A fine-scale genetic map of the Japanese population.

Genetic maps are fundamental resources for linkage and association studies. A fine-scale genetic map can be constructed by inferring historical recombination events from the genome-wide structure of linkage disequilibrium-a non-random association of alleles among loci-by using population-scale sequencing data. We constructed a fine-scale genetic map and identified recombination hotspots from 10 092 551 bi-allelic high-quality autosomal markers segregating among 150 unrelated Japanese individuals whose genotypes were determined by high-coverage (30×) whole-genome sequencing, and the genotype quality was carefully controlled by using their parents' and offspring's genotypes. The pedigree information was also utilized for haplotype phasing. The resulting genome-wide recombination rate profiles were concordant with those of the worldwide population on a broad scale, and the resolution was much improved. We identified 9487 recombination hotspots and confirmed the enrichment of previously known motifs in the hotspots. Moreover, we demonstrated that the Japanese genetic map improved the haplotype phasing and genotype imputation accuracy for the Japanese population. The construction of a population-specific genetic map will help make genetics research more accurate.

Heterozygous calcyclin-binding protein/Siah1-interacting protein (CACYBP/SIP) gene pathogenic variant linked to a dominant family with paucity of interlobular bile duct.

Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1-2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated ß-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and ß-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of ß-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the ß-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.

ALOX12 mutation in a family with dominantly inherited bleeding diathesis.

The arachidonic acid (AA) cascade plays a significant role in platelet aggregation. AA released from membrane phospholipids is metabolized by cyclooxygenase (COX) pathway to thromboxane A2 (TXA2) or by 12S-lipoxygenase (ALOX12) to 12-hydroperoxyeicosatetraenoic acid (12-HPETE). In contrast to a well-known role of the COX pathway in platelet aggregation, the role of ALOX12 is not well understood. Platelets of ALOX12-deficient mice exhibit increased sensitivity for ADP-induced aggregation. However, recent evidence strongly suggests a significant role of ALOX12 in platelet aggregation and calcium signaling. 12-HPETE potentiates thrombin- and thromboxane-induced platelet aggregation, and calcium signaling. Inhibition experiments of ALOX12 demonstrated decreased platelet aggregation and calcium signaling in stimulated platelets. We studied a family with a dominantly inherited bleeding diathesis using next-generation sequencing analysis. Platelet aggregation studies revealed that the proband's platelets had defective aggregation responses to ADP, TXA2 mimetic U46619, collagen, and AA, normal affinity of TXA2 receptor for U46619, and normal induction of GTPase activity upon stimulation with U46619. However, the production of inositol 1,4,5-triphosphate (IP3) was only increased up to 30% of the control upon U46619 stimulation, suggesting a defect in phospholipase C-ß2 (PLCB2) activation downstream from TXA2 receptors. Affected family members had no mutation of PLCB2, but had a heterozygous c.1946A > G (p.Tyr649Cys) mutation of ALOX12. ALOX12 activity in platelets from the affected members was decreased to 25-35% of the control. Our data strongly suggested that a heterozygous c.1946A > G ALOX12 mutation was a disease-causing mutation; however, further experiments are required to confirm the pathogenesis of ALOX12 mutation in platelet aggregation.

Genome-wide association study identifies new loci for albuminuria in the Japanese population.

BACKGROUND: Urinary albumin excretion (UAE) is a risk factor for cardiovascular diseases, metabolic syndrome, chronic kidney disease, etc. Only a few genome-wide association studies (GWAS) for UAE have been conducted in the European population, but not in the Asian population. Here we conducted GWAS and identified several candidate genes harboring single nucleotide polymorphisms (SNPs) responsible for UAE in the Japanese population. METHODS: We conducted GWAS for UAE in 7805 individuals of Asian ancestry from health-survey data collected by Tohoku Medical Megabank Organization (ToMMo) and Iwate Tohoku Medical Megabank Organization (IMM). The SNP genotype data were obtained with a SNP microarray. After imputation using a haplotype panel consisting of 2000 genome sequencing, 4,962,728 SNP markers were used for the GWAS. RESULTS: Eighteen SNPs at 14 loci (GRM7, EXOC1/NMU, LPA, STEAP1B/RAPGEF5, SEMA3D, PRKAG2, TRIQK, SERTM1, TPT1-AS1, OR5AU1, TSHR, FMN1/RYR3, COPRS, and BRD1) were associated with UAE in the Japanese individuals. A locus with particularly strong associations was observed on TSHR, chromosome 14 [rs116622332 (p = 3.99 × 10-10)]. CONCLUSION: In this study, we successfully identified UAE-associated variant loci in the Japanese population. Further study is required to confirm this association.

A mutation of COX6A1 causes a recessive axonal or mixed form of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6delCACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.

