 Japanese subjects. Quantification of the methylation was performed by pyrosequencing after DNA bisulfite conversion.Methylation at SNP-420 was highest in the C/C genotype (36.9 ± 5.7%), followed by C/G (21.4 ± 3.5%) and G/G (2.9 ± 1.4%; P0.001). When assessed in each genotype, methylation at SNP-420 was inversely associated with plasma resistin in the C/C (β = -0.134, P0.001) or C/G (β = -0.227, P0.001) genotype. In THP-1 human monocytes intrinsically having the C/C genotype, a demethylating reagent, 5-aza-dC, decreased the methylation at SNP-420 and increased RETN messenger RNA. SNP+1263 (rs3745369), located in the 3' untranslated region of RETN, was also associated with methylation at SNP-420. In addition, highly sensitive C-reactive protein was inversely associated with methylation at SNP-420 in the C/C genotype, whereas body mass index was positively associated.Plasma resistin was inversely associated with the extent of methylation at SNP-420 mainly dependent on the SNP-420 genotype. The association can also be explained partially independent of SNP-420 genotypes. SNP-420 could have dual, genetic and epigenetic effects on plasma resistin.

We improved a previously developed quantitative malaria diagnostic system based on fluorescent Blue-ray optical device. Here, we first improved the diagnostic system to enable fully automated operation and the field application was evaluated in Kenya. We detected Plasmodium falciparum in blood samples collected from 288 individuals aged 1-16 years using nested polymerase chain reaction (nPCR), rapid diagnostic test (RDT), and automated system. Compared to RDT, the automated system exhibited a higher sensitivity (100%; 95% confidence interval [CI], 93.3–100%) and specificity (92.8%; 95%CI, 88.5–95.8%). The limit of detection was 0.0061%. Linear regression analysis revealed a correlation between the automated system and microscopic examination for detecting parasitemia (adjusted R2 value=0.63, P=1.13×10−12). The automated system exhibited a stable quantification of parasitemia and a higher diagnostic accuracy for parasitemia than RDT. This indicates the potential of this system as a valid alternative to conventional methods used at local health facilities, which lack basic infrastructure.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants’ genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites’ drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance., Author summary Artemisinin is the most widely used drug against Plasmodium falciparum malaria. In southeast Asia, parasites have evolved genetic changes making them resistant to artemisinin. The elimination of resistant strains is a global priority, since their global spread could result in massive loss of lives. In Papua New Guinea, we found three patients infected with parasites carrying the most widespread resistant variant in southeast Asia, and they were confirmed to be artemisinin resistant. We established that the mutations were not imported from southeast Asia, and found other drug resistance variants in their genetic background, including some shared with parasites in Indonesia. This indicates that artemisinin resistance has emerged in New Guinea separately from southeast Asia, not by a chance event, but by a gradual process of evolution which may still be ongoing undetected on the island. These resistant strains could undermine malaria local control efforts, and constitute a global threat.

HIV-1 viral protein R (Vpr) plays important roles in HIV-1 replication. Despite the identification of a number of HLA class I-associated immune escape mutations; it is yet known whether immune-driven Vpr polymorphisms are associated with disease outcome. Hereby, we comprehensively analyzed Vpr sequence polymorphisms and their association with disease outcome and host HLA genotypes, by using plasma viral RNA isolated from 444 HLA-typed, treatment-naive, chronically HIV-1 infected individuals. Vpr amino acid residues at positions 13, 37, 45, 55, 63, 77, 84, 85, 86, and 93 were significantly associated with patients' plasma viral load and/or CD4 count. Further analysis revealed Ala at position 55 was significantly associated with lower plasma viral load; and Thr at position 63 was significantly associated with lower plasma viral load and higher CD4 count. Also, the number of amino acid residues at the two positions, located in a functionally important α-helical domain, correlated inversely with plasma viral load and positively with CD4 count. Moreover, a phylogenetically corrected method revealed residues at positions 55 and 63 are associated with patients' HLA genotypes. Taken together, our results suggest that Vpr polymorphisms at functionally important and immune-reactive sites may contribute, at least in part, to viral replication and disease outcome in vivo. J. Med. Virol. 89:123-129, 2017. © 2016 Wiley Periodicals, Inc.

