Rapid and comprehensive diagnostic method for repeat expansion diseases using nanopore sequencing.

We developed a diagnostic method for repeat expansion diseases using a long-read sequencer to improve currently available, low throughput diagnostic methods. We employed the real-time target enrichment system of the nanopore GridION sequencer using the adaptive sampling option, in which software-based target assignment is available without prior sample enrichment, and built an analysis pipeline that prioritized the disease-causing loci. Twenty-two patients with various neurological and neuromuscular diseases, including 12 with genetically diagnosed repeat expansion diseases and 10 manifesting cerebellar ataxia, but without genetic diagnosis, were analyzed. We first sequenced the 12 molecularly diagnosed patients and accurately confirmed expanded repeats in all with uniform depth of coverage across the loci. Next, we applied our method and a conventional method to 10 molecularly undiagnosed patients. Our method corrected inaccurate diagnoses of two patients by the conventional method. Our method is superior to conventional diagnostic methods in terms of speed, accuracy, and comprehensiveness.

Repeat conformation heterogeneity in cerebellar ataxia, neuropathy, vestibular areflexia syndrome.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, slow-progressing multisystem neurodegenerative disorder. Biallelic AAGGG repeat expansion in RFC1 has been identified as causative of this disease, and repeat conformation heterogeneity (ACAGG repeat) was also recently implied. To molecularly characterize this disease in Japanese patients with adult-onset ataxia, we accumulated and screened 212 candidate families by an integrated approach consisting of flanking PCR, repeat-primed PCR, Southern blotting and long-read sequencing using Sequel II, GridION or PromethION. We identified 16 patients from 11 families, of whom seven had ACAGG expansions [(ACAGG)exp/(ACAGG)exp] (ACAGG homozygotes), two had ACAGG and AAGGG expansions [(ACAGG)exp/(AAGGG)exp] (ACAGG/AAGGG compound heterozygotes) and seven had AAGGG expansions [(AAGGG)exp/(AAGGG)exp] (AAGGG homozygotes). The overall detection rate was 5.2% (11/212 families including one family having two expansion genotypes). Long-read sequencers revealed the entire sequence of both AAGGG and ACAGG repeat expansions at the nucleotide level of resolution. Clinical assessment and neuropathology results suggested that patients with ACAGG expansions have similar clinical features to previously reported patients with homozygous AAGGG expansions, although motor neuron involvement was more notable in patients with ACAGG expansions (even if one allele was involved). Furthermore, a later age of onset and slower clinical progression were implied in patients with ACAGG/AAGGG compound heterozygous expansions compared with either ACAGG or AAGGG homozygotes in our very limited cohort. Our study clearly shows the occurrence of repeat conformation heterogeneity, with possible different impacts on the affected nervous systems. The difference in disease onset and progression between compound heterozygotes and homozygotes might also be suspected but with very limited certainty due to the small sample number of cases in our study. Studies of additional patients are needed to confirm this.

Population Pharmacokinetics and Pharmacodynamics of Oral Propranolol in Pediatric Patients With Infantile Hemangioma.

This study aimed to characterize the population pharmacokinetics and exposure-response relationship of propranolol (Hemangiol® Syrup for Pediatric) in infants with infantile hemangioma. Using nonlinear mixed-effects modeling with 63 pooled sets of plasma concentration-time data from 32 Japanese patients aged 35-150 days, we described the disposition of propranolol adequately by a 1-compartment model with first-order absorption. The estimated population mean apparent clearance and apparent central volume of distribution were 9.34 L/h and 146 L, respectively. Body weight and postnatal age influenced the population pharmacokinetic model. The clinical end points-success (complete or nearly complete resolution of the target hemangioma) and failure-at weeks 12 and 24 were characterized by logistic regression using the area under the concentration-time curve (AUC), estimated from the final population pharmacokinetic model, as an exposure predictor. The logistic regression showed that a higher AUC was associated with a higher probability of successful treatment. At each exposure level, probability of successful treatment was correlated with gestational age and treatment duration. Model-predicted probabilities of successful treatment were consistent with actual results in the clinical trial. Simulations using several dosing regimens indicated that oral propranolol at 3 mg/kg per day was effective and would be appropriate for treating Japanese infants. These simulation results can support optimization of dosing regimens, such as selecting amounts, treatment durations, and dosing intervals, for clinical use.

Cerebellar ataxia-dominant phenotype in patients with ERCC4 mutations.