Analysis of the genes responsible for steroid-resistant nephrotic syndrome and/or focal segmental glomerulosclerosis in Japanese patients by whole-exome sequencing analysis.

Steroid-resistant nephrotic syndrome (SRNS) represents glomerular disease resulting from a number of different etiologies leading to focal segmental glomerulosclerosis (FSGS). Recently, many genes causing SRNS/FSGS have been identified. These genes encode the proteins associated with the formation and/or maintenance of glomerular filtration barrier. Next-generation sequencing is used to analyze large numbers of genes at lower costs. To identify the genetic background of Japanese patients, we studied 26 disease-causing genes using whole-exome sequencing analysis in 24 patients with SRNS and/or FSGS from 22 different Japanese families. We finally found eight causative gene mutations, four recessive and four dominant gene mutations, including three novel mutations, in six patients from five different families, and one novel predisposing mutation in two patients from two different families. Causative gene mutations have only been identified in ~20% of families and further analysis is necessary to identify the unknown disease-causing gene. Identification of the disease-causing gene would support clinical practices, including the diagnosis, understanding of pathogenesis and treatment.

Whole-exome sequencing confirmation of a novel heterozygous mutation in RUNX1 in a pregnant woman with platelet disorder.

We describe a successful pregnancy and delivery in a patient with platelet disorder. Prophylactic platelet transfusions ensured that there were no bleeding complications during and after cesarean section. Following delivery, we performed whole exome sequencing, using next generation sequencing, to analyze the DNA samples of the patient and her family, and to identify the disease-causing mutation or variant. To identify de-novo mutations systematically, we also analyzed DNA isolated from the parents of the patient and the neonate. We successfully identified a causative novel mutation c.419 G > A (p.S140N) in RUNX1 in the patient and the neonate. Mutations of RUNX1 have been reported to be associated with familial platelet disorder and with a predisposition for myelodysplasia and/or acute myeloid leukemia. The patient and the neonate require careful long-term hematological follow-up. Identification of mutations by a through whole-exome analysis using next-generation sequencing may be useful in the determination of a long-term follow-up schedule for the patient.

Simultaneous determination of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan glycosaminoglycan disaccharides by high-performance liquid chromatography using a reverse-phase column with adamantyl groups.

Glycosaminoglycans (GAGs), which are one of the major components of proteoglycans, play a pivotal role in physiological processes such as signal transduction, cell adhesion, growth, and differentiation. Characterization of GAGs is challenging due to the tremendous structural diversity of heteropolysaccharides with numerous sulfate or carboxyl groups. In this present study, we examined the analysis of 2-aminobenzamide (2-AB) labeled GAG disaccharides by high-performance liquid chromatography (HPLC) using a reverse-phase (RP)-column with adamantyl groups. Under the analytical conditions, 17 types of 2-AB labeled GAG disaccharides derived from heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan were sequentially separated in a single analysis. The analysis time was fast with high retention time reproducibility. Moreover, the RP-HPLC column with adamantyl groups allowed the quantification of GAGs in various biological samples, such as serum, cultured cells, and culture medium.

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice.

TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1), which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.

Effect of renal impairment on the pharmacokinetics of memantine.

The effect of renal impairment on the pharmacokinetics of a single oral dose of memantine (10 mg) was determined in Japanese subjects. Subjects were assigned to four groups based on baseline creatinine clearance (CL(CR)): normal renal function (> 80 mL/min, n = 6), and mild (50 to ≤ 80 mL/min, n = 6), moderate (30 to < 50 mL/min, n = 6), and severe renal impairment (5 to < 30 mL/min, n = 7). Mean memantine maximum plasma concentration (C(max)) was similar in the groups (12.66, 17.25, 15.75, and 15.83 ng/mL, respectively), as was mean time to C(max) (6.2, 5.2, 4.3, and 5.4 h, respectively). However, exposure to memantine determined from mean area under the plasma concentration-time curve was 1.62-, 1.97-, and 2.33-times higher in subjects with mild, moderate, and severe renal impairment, respectively, as compared to controls with normal renal function. Mean memantine plasma elimination half-life increased according to increasing renal impairment (61.15, 83.00, 100.13, and 124.31 h, respectively), while mean cumulative urinary recovery of unchanged memantine in 72 h after dosing decreased according to increasing renal impairment (33.68%, 33.47%, 23.60%, and 16.17%, respectively). These results are the same as those in the previous study on caucasian individuals, when compared per body weight. It is suggested that the dose of memantine should be halved in patients with renal impairment.

Expression and Localization of TRK-Fused Gene Products in the Rat Brain and Retina.

