横井 克幸

Huntington disease (HD) is caused by inherited CAG expansions, which continue expanding somatically in affected brain regions to hasten disease onset and progression. Therapeutically diminishing somatic expansions is expected to be clinically beneficial. However, it is not known if interventionally modifying somatic CAG expansions will actually modify in vivo clinically-relevant phenotypes, what the therapeutic window is, or which phenotypes will be altered. Here we show that acute (6-week) delivery of the contraction-inducing slipped-CAG DNA ligand naphthyridine-azaquinolone to young (4-week-old) (CAG)120 HD mice, induces contractions throughout brain regions, improves motor function (locomotion, balance, coordination, muscle strength), molecular disease landmarks (mHTT aggregates, nuclear envelope morphology, nucleocytoplasmic mRNA transport, transcriptomic dysregulation, neuroinflammation), and neurodegeneration. Beneficial effects of modifying somatic expansions were also evident in muscle and blood, where blood CAG instability correlated with brain instability and blood serum had diminished levels of neurofilament light (a biomarker for neurodegeneration) - offering blood as having elements of target engagement and efficacy. These data support that targeting somatic repeat expansions can be a rapid disease-modifying therapeutic avenue for HD and possibly other repeat expansion diseases. Our findings support an etiologic pathway interconnected to somatic CAG expansions that will inform the design of clinical trials expecting clinical benefit by modulating somatic expansions.

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immunostimulatory or damaged DNA is unknown. Here, we show C9orf72Exp in pre-symptomatic and amyotrophic lateral sclerosis-frontotemporal dementia patient cells and brains cause the folate-sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33 kb of C9orf72 as highly compacted chromatin embedded in an 8.2 Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72Exp patient contained a highly rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp repeat instability and chromosomal fragility are sensitive to folate deficiency. Age-dependent repeat instability, chromosomal fragility and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72Exp mice, implicating C9orf72Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.

症例は5歳女児で、発熱、悪心、胸痛を主訴とした。砂遊びによる両手指の慢性湿疹に対して外用加療中であり、近医受診後も発熱が持続し、入院時には胸骨直上に辺縁不明瞭な発赤、腫脹を認め、CT検査で胸骨体の両側に低吸収帯を認めた。感染症の疑いで抗菌薬治療を開始し、血液培養でメチシリン耐性黄色ブドウ球菌(MRSA)が同定されたため、バンコマイシン(VCM)を併用した。その結果、解熱と胸痛の軽快が得られ、MRSAによる原発性胸骨骨髄炎と診断した。その後はCRP陰性化を確認してリネゾリド内服に変更し、VCMと併せて計6週間の抗MRSA薬による治療を行ったところ、炎症の再燃や血小板減少は認めなかった。本症例では手指の慢性湿疹が感染経路と考えられ、慢性湿疹を背景に持つ患児では薬剤耐性菌による全身性の感染症に留意する必要があると考えられた。

Mutations in transport and Golgi organization 2 homolog (TANGO2) have recently been described as a cause of an autosomal recessive syndrome characterized by episodes of metabolic crisis associated with rhabdomyolysis, cardiac arrhythmias, and neurodegeneration. Herein, we report a case of a one-and-a-half-year-old Japanese girl, born to nonconsanguineous parents, who presented with metabolic crisis characterized by hypoglycemia with hypoketonemia, rhabdomyolysis, lactic acidosis, and prolonged corrected QT interval (QTc) at the age of 6 months. Acylcarnitine analysis during the episode of crisis showed prominent elevation of C14:1, suggesting very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. In addition, worsening rhabdomyolysis was observed after intravenous administration of L-carnitine. VLCAD deficiency was initially suspected; however, the enzyme activity in lymphocytes was only mildly decreased at the gene carrier level, and no mutation in the VLCAD gene (ADADVL) was detected. Subsequently, acylcarnitine analysis was nonspecific at 17-h fasting and almost normal during the stable phase. Eventually, a trio whole-exome sequencing revealed a compound heterozygous variant of two novel variants in the TANGO2 gene, a missense variant, and a deletion of exon 7. This is the first case of TANGO2 deficiency in Asians. Our case suggests that elevated C14:1 may be seen in severe metabolic crises and that the use of L-carnitine should be avoided during metabolic crises.