A missense variant (rs373863828:G A; p.Arg457Gln) of the CREBRF gene is strongly associated with a higher body mass index (BMI; kg/mThe aim of this study is to examine the association of rs373863828:G A with levels of serum lipids in four Pacific populations.A total of 613 adult subjects were recruited from Tonga (Polynesians) and the Solomon Islands (Melanesians and Micronesians). Multiple regression analyses adjusted for age and sex were performed to examine the association of rs373863828 with levels of serum lipids in each population.A significant association of rs373863828:G A with lower level of HDL-cholesterol was detected in the Tonga population (β = -3.32 and p-value = 0.030). The expected change in HDL-cholesterol with respect to a single copy of the rs373863828-A allele was 3.32 mg/dL. However, the association between rs373863828-A and lower levels of HDL-cholesterol was not significant after further adjustment for BMI in the Tonga population (β = -2.32 and p-value = 0.13).The rs373863828-A allele may not directly affect the level of serum HDL-cholesterol independent of BMI. To confirm the present findings, association studies with large sample sizes and functional analyses are required.

Parasite virulence, an important factor contributing to the severity of Plasmodium falciparum infection, varies among P. falciparum strains. Relatively little is known regarding markers of virulence capable of identifying strains responsible for severe malaria. We investigated the effects of genetic variations in the P.f. merozoite surface protein 2 gene (msp2) on virulence, as it was previously postulated as a factor. We analyzed 300 msp2 sequences of single P. falciparum clone infection from patients with uncomplicated disease as well as those admitted for severe malaria with and without cerebral disease. The association of msp2 variations with disease severity was examined. We found that the N allele at codon 8 of Block 2 in the FC27-like msp2 gene was significantly associated with severe disease without cerebral complications (odds ratio = 2.73, P = 0.039), while the K allele at codon 17 of Block 4 in the 3D7-like msp2 gene was associated with cerebral malaria (odds ratio = 3.52, P = 0.024). The data suggests possible roles for the associated alleles on parasite invasion processes and immune-mediated pathogenicity. Multiplicity of infection was found to associate with severe disease without cerebral complications, but not cerebral malaria. Variations in the msp2-FC27-block 2-8N and 3D7-block 4-17K allele appear to be parasite virulence markers, and may be useful in determining the likelihood for severe and cerebral malaria. Their interactions with potential host factors for severe diseases should also be explored.

Significant associations of HLA-DP alleles with chronic hepatitis B (CHB) infection are evident in Asian and Arabian populations, including Japanese, Han Chinese, Korean, and Saudi Arabian populations. Here, significant associations between CHB infection and five DPB1 alleles (two susceptibility alleles, DPB1(*) 05:01 and (*) 09:01, and three protective alleles, DPB1(*) 02:01, (*) 04:01, and (*) 04:02) were confirmed in a population comprising of 2582 Japanese individuals. Furthermore, odds ratios for CHB were higher for those with both DPB1 susceptibility alleles than for those with only one susceptibility allele; therefore, effects of susceptibility alleles were additive for risk of CHB infection. Similarly, protective alleles showed an additive effect on protection from CHB infection. Moreover, heterozygotes of any protective allele showed stronger association with CHB than did homozygotes, suggesting that heterozygotes may bind a greater variety of hepatitis B-derived peptides, and thus present these peptides more efficiently to T-cell receptors than homozygotes. Notably, compound heterozygote of the protective allele (any one of DPB1*02:01, *04:01, and *04:02) and the susceptible allele DPB1*05:01 was significantly associated with protection against CHB infection, which indicates that one protective HLA-DPB1 molecule can provide dominant protection. Identification of the HLA-DPB1 genotypes associated with susceptibility to and protection from CHB infection is essential for future analysis of the mechanisms responsible for immune recognition of hepatitis B virus antigens by HLA-DPB1 molecules.