The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It has been reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. However, no information regarding the localization of Tfg in rat tissues is available. In this study, we investigated the expression of Tfg mRNA in normal rat tissues using reverse transcription-polymerase chain reaction (RT-PCR). We also produced an antibody against Tfg gene products and examined the localization of TFG in the rat brain and retina. The RT-PCR experiments demonstrated that two types of Tfg mRNA were expressed in rat tissues: the conventional form of Tfg (cTfg) and a novel variant form, retinal Tfg (rTfg). RT-PCR analyses demonstrated that cTfg was ubiquitously expressed in rat tissues, while rTfg was predominantly expressed in the brain and retina. Western blot analysis demonstrated two bands with molecular weights of about 30 kDa and 50 kDa in the rat brain. Immunohistochemistry indicated that TFG proteins were predominantly expressed by neurons in the brain. In the rat retina, intense TFG-immunoreactivity was detected in the layer of rods and cones and the outer plexiform layer.

Free-wall perforation of the left ventricle after acute myocardial infarction complicated with pericardial tamponade that mimicked ascending aortic dissection: A case report.

Life-threatening cardiac events may be misdiagnosed as acute aortic dissection because of notable symptom mimicry. We report the case of a 72-year-old male patient with presentations presumed to be of aortic origin. However, surgery revealed posterior free-wall perforation in the left ventricle caused by the occlusion of an obtuse marginal branch.

Impact of GERAADA score in patients with acute type A aortic dissection.

BACKGROUND: Despite continuous developments and advances in the perioperative management of patients suffering from acute aortic dissection type A (AADA), the associated postoperative morbidity and mortality remain high and strongly depend on the preoperative clinical status. The associated postoperative mortality is still hard to predict prior to the surgical procedure. The so-called German Registry of Acute Aortic Dissection Type A (GERAADA) score uses very basic and easily retrievable parameters and was specifically designed for predicting the 30-day mortality rate in patients undergoing surgery for AADA. This study evaluated impact of the GERAADA score in the authors' institutional results. METHODS: Among 101 acute type A aortic dissection patients treated at our hospital during August 2015-March 2021, the GERAADA was calculated individually and retrospectively. Predicted and actual mortalities were assessed, and independent predicted factors were searched. The primary endpoint was defined as comparison of GERAADA scores and early mortality, and the secondary endpoints were defined as comparison of GERAADA scores and other postoperative results, and comparison of preoperative factors and postoperative results regardless to GERAADA scores. RESULTS: While the overall 30-day mortality for the entire study cohort calculated by the GERAADA score was 14.3 (8.1-77.6)%, the actual mortality rate was 6%. However, the GERAADA score was significantly high in some postoperative complications and showed significant correlation with some peri- and post-operative factors. In addition, factors not belonging to GERAADA score such as time from onset to arrival at the hospital, time from onset to arrival at the operation room, spouse presence, and hemodialysis were significantly associated with 30-day mortality. CONCLUSIONS: Although the actual mortality was lower than predicted, GERAADA score may impact on the postoperative course. In addition, it would be desirable to add parameters such as the time from onset to arrival, family background, and hemodialysis for further accuracy.

Effect of intensive care provided by nurse practitioners for postoperative patients: A retrospective observational before-and-after study.

Nurse practitioners are increasingly now members of intensive care teams in Japan, but no data exist about their effect on the outcomes for critically ill patients. This study aimed to compare the outcomes of postoperative patients on mechanical ventilators before and after the participation of nurse practitioners in intensive care teams. We retrospectively identified 387 patients who underwent postoperative mechanical ventilation at a University Hospital in Japan, using data from medical records from 1 April 2015 to 31 March 2017. We extracted data and compared patients' length of stay in the intensive care unit and the hospital, mechanical ventilation days, postoperative rehabilitation start date, rehabilitation prescription, intensive care unit and hospital mortality, and intensive care unit readmission. Multiple regression analysis was used to analyze the factors affecting length of stay in the intensive care unit. Patients who received care from nurse practitioners and physicians had significantly shorter stays in intensive care (4.8 ± 4.8 days versus 6.7 ± 10.3 days, p < 0.021). Mechanical ventilation days, total length of hospital stay, rehabilitation prescription, mortality in intensive care and hospital, and readmission to intensive care were all similar to those who received care only from physicians. The multiple regression analysis suggests that participation of nurse practitioners in intensive care reduced the length of stay in the unit by 2.6 days (p = 0.003). These findings could help to increase use of non-physician healthcare providers in intensive care. Our results demonstrated that it is both effective and safe for nurse practitioners to participate in intensive care teams that provide care for postoperative patients receiving mechanical ventilation.

Surgical treatment for brachiocephalic artery aneurysm with Takayasu arteritis using isolated cerebral perfusion: a case study.