Maple syrup urine disease (MSUD) is a rare autosomal recessive inherited disorder of branched-chain amino acid metabolism caused by mutations in BCKDHA, BCKDHB, and DBT that encode the E1α, E1β, and E2 subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. Various MSUD-causing variants have been described; however, no structural rearrangements in BCKDHA have been reported to cause the classic MSUD phenotype. Here, we describe the classic patient with MSUD with compound heterozygous pathogenic variants in BCKDHA: a missense variant (NM_000709.3:c.757G > A, NP_000700.1:p.Ala253Thr) and a paracentric inversion disrupting Intron 1 of BCKDHA, which was identified by whole-genome sequencing and validated by fluorescence in situ hybridization. Using the sequence information of the breakpoint junction, we gained mechanistic insight into the development of this structural rearrangement. Furthermore, the establishment of junction-specific polymerase chain reaction could facilitate identification of the variant in case carrier or future prenatal/preimplantation tests are necessary.

Thrombotic microangiopathy (TMA) is a disease that causes organ damage due to microvascular hemolytic anemia, thrombocytopenia, and microvascular platelet thrombosis. Streptococcus pneumoniae-associated TMA (spTMA) is a rare complication of invasive pneumococcal infection. In addition, atypical hemolytic uremic syndrome (aHUS) is TMA associated with congenital or acquired dysregulation of complement activation. We report the case of a nine-month-old boy with refractory nephrotic syndrome complicated by spTMA in the setting of heterozygous complement factor-I (CFI) gene mutation and CFHR3-CFHR1 deletion. He repeatedly developed thrombocytopenia, anemia with schistocytes, hypocomplementemia, and abnormal coagulation triggered by infection, which manifested clinically with convulsions and an intraperitoneal hematoma. Eculizumab (a monoclonal humanized anti-C5 antibody) provided transient symptomatic benefit including improvement in thrombocytopenia; however, he developed unexplained cardiac arrest and was declared brain dead a few days later. In this report, we highlight the diagnostic challenges of this case and the causal relationship between spTMA and complement abnormalities and consider the contribution of heterozygous mutation of CFI and CFHR3-CFHR1 deletion.

Maturity-onset diabetes of the young (MODY) is a form of diabetes mellitus characterized by autosomal dominant inheritance, early onset, and the absence of pancreatic autoimmune markers. MODY-causing mutations have been identified in 14 genes, and carboxyl ester lipase (CEL) has been implicated in MODY8. We report a Japanese patient with MODY who harbored a heterogeneous mutation in CEL exon 2 (NM_001807.4:c.146_147delCT; NP_001798.2:p.Ser49CysfsTer52). A 13-year-old girl experienced her first episode of diabetic ketoacidosis, during which her endogenous insulin secretion was poor. However, her insulin secretion had apparently recovered 2 months after the commencement of insulin treatment, and no further treatment was required for the following 2 years. Diabetic ketoacidosis recurred when the patient was 15 years old, when her insulin secretion was again poor. Since that time, the patient, who is now 18 years old, has been undergoing continuous insulin treatment. The large fluctuations in her insulin secretory capacity led us to suspect MODY. MODY8 patients that carry a mutation in the variable number of tandem repeats in the last exon of the CEL gene typically show pancreatic exocrine dysfunction. However, in the present case, which features premature termination, there is no involvement of exocrine dysfunction, potentially demonstrating a genotype-phenotype correlation.

