Takashi Matsukawa

Mutations in COQ2 in familial and sporadic multiple-system atrophy the multiple-system atrophy research collaboration

BACKGROUNDnMultiple-system atrophy is an intractable neurodegenerative disease characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Although multiple-system atrophy is widely considered to be a nongenetic disorder, we previously identified multiplex families with this disease, which indicates the involvement of genetic components.nnnMETHODSnIn combination with linkage analysis, we performed whole-genome sequencing of a sample obtained from a member of a multiplex family in whom multiple-system atrophy had been diagnosed on autopsy. We also performed mutational analysis of samples from members of five other multiplex families and from a Japanese series (363 patients and two sets of controls, one of 520 persons and one of 2383 persons), a European series (223 patients and 315 controls), and a North American series (172 patients and 294 controls). On the basis of these analyses, we used a yeast complementation assay and measured enzyme activity of parahydroxybenzoate-polyprenyl transferase. This enzyme is encoded by the gene COQ2 and is essential for the biosynthesis of coenzyme Q10. Levels of coenzyme Q10 in lymphoblastoid cells and brain tissue were measured on high-performance liquid chromatography.nnnRESULTSnWe identified a homozygous mutation (M78V-V343A/M78V-V343A) and compound heterozygous mutations (R337X/V343A) in COQ2 in two multiplex families. Furthermore, we found that a common variant (V343A) and multiple rare variants in COQ2, all of which are functionally impaired, are associated with sporadic multiple-system atrophy. The V343A variant was exclusively observed in the Japanese population.nnnCONCLUSIONSnFunctionally impaired variants of COQ2 were associated with an increased risk of multiple-system atrophy in multiplex families and patients with sporadic disease, providing evidence of a role of impaired COQ2 activities in the pathogenesis of this disease. (Funded by the Japan Society for the Promotion of Science and others.).

Genomic Landscape of Esophageal Squamous Cell Carcinoma in a Japanese Population

BACKGROUND & AIMSnEsophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in Japan. Smoking and drinking alcohol are environmental risk factors for ESCC, whereas single nucleotide polymorphisms in ADH1B and ALDH2, which increase harmful intermediates produced by drinking alcohol, are genetic risk factors. We conducted a large-scale genomic analysis of ESCCs from patients in Japan to determine the mutational landscape of this cancer.nnnMETHODSnWe performed whole-exome sequence analysis of tumor and nontumor esophageal tissues collected from 144 patients with ESCC who underwent surgery at 5 hospitals in Japan. We also performed single-nucleotide polymorphism array-based copy number profile and germline genotype analyses of polymorphisms in ADH1B and ALDH2. Polymorphisms in CYP2A6, which increase harmful effects of smoking, were analyzed. Functions of TET2 mutants were evaluated in KYSE410 and HEK293FT cells.nnnRESULTSnA high proportion of mutations in the 144 tumor samples were C to T substitution in CpG dinucleotides (called the CpG signature) and C to G/T substitutions with a flanking 5 thymine (called the APOBEC signature). Based on mutational signatures, patients were assigned to 3 groups, which associated with environmental (drinking and smoking) and genetic (polymorphisms in ALDH2 and CYP2A6) factors. Many tumors contained mutations in genes that regulate the cell cycle (TP53, CCND1, CDKN2A, FBXW7); epigenetic processes (MLL2, EP300, CREBBP, TET2); and the NOTCH (NOTCH1, NOTCH3), WNTxa0(FAT1, YAP1, AJUBA) and receptor-tyrosine kinase-phosphoinositide 3-kinase signaling pathways (PIK3CA, EGFR, ERBB2). Mutations in EP300 and TET2 correlated with shorter survival times, and mutations in ZNF750 associated with an increased number ofxa0mutations of the APOBEC signature. Expression of mutant forms of TET2 did not increase cellular levels of 5-hydroxymethylcytosine in HEK293FT cells, whereas knockdown of TET2 increased the invasive activity of KYSE410 ESCC cells. Computational analyses associated the mutations in NFE2L2 we identified with transcriptional activation of its target genes.nnnCONCLUSIONSnWe associated environmental and genetic factors with base substitution patterns of somatic mutations and provide a registry of genes and pathways that are disrupted in ESCCs. These findings might be used to design specific treatments for patients with esophageal squamous cancers.