UTokyo Research掲載「免疫タンパク質の不安定さが、自己免疫疾患のかかりやすさに関係」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/autoimmunity-associated-genes-encode-exceptionally-unstable-proteins/, UTokyo Research "Autoimmunity associated genes encode exceptionally unstable proteins" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/autoimmunity-associated-genes-encode-exceptionally-unstable-proteins/

It has been suggested that a ‘thrifty’ genotype hypothesis can account for high prevalence of obesity in the island populations of Oceania. A recent genome-wide association study revealed that a missense variant, rs373863828-A (p.Arg457Gln), of the CREBRF gene (encoding CREB3 regulatory factor) was associated with an excessive increase in body mass index (BMI) in Samoans. In the present study, the association of rs373863828-A with an increase in BMI was examined in four Austronesian (AN)-speaking populations in Oceania. We found that rs373863828-A was frequently observed (frequency of 0.15) in Tongans (Polynesians), and was strongly associated with higher BMI (P=6.1 × 10−4). A single copy of the rs373863828-A allele increased BMI by 3.09 kg m−2 after adjustment of age and sex. No significant association was detected in the other three AN-speaking populations (Melanesians and Micronesians) living in Solomon Islands. This was probably due to the low allele frequency (0.02–0.06) of rs373863828-A as well as small sample size. The rs373863828-A allele was not found in both AN-speaking and non-AN-speaking Melanesians living in Papua New Guinea. Our results suggest that rs373863828-A of CREBRF, a promising thrifty variant, arose in recent ancestors of AN-speaking Polynesians.

Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a 'malaria mutator'), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3' → 5' exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175-178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.

Background Dengue patients present a range of symptoms: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). It is not clear whether this variability is due to their genetic background. Here we tested polymorphisms of interleukin 1 beta (IL1B) and interleukin 1 receptor antagonist (IL1RA) genes for association with DSS in the Thai population. Methods Polymorphisms of IL1B -31C/T (rs1143627) and IL1RA 86-base-pair tandem repeat were analyzed in 871 patients (DF = 384, DHF = 413, and DSS = 74). Results IL1B -31C and IL1RA 2/4 genotype were associated with DSS (IL1B -31C: DSS vs DHF: P = .0061, odds ratio [OR, 95% confidence interval {CI}], 3.49 [1.36-8.95]; DSS vs DF: P = .027, OR [95% CI], 2.81 [1.12-7.06]; IL1RA 2/4: DSS vs DHF: P = .017, OR [95% CI], 1.94 [1.12-3.40]; DSS vs DF: P = .024, OR [95% CI], 1.90 [1.07-3.4]). No difference was found between DF and DHF. Logistic regression analysis revealed that IL1B -31C and IL1RA 2/4 genotypes were each independently associated with DSS. Conclusions Patients with IL1B -31C carrier, or IL1RA 2/4 genotype carry a risk for DSS, implying that IL1B may play a role in pathogenesis of DSS.

Background During Plasmodium falciparum infection, microRNA expression alters in brain tissue of mice with cerebral malaria compared to noninfected controls. MicroRNA regulates gene expression post‐transcriptionally to influence biological processes. Cerebral malaria pathology caused mainly by the immunological disorder. We hypothesize that single‐nucleotide polymorphism in a microRNA influences microRNA biogenesis or target gene recognition and altering susceptibility to cerebral malaria. Methods We performed a literature search based on immunological mechanism and applied microRNA‐related single‐nucleotide polymorphisms database to examine candidate microRNA SNPs possibly responsible for cerebral malaria. MicroRNA‐27a and microRNA‐146a are supposed to involve in cerebral malaria pathology. To assess the relationship of microRNA SNP to cerebral malaria outcome, we performed TaqMan Genotyping Assays in 110 cerebral malaria and 207 uncomplicated malaria cases for three candidate microRNA SNPs (rs895819 of microRNA‐27a, rs57095329 and rs2910164 of microRNA‐146a). Results Our study detected no significant difference in genotype and allele frequency of individual microRNA SNPs as well as in haplotypes of microRNA‐146a between these two groups of malaria patients in Thailand. Hardy–Weinberg disequilibrium of rs57095329 in the cerebral malaria group showed a heterozygous excess which might be due to natural selection. Conclusion Our data supported that the candidate microRNA SNPs have no major role to develop cerebral malaria.

