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Context Nonalcoholic fatty liver disease (NAFLD) is becoming a major issue worldwide, even in children. Multiple parallel hits hypothesis has been suggested as progress of NAFLD, but the mechanism of NAFLD is not completely understood. β-Tubulin is essential in mitoses, neuronal migration, and axon guidance during neuronal development. Pathogenic variants in the TUBB3 gene were shown to be associated with a wide spectrum of neurological abnormalities, but not accompanied by hepatic complications, such as NAFLD. Objective This work aims to examine the association between TUBB3 mutation and nonalcoholic steatohepatitis (NASH). Methods An 11-year-old girl has been followed up as having atypical Möbius syndrome since infancy, as she was born with bilateral ptosis, paralytic strabismus, and facial weakness. At age 7 years, she was diagnosed with TUBB3 E410K syndrome by whole-exome sequencing. At age 10 years, her blood examination revealed elevated liver transaminase levels, which persisted for almost 2 years. She underwent liver biopsy, the results of which were suggestive of NASH. Results The expression of TUBB3 was absent, but that of tyrosine hydroxylase (TH) was present in the parenchymal nerve fibers of the liver. On the other hand, in comparison with an autopsy case of NASH and a normal control, these showed coexpression of TUBB3 and TH in the liver. Conclusion We report the first case of TUBB3 E410K syndrome accompanied by NASH. This case suggests that the TUBB3 mutation may be associated with the pathogenesis and progression of NASH in humans.

Long-term treatment of Parkinson’s disease (PD) by levodopa leads to motor complication “wearing-off”. Zonisamide is a nondopaminergic antiparkinsonian drug that can improve “wearing-off” although response to the treatment varies between individuals. To clarify the genetic basis of zonisamide responsiveness, we conducted a genome-wide association study (GWAS) on 200 PD patients from a placebo-controlled clinical trial, including 67 responders whose “off” time decreased ≥1.5 h after 12 weeks of zonisamide treatment and 133 poor responders. We genotyped and evaluated the association between 611,492 single nucleotide polymorphisms (SNPs) and “off” time reduction. We also performed whole-genome imputation, gene- and pathway-based analyses of GWAS data. For promising SNPs, we examined single-tissue expression quantitative trait loci (eQTL) data in the GTEx database. SNP rs16854023 (Mouse double minute 4, MDM4) showed genome-wide significant association with reduced “off” time (PAdjusted = 4.85 × 10−9). Carriers of responsive genotype showed >7-fold decrease in mean “off” time compared to noncarriers (1.42 h vs 0.19 h; P = 2.71 × 10−7). In silico eQTL data indicated that zonisamide sensitivity is associated with higher MDM4 expression. Among the 37 pathways significantly influencing “off” time, calcium and glutamate signaling have also been associated with anti-epileptic effect of zonisamide. MDM4 encodes a negative regulator of p53. The association between improved motor fluctuation and MDM4 upregulation implies that p53 inhibition may prevent dopaminergic neuron loss and consequent motor symptoms. This is the first genome-wide pharmacogenetics study on antiparkinsonian drug. The findings provide a basis for improved management of “wearing-off” in PD by genotype-guided zonisamide treatment.