Autosomal recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous neurological disorders. Through whole-exome sequencing of Japanese ARCA patients, we identified three index patients from unrelated families who had biallelic mutations in ERCC4. ERCC4 mutations have been known to cause xeroderma pigmentosum complementation group F (XP-F), Cockayne syndrome, and Fanconi anemia phenotypes. All of the patients described here showed very slowly progressive cerebellar ataxia and cognitive decline with choreiform involuntary movement, with young adolescent or midlife onset. Brain MRI demonstrated atrophy that included the cerebellum and brainstem. Of note, cutaneous symptoms were very mild: there was normal to very mild pigmentation of exposed skin areas and/or an equivocal history of pathological sunburn. However, an unscheduled DNA synthesis assay of fibroblasts from the patient revealed impairment of nucleotide excision repair. A similar phenotype was very recently recognized through genetic analysis of Caucasian cerebellar ataxia patients. Our results confirm that biallelic ERCC4 mutations cause a cerebellar ataxia-dominant phenotype with mild cutaneous symptoms, possibly accounting for a high proportion of the genetic causes of ARCA in Japan, where XP-F is prevalent.

Efficacy and safety of oral propranolol for infantile hemangioma in Japan.

BACKGROUND: There have been few reports on the efficacy and safety of oral propranolol at 3 mg/kg/day for infantile hemangioma (IH) in Japanese patients. METHODS: A multicenter, open-label phase III study was conducted to evaluate the efficacy and safety of oral propranolol solution in Japanese infants aged 35-150 days with proliferating IH. Thirty-two patients were enrolled in the study, received propranolol solution for 24 weeks at 3 mg/kg/day, and completed the study. RESULTS: The success rate (complete or nearly complete resolution) at week 24 (primary endpoint) was 78% (95%CI: 60-91%). The improvement rate since the previous visit was 100% (32/32) after week 5. Overall, the IH surface area, maximum diameter, and color intensity all decreased over time. Consistency in assessment between the centralized and the investigator on-site assessments was observed in 26 patients. Of the 32 patients, 11 needed further treatment other than the study drug. The incidence of adverse events (AE) and drug-related AE was 97% and 31%, respectively. AE that occurred in ≥two patients were either typical of propranolol use (such as blood pressure decrease) or common events in infants. AE that resulted in dose reduction were observed in two patients, but no serious AE or AE that led to study drug discontinuation were observed. CONCLUSION: Oral propranolol solution at 3 mg/kg/day is effective and safe in Japanese IH patients.

Exome sequencing reveals a homozygous SYT14 mutation in adult-onset, autosomal-recessive spinocerebellar ataxia with psychomotor retardation.

Autosomal-recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous disorders associated with diverse neurological and nonneurological features that occur before the age of 20. Currently, mutations in more than 20 genes have been identified, but approximately half of the ARCA patients remain genetically unresolved. In this report, we describe a Japanese family in which two siblings have slow progression of a type of ARCA with psychomotor retardation. Using whole-exome sequencing combined with homozygosity mapping, we identified a homozygous missense mutation in SYT14, encoding synaptotagmin XIV (SYT14). Expression analysis of the mRNA of SYT14 by a TaqMan assay confirmed that SYT14 mRNA was highly expressed in human fetal and adult brain tissue as well as in the mouse brain (especially in the cerebellum). In an in vitro overexpression system, the mutant SYT14 showed intracellular localization different from that of the wild-type. An immunohistochemical analysis clearly showed that SYT14 is specifically localized to Purkinje cells of the cerebellum in humans and mice. Synaptotagmins are associated with exocytosis of secretory vesicles (including synaptic vesicles), indicating that the alteration of the membrane-trafficking machinery by the SYT14 mutation may represent a distinct pathomechanism associated with human neurodegenerative disorders.

Ion-trap mass spectrometry unveils the presence of isomeric oligosaccharides in an analyte: stage-discriminated correlation of energy-resolved mass spectrometry.

Mass spectrometry, especially tandem mass spectrometry, has been widely used in the field of analytical sciences for handling biological and chemical samples. The technique resolves molecular and fragment ions based on the mass to charge ratio. Energy-resolved mass spectrometry (ERMS) further provides an activation energy-related factor in the dissociation reaction. Therefore, it is a very powerful technique that can discriminate isomeric compounds. Despite the power of ERMS, useful information cannot be obtained when an analyte contains structural isomers. Carbohydrates carry multiple chiral centers, thus oligomers of monosaccharides can form a vast number of structural isomers. We decided to use such species in our endeavors to establish a method of identifying the 'purity' of an analyte solely based on mass spectrometry. In the present paper, we describe a stage-discriminated spectral correlation of ERMS, which not only enables identification of the presence of contaminants in an analyte, but also provides information regarding the 'purity' of fragment ions.

Discrimination of 16 structural isomers of fucosyl galactoside based on energy-resolved mass spectrometry.