We investigated the effects of carbohydrate counting (CC) on glycemic control and the quality of life in Japanese patients with adult-onset type 1 diabetes as a pilot study. Seven patients with type 1 diabetes (3 males and 4 females) aged 62.0 ± 11.9 years were instructed in CC with subsequent insulin dose adjustments. We measured the HbA1c, glycated albumin, body mass index (BMI) and lipid profile, recorded daily energy intake and insulin dose, and administered the Diabetes Satisfaction Questionnaire (DTSQ) and the shortened version of the Diabetes Diet-Related Quality-of-Life (sDDRQOL) scale questionnaire at baseline, 3 or 6 months, and 12 months. Glycated albumin but not HbA1c was significantly reduced at 6 months (P = 0.021), and both HbA1c and glycated albumin were significantly reduced at 12 months (P = 0.004 and P = 0.003, respectively) compared with those at baseline. The total satisfaction score of the DTSQ and burden of diet therapy score of the sDDRQOL scale were significantly improved at 3 and 12 months compared with those at baseline (P = 0.047 and P = 0.034 in the former and P = 0.049 and P = 0.044 in the latter). BMI, lipid profile, daily energy intake and insulin dose at 12 months were not different compared with those at baseline. This study suggests that CC may improve glycemic control as well as the quality of life without worsening lipid profiles or increasing the BMI in patients with type 1 diabetes.

Human essential hypertension is partly caused by genetic factors. Angiotensinogen (AGT), G-protein β3-subunit (GNB3) and cytochrome P450 3A5 (CYP3A5) are candidate hypertension susceptibility genes and risk alleles at these loci have been thought to arise owing to human adaptation to climatic changes following the migration out-of-Africa. This study aimed to reveal the frequencies of hypertension-susceptibility genotypes in Pacific Island populations and associations of these single-nucleotide polymorphisms (SNPs) to hypertension. Genotyping was conducted for 804 individuals from Melanesian, Micronesian and Polynesian populations at SNPs in the genes encoding AGT (rs699, rs5049 and rs5051), GNB3 (rs5443) and CYP3A5*1/*3 (rs776746). Associations between these SNPs and hypertension were tested for 383 Melanesian Solomon Islanders. We found that the A/A genotype at rs5049 was a risk factor for hypertension (P=0.025) in the Melanesian Solomon Islanders; three SNPs for AGT were in linkage disequilibrium. The ancestral alleles of rs699, rs5051 and rs776746, and the derived allele of rs5443 were as frequent in the populations surveyed here as in other equatorial populations. Although other polymorphisms associated with hypertension and additional populations remain to be studied, these findings suggest that the Pacific Islanders' susceptibility to hypertension arose because of human migration and adaptation.

Resistin is an adipokine secreted from adipocytes in mice. We previously reported that a single nucleotide polymorphism (SNP) –420 (rs1862513) in the human resistin gene (RETN), is correlated with plasma resistin. Decorin is a multifunctional proteoglycan, and its isoform, lacking 14 amino acids from the N terminal region of mature core decorin, recently was identified as a resistin receptor in mice. To examine whether SNPs in the vicinity of the human decorin gene (DCN) are associated with plasma resistin, we cross-sectionally analyzed six tag SNPs selected around DCN in the same linkage disequilibrium block in 2,078 community-dwelling Japanese subjects. Plasma resistin was associated with the rs7139228, rs7956537, rs516115, and rs3138167 genotypes in DCN. A multiple regression analysis revealed that the genotype of rs7308752 (G/G) or rs516115 (C/C) was associated with decreased plasma resistin after adjusted for age, sex, BMI, and the RETN SNP rs1862513. The effect of rs7139228 and rs1862513 seemed to be additive without synergistic interaction. Therefore, plasma resistin was associated with some tag SNPs around DCN in the general Japanese population. The possibility that human decorin is a human resistin receptor should be pursued.

Myxovirus resistance A (MxA) is a major interferon (IFN)-inducible antiviral protein. Promoter single-nucleotide polymorphisms (SNPs) of MxA near the IFN-stimulated response element (ISRE) have been frequently associated with various viral diseases, including emerging respiratory infections. We investigated the expression profile of MxA transcripts with distinct first exons in human bronchial epithelial cells. For primary culture, the bronchial epithelium was isolated from lung tissues with different genotypes, and total RNA was subjected to real-time reverse transcription polymerase chain reaction. The previously reported MxA transcript (T1) and a recently registered transcript with a distinct 5′ first exon (T0) were identified. IFN-β and polyinosinic–polycytidylic acid induced approximately 100-fold higher expression of the T1 transcript than that of the T0 transcript, which also had a potential ISRE motif near its transcription start site. Even without inducers, the T1 transcript accounted for approximately two thirds of the total expression of MxA, levels of which were significantly associated with its promoter and exon 1 SNPs (rs17000900, rs2071430, and rs464138). Our results suggest that MxA observed in respiratory viral infections is possibly dominated by the T1 transcript and partly influenced by relevant 5′ SNPs. Electronic supplementary material The online version of this article (doi:10.1007/s00251-012-0663-8) contains supplementary material, which is available to authorized users.