ObjectiveInterindividual variations in responsiveness to zonisamide in patients with Parkinson’s disease (PD) have been observed in clinical settings. To decipher the molecular mechanisms determining the efficacy of zonisamide, we conducted whole transcriptome sequencing analysis of patients with PD.MethodsWe selected 23 super-responders (SRs) and 25 non-responders (NRs) to zonisamide from patients with PD who had participated in a previous clinical trial for the approval of zonisamide for the treatment of ‘wearing-off’. Whole transcriptome analysis of peripheral blood was conducted on samples taken before and 12 weeks after zonisamide treatment. We performed differential gene expression analysis to compare between the SRs and NRs at each time point.ResultsDifferentially expressed genes in the pre-treatment samples were significantly enriched for glutamatergic synapses and insulin-like growth factor binding (Padj=7.8 × 10−3and 0.029, respectively). The gene sets associated with these functions changed more dynamically by treatment in SRs than NRs (p=7.2 × 10−3and 8.2 × 10−3, respectively).ConclusionsOur results suggest that the efficacy of zonisamide in PD patients is associated with glutamate-related synaptic modulation and p53-mediated dopaminergic neural loss. Their transcriptomic differences could be captured before treatment, which would lead to the realisation of future personalised treatment.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuron loss. At present, there are no drugs that stop the progression of PD. As with other multifactorial genetic disorders, genome-wide association studies (GWASs) found multiple risk loci for PD, although their clinical significance remains uncertain. Here, we report the identification of candidate drugs for PD by a method using GWAS data and in silico databases. We identified 57 Food and Drug Administration-approved drug families as candidate neuroprotective drugs for PD. Among them, dabrafenib, which is known as a B-Raf kinase inhibitor and is approved for the treatment of malignant melanoma, showed remarkable cytoprotective effects in neurotoxin-treated SH-SY5Y cells and mice. Dabrafenib was found to inhibit apoptosis, and to enhance the phosphorylation of extracellular signal-regulated kinase (ERK), and inhibit the phosphorylation of c-Jun NH2-terminal kinase. Dabrafenib targets B-Raf, and we confirmed a protein–protein interaction between B-Raf and Rit2, which is coded by RIT2, a PD risk gene in Asians and Caucasians. In RIT2-knockout cells, the phosphorylation of ERK was reduced, and dabrafenib treatment improved the ERK phosphorylation. These data indicated that dabrafenib exerts protective effects against neurotoxicity associated with PD. By using animal model, we confirmed the effectiveness of this in silico screening method. Furthermore, our results suggest that this in silico drug screening system is useful in not only neurodegenerative diseases but also other common diseases such as diabetes mellitus and hypertension.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized by obesity, mental impairment, rod-cone dystrophy, polydactyly, male hypogonadism, and renal abnormalities. This disorder is caused by mutations in BBS1-21. Alström syndrome (AS), caused solely by mutations in ALMS1, is another genetic obesity syndrome clinically similar to BBS. We previously conducted the first nationwide survey of BBS in Japan and found four patients with genetically definite BBS. In this study, exome analyses were performed on new patients whose symptoms fulfilled the diagnostic criteria for BBS. We identified one reported heterozygous mutation in BBS1 (p.R429*) in one patient, two novel mutations (p.L493R and p.H719Y) in BBS20 in a second patient, and one novel mutation (p.Q920*) and one reported mutation (p.R2928*) in ALMS1 in a third patient, who was subsequently diagnosed with AS. The first patient with BBS was previously considered to have digenic heterozygous mutations in BBS1 and BBS4. RT-PCR and long-range genomic PCR analyses identified a new heterozygous mutation in BBS1, the deletion of exons 10 and 11. Thus, this patient was compound heterozygous for mutations in BBS1. Many studies have described digenic heterozygous mutations in BBS. However, undetected mutations might have existed in either one of the mutated genes.

Long-term treatment of Parkinson’s disease (PD) by levodopa leads to motor complication “wearing-off”. Zonisamide is a nondopaminergic antiparkinsonian drug that can improve “wearing-off” although response to the treatment varies between individuals. To clarify the genetic basis of zonisamide responsiveness, we conducted a genome-wide association study (GWAS) on 200 PD patients from a placebo-controlled clinical trial, including 67 responders whose “off” time decreased ≥1.5 h after 12 weeks of zonisamide treatment and 133 poor responders. We genotyped and evaluated the association between 611,492 single nucleotide polymorphisms (SNPs) and “off” time reduction. We also performed whole-genome imputation, gene- and pathway-based analyses of GWAS data. For promising SNPs, we examined single-tissue expression quantitative trait loci (eQTL) data in the GTEx database. SNP rs16854023 (Mouse double minute 4, MDM4) showed genome-wide significant association with reduced “off” time (PAdjusted = 4.85 × 10−9). Carriers of responsive genotype showed >7-fold decrease in mean “off” time compared to noncarriers (1.42 h vs 0.19 h; P = 2.71 × 10−7). In silico eQTL data indicated that zonisamide sensitivity is associated with higher MDM4 expression. Among the 37 pathways significantly influencing “off” time, calcium and glutamate signaling have also been associated with anti-epileptic effect of zonisamide. MDM4 encodes a negative regulator of p53. The association between improved motor fluctuation and MDM4 upregulation implies that p53 inhibition may prevent dopaminergic neuron loss and consequent motor symptoms. This is the first genome-wide pharmacogenetics study on antiparkinsonian drug. The findings provide a basis for improved management of “wearing-off” in PD by genotype-guided zonisamide treatment.

A Japanese woman aged in her late 30s with severe insulin resistance and bodily features including a triangular face, prominent forehead, small chin, large and low‐set ears, and ocular depression was investigated. A similar phenotype was not observed in other family members with the exception of her son, suggesting that the condition was caused by a de novo mutation that was transmitted from mother to son. Exome analysis showed the presence in the proband and her son of a c.1945C>T mutation in PIK3R1, a common mutation associated with SHORT (short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay) syndrome. Administration of a sodium–glucose cotransporter 2 inhibitor lowered the proband's hemoglobin A1c level and allowed a reduction in her insulin dose without treatment‐related adverse events including ketoacidosis, exaggerated loss of body mass or hypoglycemia. Sodium–glucose cotransporter 2 inhibitors might thus offer an additional option for the treatment of genetic syndromes of severe insulin resistance.