Glycans, a family of compounds often attached to proteins and ceramides, are diverse molecules involved in a wide range of biological functions. Their structural analysis is necessary and is often carried out at the microscale level. Methods based on mass spectrometry are therefore used, although they do not provide information regarding isomeric structures often found in glycan structures. If one finds "factors" characteristic of a certain isomer, this information can be used to elucidate an unknown oligosaccharide sequence. One potential technique is to use energy-resolved mass spectrometry (ERMS) that has been used to distinguish a pair of isomeric compounds. Thus, compounds in a combinatorial library might be effectively used for this purpose. We analyzed a set of 16 isomeric disaccharides, the structures of which consisted of all possible combinations of anomeric configurations and interglycosidic linkage positions. All of the compounds were distinguished based on ERMS where normal collision-induced dissociation could distinguish only seven compounds. Furthermore, it was shown that alpha-glycosidic linkages of fucose were more reactive than the beta-isomers and the secondary glycosides were more reactive than the primary glycosides.

Synthesis of a library of fucopyranosyl-galactopyranosides consisting of a complete set of anomeric configurations and linkage positions.

A library composed of a complete set of fucopyranosyl-galactopyranosides was synthesized. A perbenzylated phenylthio fucopyranoside and a series of tri-O-benzyl-galactopyranosyl fluorides having single hydroxyl groups at the 2-, 3-, 4-, and 6-positions were used as the glycosyl donor and glycosyl acceptors, respectively. The chosen set of functionalities at the anomeric centers enabled rapid access to the oligosaccharides based on chemoselective activation. The first coupling reaction was achieved by the action of dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST). The resulting disaccharide fluoride was readily activated by hafnocene bistrifluoromethanesulfonate [Cp2Hf(OTf)2] and glycosidated with n-octanol.

A method of orthogonal oligosaccharide synthesis leading to a combinatorial library based on stationary solid-phase reaction.

A new, efficient synthesis of oligosaccharides, which involves solid-phase reactions without mixing in combination with an orthogonal-glycosylation strategy, is described. Despite a great deal of biological interest, the combinatorial chemistry of oligosaccharides is an extremely difficult subject. The problems include 1) lengthy synthetic protocols required for the synthesis and 2) the variety of glycosylation conditions necessary for individual reactions. These issues were addressed and solved by using the orthogonal-coupling protocol and the application of a temperature gradient to provide appropriate conditions for individual reactions. Furthermore, we succeeded in carrying out solid-phase reactions with neither mechanical mixing nor flow. In this report, the synthesis of a series of trisaccharides, namely, alpha/beta-L-Fuc-(1-->6)-alpha/beta-D-Gal-(1-->2/3/4/6)-alpha/beta-D-Glc-octyl, is reported to demonstrate the eligibility of the synthetic method in combinatorial chemistry.

Development of highly potent D-glucosamine-based chiral fluorescent labeling reagents and a microwave-assisted beta-selective glycosidation of a methyl glycoside reagent.

We synthesized new chiral fluorescence labeling reagents having a 2,3-anthracenedicarboximide group from D-glucosamine, and it was possible to introduce target alcohols at the anomeric positions of the reagents with beta-selectivity by glycosidations. Especially, it was possible to use methyl glycoside reagent as a glycosyl donor with a Lewis acid and microwave irradiation, and it gave selectively beta-glycoside while the reaction without microwave irradiation gave alpha- and beta-mixed glycosides. Those reagents showed very high chiral discrimination ability, and they made it possible to separate the eight stereoisomers of 4,8,12,16-tetramethylheptadecanol by HPLC after derivatizations.

A rapid diagnostic method for a retrotransposal insertional mutation into the FCMD gene in Japanese patients with Fukuyama congenital muscular dystrophy.

Fukuyama-type congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy in combination with central nervous system (CNS) abnormalities. Differential diagnosis of FCMD from Duchenne and Becker muscular dystrophies (DMD/BMD) or other types of congenital muscular dystrophy is occasionally difficult, because of their phenotypic similarity. The gene (FCMD) responsible for FCMD at 9q31 was isolated in 1998. In Japan, most FCMD-bearing chromosomes (87%) have a 3-kb retrotransposal insertion into the 3'-untranslated region (UTR) of the gene that could be derived from a single ancestral founder. Nine non-founder mutations have been identified in Japanese FCMD patients. Severe phenotype was significantly more frequent in patients who were compound heterozygotes for a point mutation and the founder mutation, than in homozygotes for the founder mutation. We developed a PCR-based diagnostic method for a rapid detection of the retrotransposal insertion mutation. Using this system, we screened 18 FCMD patients, and found 16 homozygotes and two heterozygotes for the insertion. We also evaluated the carrier frequency in the normal Japanese population. Six of 676 persons were recognized as a heterozygous carrier. Furthermore, we found three homozygotes for the FCMD founder mutation among 97 patients who had been said to have probable DMD/BMD without any DMD mutations. On the other hand, there were no FCMD homozygotes but four heterozygous carriers among 335 patients with DMD mutations. The diagnostic method we developed will provide a rapid and reliable diagnosis of FCMD, which can bring important information in genetic counseling, such as the accurate mode of inheritance, recurrence risk and a life expectancy.