ERBB4 Mutations that Disrupt the Neuregulin-ErbB4 Pathway Cause Amyotrophic Lateral Sclerosis Type 19

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3-5 years from onset. Familial ALS (FALS) comprises 5%-10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions.

Development of a high-throughput microarray-based resequencing system for neurological disorders and its application to molecular genetics of amyotrophic lateral sclerosis.

BACKGROUNDnComprehensive resequencing of the causative and disease-related genes of neurodegenerative diseases is expected to enable (1) comprehensive mutational analysis of familial cases, (2) identification of sporadic cases with de novo or low-penetrant mutations, (3) identification of rare variants conferring disease susceptibility, and ultimately (4) better understanding of the molecular basis of these diseases.nnnOBJECTIVEnTo develop a microarray-based high-throughput resequencing system for the causative and disease-related genes of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases.nnnDESIGNnValidation of the system was conducted in terms of the signal-to-noise ratio, accuracy, and throughput. Comprehensive gene analysis was applied for patients with ALS. Subjects Ten patients with familial ALS, 35 patients with sporadic ALS, and 238 controls.nnnRESULTSnThe system detected point mutations with 100% accuracy and completed the resequencing of 270 kilobase pairs in 3 working days with greater than 99.9% accuracy of base calls, or the determination of base(s) at each position. Analysis of patients with familial ALS revealed 2 SOD1 mutations. Analysis of the 35 patients with sporadic ALS revealed a previously known SOD1 mutation, S134N, a novel putative pathogenic DCTN1 mutation, R997W, and 9 novel variants including 4 nonsynonymous heterozygous variants consisting of 2 in ALS2, 1 in ANG, and 1 in VEGF that were not found in the controls.nnnCONCLUSIONnThe DNA microarray-based resequencing system is a powerful tool for high-throughput comprehensive analysis of causative and disease-related genes. It can be used to detect mutations in familial and sporadic cases and to identify numerous novel variants potentially associated with genetic risks.

Variants associated with Gaucher disease in multiple system atrophy

Glucocerebrosidase gene (GBA) variants that cause Gaucher disease are associated with Parkinson disease (PD) and dementia with Lewy bodies (DLB). To investigate the role of GBA variants in multiple system atrophy (MSA), we analyzed GBA variants in a large case–control series.

CSF1R mutations identified in three families with autosomal dominantly inherited leukoencephalopathy

Genetic and phenotypic heterogeneities are considerably high in adult‐onset leukoencephalopathy, in which comprehensive mutational analyses of the candidate genes by conventional methods are too laborious. We applied exome sequencing to conduct a comprehensive mutational analysis of genes for autosomal dominant leukoencephalopathies. Genomic DNA samples from four patients of three families with autosomal dominantly inherited adult‐onset leukodystrophy were subjected to exome sequencing. On the basis of the results, 21 patients with adult‐onset sporadic leukodystrophy and one patient with pathologically proven HDLS were additionally screened for CSF1R mutations. Exome sequencing identified heterozygous CSF1R mutations (p.I794T and p.R777W) in two families. I794T has recently been reported as a causative mutation for hereditary diffuse leukoencephalopathy with spheroids (HDLS), and R777W is a novel mutation. Although mutational analysis of CSF1R in 21 sporadic cases revealed no mutations, another novel CSF1R mutation, p.C653Y, was identified in one patient with autopsy‐proven HDSL. These variants were located in the PTK domain where the causative mutations cluster. Functional prediction of the mutant CSF1R as well as cross‐species conservation of the affected amino acids supports the notion that these variants are pathogenic for HDLS. Exome sequencing is useful for a comprehensive mutational analysis of causative genes for hereditary leukoencephalopathies, and CSF1R should be considered a candidate gene for patients with autosomal dominant leukoencephalopathies.

Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes

Adrenoleukodystrophy (ALD) is an X-linked disorder affecting primarily the white matter of the central nervous system occasionally accompanied by adrenal insufficiency. Despite the discovery of the causative gene, ABCD1, no clear genotype–phenotype correlations have been established. Association studies based on single nucleotide polymorphisms (SNPs) identified by comprehensive resequencing of genes related to ABCD1 may reveal genes modifying ALD phenotypes. We analyzed 40 Japanese patients with ALD. ABCD1 and ABCD2 were analyzed using a newly developed microarray-based resequencing system. ABCD3 and ABCD4 were analyzed by direct nucleotide sequence analysis. Replication studies were conducted on an independent French ALD cohort with extreme phenotypes. All the mutations of ABCD1 were identified, and there was no correlation between the genotypes and phenotypes of ALD. SNPs identified by the comprehensive resequencing of ABCD2, ABCD3, and ABCD4 were used for association studies. There were no significant associations between these SNPs and ALD phenotypes, except for the five SNPs of ABCD4, which are in complete disequilibrium in the Japanese population. These five SNPs were significantly less frequently represented in patients with adrenomyeloneuropathy (AMN) than in controls in the Japanese population (pu2009=u20090.0468), whereas there were no significant differences in patients with childhood cerebral ALD (CCALD). The replication study employing these five SNPs on an independent French ALD cohort, however, showed no significant associations with CCALD or pure AMN. This study showed that ABCD2, ABCD3, and ABCD4 are less likely the disease-modifying genes, necessitating further studies to identify genes modifying ALD phenotypes.

Continuous epidural administration of midazolam and bupivacaine for postoperative analgesia.

Background: Midazolam has been reported to have a spinally mediated analgesic effect. Clinically, single‐shot epidural or spinal administration of midazolam has been shown to have an analgesic effect on perioperative pain. In this study, we investigated the analgesic effect of continuous epidural administration of midazolam with bupivacaine on postoperative pain.

Adult-onset leukoencephalopathies with vanishing white matter with novel missense mutations in EIF2B2, EIF2B3, and EIF2B5.

Leukoencephalopathy with vanishing white matter (VWM) is a type of leukoencephalopathy with autosomal recessive inheritance. Magnetic resonance imaging (MRI) reveals diffuse leukoencephalopathy with lesions having cerebrospinal fluid (CSF)-like signals. The clinical presentations include progressive cerebellar ataxia, spasticity, and mental decline. The course is chronic progressive with episodes of rapid deterioration following a minor head trauma. Mutations in the five gene-encoding subunits of the translation initiation factor eIF2B, EIF2B1-5, have been identified as the causative mutations for VWM. Although the age at onset of VWM is usually 2–6 years, patients with adult onset have been described. All adult-onset cases except one have been found to be associated with mutations in EIF2B5 [1]. We report cases of adult-onset VWM with novel missense mutations in EIF2B2, EIF2B3, and EIF2B5, which showed decreased eIF2B activities.

Optimal length and angle of a new lightwand device (TrachlightTM)

STUDY OBJECTIVEnTo investigate the effects of angle and length of the Trachlight lightwand and the effect of obesity on transillumination of the neck and difficulty of intubation.nnnDESIGNnProspective, randomized study.nnnSETTINGnOperating room of a university hospital.nnnPATIENTSn270 patients, aged 30 to 70 years, requiring anesthesia.nnnINTERVENTIONnThree different studies were performed. 1) One hundred twenty adults for general anesthesia to test the effect of the angle, 2) 120 patients for the length of the extrusion, and 3) 30 patients with body weight > or = 120% of the standard (Brocas formula) versus 30 with body weight < 120% of the standard to test the effect of the obesity. 1) The lightwand was bent at 20 degrees, 40 degrees, 60 degrees, or 80 degrees with 0 cm extruded from the endotracheal tube; 2) the lightwand was exposed from the endotracheal tube with -0.5, 0, 1, 2, 3, or 4 cm with 40 degrees angle; and 3) the effect of obesity was tested with 40 degrees angle and 0-cm extrusion. Endotracheal intubation was performed using the lightwand. Transillumination of the light at neck and the difficulty of intubation were assessed.nnnMEASUREMENTS AND MAIN RESULTSn1) A 20 degrees angle decreased the transillumination and an 80 degrees angle increased the number of difficult intubation. 2) Transillumination decreased with -0.5 cm and 0 cm extrusion. Difficult intubation increased with the extrusion > or = 3 cm. 3) Transillumination decreased and difficult intubation increased in the patients with body weight > or = 120% of the standard.nnnCONCLUSIONSnTo increase the success rate of tracheal intubation using the lightwand, the lightwand should be bent in 40 degrees to 60 degrees, with the extrusion of 1 to 2 cm from the tracheal tube. For obese patients with body weight > or = 120% of the standard, the lightwand was not useful.