Genetic factors are known to have an important role in intracranial aneurysm (IA) pathogenesis. The purpose of this study is to identify single-nucleotide polymorphisms (SNPs) that are associated with IA in Japanese population. A total of 2050 IA patients and 1835 controls recruited in Biobank Japan, The University of Tokyo were used in this study. In all, 45 SNPs in 24 genes encoding proteins, which have been considered to be possible risk factors to IA pathogenesis, were genotyped using multiplex PCR-invader assay. Association analysis was evaluated by logistic regression analysis before and after adjustment of age, smoking and hypertension status. This case-control association study revealed a SNP, rs6460071 located on LIMK1 gene (P = 0.00069) to be significantly associated with increased risk of IA. In addition, two SNPs, rs243847 (P = 0.00086) and rs243865 (P = 0.00090), on matrix metallopeptidase 2 (MMP2) gene and one SNP rs1799724 (P = 0.0026) on tumor necrosis factor-α (TNF-α) gene, are marginally associated with IA in male- and female-specific manner, respectively. In conclusion, a large-scale case-control association study was conducted to verify genetic variations associated with IA in Japanese population. This study gave insights on the importance of stratified analysis between genders, and suggested that the underlying mechanism of IA pathogenesis might differ between females and males.

Warfarin is a commonly used anticoagulant, whose dose needs to be determined for each individual patient owing to large inter-individual variability in its therapeutic dose. Although several clinical and genetic variables influencing warfarin dose have been identified, uncovering additional factors are critically important for safer use of warfarin. Through a genome-wide association study, we identified single-nucleotide polymorphism (SNP) rs2108622 [cytochrome P450, family 4, subfamily F, polypeptide 2 (CYP4F2)] as a genetic determinant of warfarin responsiveness for Japanese. Stratifying subjects who have been pre-classified according to the genotypes of SNP rs10509680 [cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9)] and SNP rs9923231 [vitamin K epoxide reductase complex subunit 1 (VKORC1)], based on their genotypes of rs2108622 allowed identification of subjects who require higher dose of warfarin. Incorporating genotypes of rs2108622 into a warfarin dosing algorithm that considers age, body surface area, status of amiodarone co-administration and genotypes of SNPs in the CYP2C9 and VKORC1 genes improved the model's predictability to 43.4%. In this study, the association of CYP4F2 with warfarin dose of the Japanese has been established for the first time. Besides, a warfarin dosing algorithm that incorporates genotypes of rs2108622 and amiodarone co-administration status was suggested for the Japanese. Our study also implied that common SNPs other than those in the CYP2C9, VKORC1 and CYP4F2 genes that show strong effect on the therapeutic warfarin dose might not exist.

Gamma-glutamyl carboxylase (GGCX) plays an important role in blood coagulation through post-translational carboxylation of vitamin K-dependent blood-clotting proteins. This carboxylation process is impaired in the presence of warfarin, a vitamin K antagonist. Recent studies on GGCX have provided insights into association of polymorphisms in this gene, with inter-individual differences in the required warfarin maintenance dose. In order to provide a useful resource for further elucidating this association, we here report a high-resolution single nucleotide polymorphism (SNP) and haplotype maps of an 18-kb genomic region corresponding to the GGCX locus in the Japanese population. Among 41 SNPs, seven insertion/deletion polymorphisms, and a microsatellite polymorphism that we detected by direct sequencing of the DNAs of 96 Japanese individuals who were treated with warfarin, 32 genetic variations have not been reported. Using genotype information from 12 SNPs and the EM algorithm, we estimated haplotypes for this genomic region. Subsequently, we investigated associations of each of these polymorphisms with the warfarin maintenance-dose requirements of 828 Japanese patients, including the 96 patients that were used for DNA sequencing. We found no significant association between the polymorphisms in GGCX and the dose requirement.

Gallbladder cancer (GC) is a relatively uncommon cancer with higher incidence in certain areas including Japan. Because of the difficulty in diagnosis, prognosis of GC is very poor. To identify genetic determinants of GC, we conducted a genome-wide association study (GWAS) in 41 GC patients and 866 controls. Association between each single-nucleotide polymorphism (SNP) with GC susceptibility was evaluated by multivariate logistic regression analysis conditioned on age and gender of subjects. SNPs that showed suggestive association (P

Human intelligence, as measured by intelligence quotient (IQ) tests, demonstrates one of the highest heritabilities among human quantitative traits. Nevertheless, studies to identify quantitative trait loci responsible for intelligence face challenges because of the small effect sizes of individual genes. Phenotypically discordant monozygotic (MZ) twins provide a feasible way to minimize the effects of irrelevant genetic and environmental factors, and should yield more interpretable results by finding epigenetic or gene expression differences between twins. Here we conducted array-based genome-wide DNA methylation and gene expression analyses using 17 pairs of healthy MZ twins discordant intelligently. ARHGAP18, related to Rho GTPase, was identified in pair-wise methylation status analysis and validated via direct bisulfite sequencing and quantitative RT-PCR. To perform expression profile analysis, gene set enrichment analysis (GSEA) between the groups of twins with higher IQ and their co-twins revealed up-regulated expression of several ribosome-related genes and DNA replication-related genes in the group with higher IQ. To focus more on individual pairs, we conducted pair-wise GSEA and leading edge analysis, which indicated up-regulated expression of several ion channel-related genes in twins with lower IQ. Our findings implied that these groups of genes may be related to IQ and should shed light on the mechanism underlying human intelligence.