Background Dengue hemorrhagic fever (DHF) is a severe life-threatening form of dengue infection. Low platelet count is one of the characteristic clinical manifestations in patients with severe dengue. However, little is known about genetic factors in the host that cause low platelet count in patients with dengue. Methods A previous genome-wide association study of hematological and biochemical traits identified single nucleotide polymorphisms (SNPs) associated with low platelet count in healthy subjects. To examine the possible association of these SNPs with DHF, 918 Thai patients with dengue [509 patients with DHF and 409 with dengue fever (DF)] were genotyped for five SNPs: rs5745568 in BAK1, rs6141 in THPO, rs6065 in GP1BA, rs739496 in SH2B3, and rs385893 in RCL1. In addition, rs4804803 in CD209, that has been reported to be associated with dengue infection, was also genotyped to examine if rs4804803 affects the association detected in this study. Results The allele frequencies of each SNP were compared between the DHF and DF groups. Among the five SNPs, the G allele of rs5745568 in BAK1 was significantly associated with a risk for DHF [P = 0.006 and crude odd ratio (95 % confidence interval) = 1.32 (1.09–1.60)]. The association of this allele with DHF was also significant in a logistic regression analysis adjusted for age, sex, hospital (i.e., geographic region), immune status (i.e., primary or secondary infection), and virus serotype [P = 0.016 and adjusted odd ratio (95 % confidence interval) = 1.29 (1.05–1.58)]. The result was not influenced by rs4804803 [P = 0.0167 and adjusted OR (95 % CI) = 1.29 (1.05–1.58)]. No other SNPs including rs4804803 showed significant association. Conclusions The low-level constitutive production of platelets caused by the G allele of rs5745568 seems to increase the risk of bleeding in dengue infection. Our results suggest that BCL-2 homologous antagonist/killer (BAK) protein, encoded by BAK1, plays a crucial role in the pathogenesis of DHF.

Resistin is a cytokine inducing insulin resistance in mice. We previously identified single nucleotide polymorphisms (SNPs) at −420 (rs1862513) and −358 (rs3219175) located in the human resistin gene ( RETN) promoter as strong determinants for circulating resistin in the Japanese population. The objective was to identify additional functional variants for circulating resistin. We conducted a genome-wide association study in 448 Japanese subjects. A peak association signal was found on chromosome 19 where RETN is located. The top-hit SNP was SNP −358 G>A, followed by rs1423096 C>T, SNP −420 C>G, and rs10401670 C>T ( P = 5.39×10−47, 1.81×10−22, 2.09×10−16, and 9.25×10−15, respectively). Meta-analysis including another two independent general Japanese populations showed that circulating resistin was most strongly associated with SNP-358, followed by SNP-420, rs1423096, and rs10401670. Rs1423096 and rs10401670 were located in the 3′-region of RETN and were in strong linkage disequilibrium. Although these SNPs were also in linkage disequilibrium with the promoter SNPs, conditional and haplotype association analyses identified rs1423096 and rs10401670 as independent determinants for circulating resistin. Functionally, nuclear proteins specifically recognized T but not C at rs10401670 as evidenced by an electrophoretic mobility shift assay. The promoter activity of a luciferase reporter with T at either rs1423096 or rs10401670 was lower than that with C in THP-1 human monocytes. Therefore, rs1423096 and rs10401670, in addition to SNP-420 and SNP-358, were identified as possible functional variants affecting circulating resistin by the genome-wide search in the Japanese population.

The emergence and spread of artemisinin-resistant Plasmodium falciparum is of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in the Pfkelch13 gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced the Pfkelch13 propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on the Pfkelch13 propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline of Pfkelch13 polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, various Pfkelch13 mutations have been selected under artemisinin pressure.