BACKGROUND: Takayasu arteritis (TA) is a chronic inflammatory disease that induces stenosis, occlusion, or aneurysmal degeneration of the aorta and its major branches. Though rarely reported, proximal aneurysmal lesions from the aortic root to the arch are more common in Asian populations than in Western populations. In the surgical treatment of TA, anastomotic aneurysm can be problematic. Furthermore, atherosclerotic complications should be considered in surgical treatment for elderly TA patients. CASE PRESENTATION: Here, we report a case of brachiocephalic artery (BCA) aneurysm with TA for which surgical treatment was successful. Though it was solely a lesion of the brachiocephalic artery, after considering the patient's clinical background and the features of TA, we chose a partial arch replacement. Further, for avoidance of anastomotic aneurysm, both distal and proximal anastomosis were reinforced with Teflon felt strips. Preoperative computed tomography detected severe atherosclerotic changes in the arch vessels. The patient underwent partial arch replacement using isolated cerebral perfusion (ICP) for brain protection and recovered without any neurological deficits. CONCLUSIONS: In avoidance with anastomotic aneurysm, reinforcement of the anastomosis was introduced. ICP was effective for brain protection in case with severe atherosclerotic changes.

Impact of direct carotid artery perfusion in acute type A aortic dissection involving the common carotid artery.

OBJECTIVES: Acute type A aortic dissection complicated with brain ischemia is associated with significantly higher mortality risks. Even if rescued with central aortic repair, some patients develop permanent postoperative neurological deficiency postoperatively. We recently introduced direct common carotid artery perfusion for acute type A aortic dissection involving the common carotid artery. This study introduced this technique to prevent postoperative neurological deficiency by comparing brain protection strategies. METHODS: Among 91 acute type A aortic dissection patients treated at our hospital during August 2015-October 2020, the common carotid artery was involved in 19 (21%), which had > 90% stenosis in either of the carotid arteries on preoperative contrast-enhanced computed tomography. Twelve patients underwent conventional selective cerebral perfusion during August 2015-December 2018 and seven patients underwent direct carotid artery perfusion during January 2019-October 2020. We assessed patient characteristics, surgical courses, clinical outcomes, and neurological outcomes. RESULTS: The mean age was 69 (range 39-84) years; 17 patients were male (89%). Eight patients (42%) had neurological symptoms. Concomitant procedures, postoperative neurological symptoms, and late mortality were significantly associated with conventional selective cerebral perfusion. Five selective cerebral perfusion patients experienced worsened neurological symptoms and two died of broad cerebral ischemia. No direct carotid artery perfusion patient died during hospitalization or experienced worsened neurological outcomes. CONCLUSIONS: Direct carotid artery perfusion may be useful in aortic dissection with brain ischemia because it does not aggravate neurological symptoms and is not associated with intraoperative cerebral infarction. However, it may be ineffective when cerebral infarction has already developed.

Construction and integration of three de novo Japanese human genome assemblies toward a population-specific reference.

The complete human genome sequence is used as a reference for next-generation sequencing analyses. However, some ethnic ancestries are under-represented in the reference genome (e.g., GRCh37) due to its bias toward European and African ancestries. Here, we perform de novo assembly of three Japanese male genomes using > 100× Pacific Biosciences long reads and Bionano Genomics optical maps per sample. We integrate the genomes using the major allele for consensus and anchor the scaffolds using genetic and radiation hybrid maps to reconstruct each chromosome. The resulting genome sequence, JG1, is contiguous, accurate, and carries the Japanese major allele at most loci. We adopt JG1 as the reference for confirmatory exome re-analyses of seven rare-disease Japanese families and find that re-analysis using JG1 reduces total candidate variant calls versus GRCh37 while retaining disease-causing variants. These results suggest that integrating multiple genomes from a single population can aid genome analyses of that population.

Japonica Array NEO with increased genome-wide coverage and abundant disease risk SNPs.

Ethnic-specific SNP arrays are becoming more important to increase the power of genome-wide association studies in diverse population. In the Tohoku Medical Megabank Project, we have been developing a series of Japonica Arrays (JPA) for genotyping participants based on reference panels constructed from whole-genome sequence data of the Japanese population. Here, we designed a novel version of the SNP array for the Japanese population, called Japonica Array NEO (JPA NEO), comprising a total of 666,883 markers. Among them, 654,246 tag SNPs of autosomes and X chromosome were selected from an expanded reference panel of 3,552 Japanese, 3.5KJPNv2, using pairwise r2 of linkage disequilibrium measures. Additionally, 28,298 markers were included for the evaluation of previously identified disease risk markers from the literature and databases, and those present in the Japanese population were extracted using the reference panel. Through genotyping 286 Japanese samples, we found that the imputation quality r2 and INFO score in the minor allele frequency bin >2.5-5% were >0.9 and >0.8, respectively, and >12 million markers were imputed with an INFO score >0.8. From these results, JPA NEO is a promising tool for genotyping the Japanese population with genome-wide coverage, contributing to the development of genetic risk scores.