Aneurysmal subarachnoid hemorrhage (aSAH) is the most serious subtype of stroke. Genetic factors have been known to play an important role in the development of intracranial aneurysm (IA), some of which further progress to subarachnoid hemorrhage (SAH). In this study, we conducted a genome-wide association study (GWAS) to identify common genetic variants that are associated with the risk of IA, using 1383 aSAH subjects and 5484 control individuals in the Japanese population. We selected 36 single-nucleotide polymorphisms (SNPs) that showed suggestive association (P

Uterine fibroids are a common benign tumor of the female genital tract. We conducted a genome-wide association study in which 457,044 SNPs were analyzed in 1,607 individuals with clinically diagnosed uterine fibroids and 1,428 female controls. SNPs showing suggestive associations (P5 × 10(-5)) were further genotyped in 3,466 additional cases and 3,245 female controls. Three loci on chromosomes 10q24.33, 22q13.1 and 11p15.5 revealed genome-wide significant associations with uterine fibroids. The SNPs showing the most significant association in a combination analysis at each of these loci were rs7913069 (P = 8.65 × 10(-14), odds ratio (OR) = 1.47), rs12484776 (P = 2.79 × 10(-12), OR = 1.23) and rs2280543 (P = 3.82 × 10(-12), OR = 1.39), respectively. Subsequent fine mapping of these regions will be necessary to pinpoint the causal variants. Our findings should shed light on the pathogenesis of uterine fibroids.

Irinotecan is an anti-neoplastic agent that is widely used for treating colorectal and lung cancers, but often causes toxicities such as severe myelosuppression and diarrhea. In this study, we performed a two-stage case-control association study for irinotecan-induced severe myelosuppression (grades 3 and 4). In the first stage, 23 patients who developed severe myelosuppression and 58 patients who did not develop any toxicity were examined for 170 single nucleotide polymorphisms (SNPs) in 14 genes involved in the metabolism and transport of irinotecan. A total of five SNPs were identified to show the possible association with severe myelosuppression (P(Fisher)

Laing distal myopathy (LDM) is an autosomal dominant myopathy that is caused by mutations in the slow/beta cardiac myosin heavy-chain (MYH7) gene. It has been recently reported that LDM presents with a wide range of clinical manifestations. We herein report a large Chinese family with autosomal dominant myopathy. The affected individuals in the family presented with foot drop in early childhood, along with progressive distal and proximal limb weakness. Their characteristic symptoms include scapular winging and scoliosis in the early disease phase and impairment of ambulation in the advanced phase. Although limb-girdle muscle dystrophy (LGMD) was suspected initially, a definite diagnosis could not be reached. As such, we performed linkage analysis and detected four linkage regions, namely 1q23.2-24.1, 14q11.2-12, 15q26.2-26.3 and 17q24.3. Through subsequent whole exome sequencing, we found a de novo p.K1617del causative mutation in the MYH7 gene and diagnosed the disease as LDM. This is the first LDM case in China. Our patients have severe clinical manifestations that mimic LGMD in comparison with the patients with the same mutation reported elsewhere.

The extensive nucleotide diversity in drug-related genes predisposes individuals to different drug responses and is a major problem in current clinical practice and drug development. Striking allelic frequency differences exist in these genes between populations. In this study, we genotyped 240 sites known to be polymorphic in the Japanese population in each of 270 unrelated healthy individuals comprising 90 each of Malaysian Malays, Indians, and Chinese. These sites are distributed in 109 genes that are drug related, such as genes encoding drug-metabolizing enzymes and drug transporters. Allele frequency and linkage disequilibrium distributions of these sites were determined and compared. They were also compared with similar data of 752 Japanese. Extensive similarities in allele frequency and linkage disequilibrium distributions were observed among Japanese, Malaysian Chinese, and Malays. However, significant differences were observed between Japanese and Malaysian Chinese with Malaysian Indians. These four populations were grouped into two genetic clusters of different ancestries. However, a higher correlation was found between Malaysian Malays and Indians, indicating the existence of extensive admixture between them. The results also imply the possible and rational use of existing single nucleotide polymorphism databases as references to assist future pharmacogenetic studies involving populations of similar ancestry.