Urea cycle disorders (UCDs), inborn errors of hepatocyte metabolism, cause hyperammonemia and lead to neurocognitive deficits, coma, and even death. Sodium 4-phenylbutyrate (NaPB), a standard adjunctive therapy for UCDs, generates an alternative pathway of nitrogen deposition through glutamine consumption. Administration during or immediately after a meal is the approved usage of NaPB. However, we previously found that preprandial oral administration enhanced its potency in healthy adults and pediatric patients with intrahepatic cholestasis. The present study evaluated the effect of food on the pharmacokinetics and pharmacodynamics of NaPB in five patients with UCDs. Following an overnight fast, NaPB was administered orally at 75 mg/kg/dose (high dose, HD) or 25 mg/kg/dose (low dose, LD) either 15 min before or immediately after breakfast. Each patient was treated with these four treatment regimens with NaPB. With either dose, pre-breakfast administration rather than post-breakfast administration significantly increased plasma PB levels and decreased plasma glutamine availability. Pre-breakfast LD administration resulted in a greater attenuation in plasma glutamine availability than post-breakfast HD administration. Plasma levels of branched-chain amino acids decreased to the same extent in all tested regimens. No severe adverse events occurred during this study. In conclusion, preprandial oral administration of NaPB maximized systemic exposure of PB and thereby its efficacy on glutamine consumption in patients with UCDs.

<title>Abstract</title>We report a case of a 13-year<bold>-</bold>old boy with arginase 1 deficiency carrying a new variant in <italic>ARG1</italic>. Sanger sequencing identified the compound heterozygous variants: NM_000045.4: c.365G&gt;A (p.Trp122*)/c.820G&gt;A (p.Asp274Asn). Although not previously reported, the p.Asp274Asn variant is predicted to have strong pathogenicity because it is located in a highly conserved domain in the protein core and arginase activity in the patient was below measurement sensitivity.

Cancer treatment with a fluoropyrimidine (FP) is often accompanied by severe toxicity that may be dependent on the activity of catalytic enzymes encoded by the DPYD, DPYS, and UPB1 genes. Genotype-guided dose individualization of FP therapy has been proposed in western countries, but our knowledge of the relevant genetic variants in East Asian populations is presently limited. To investigate the association between these genetic variations and FP-related high toxicity in a Japanese population, we obtained blood samples from 301 patients who received this chemotherapy and sequenced the coding exons and flanking intron regions of their DPYD, DPYS, and UPB1 genes. In total, 24 single nucleotide variants (15 in DPYD, 7 in DPYS and 2 in UPB1) were identified including 3 novel variants in DPYD and 1 novel variant in DPYS. We did not find a significant association between FP-related high toxicity and each of these individual variants, although a certain trend toward significance was observed for p.Arg181Trp and p.Gln334Arg in DPYS (P = .0813 and .087). When we focused on 7 DPYD rare variants (p.Ser199Asn, p.IIe245Phe, p.Thr305Lys, p.Glu386Ter, p.Ser556Arg, p.Ala571Asp, p.Trp621Cys) which have an allele frequency of less than 0.01% in the Japanese population and are predicted to be loss-of-function mutations by in silico analysis, the group of patients who were heterozygous carriers of at least one these rare variants showed a strong association with FP-related high toxicity (P = .003). Although the availability of screening of these rare loss-of-function variants is still unknown, our data provide useful information that may help to alleviate FP-related toxicity in Japanese patients with cancer.

Background: Hypophosphatasia (HPP) is a rare inborn error of metabolism that results from a dysfunctional tissue non-specific alkaline phosphatase enzyme (TNSALP). Although genotype-phenotype correlations have been described in HPP patients, only sparse information is currently available on the genetics of mild type HPP. Methods: We investigated 5 Japanese patients from 3 families with mild HPP (patients 1 and 2 are siblings; patient 4 is a daughter of patient 5) who were referred to Fujita Health University due to the premature loss of deciduous teeth. Physical and dental examinations, and blood, urine and bone density tests were conducted. Genetic analysis of the ALPL gene was performed in all patients with their informed consent. Results: After a detailed interview and examination, we found characteristic symptoms of HPP in some of the study cases. Mobile teeth or the loss of permanent teeth were observed in 2 patients, and 3 out of 5 patients had a history of asthma. The serum ALP levels of all patients were 30% below the lower limit of the age equivalent normal range. ALPL gene analysis revealed compound heterozygous mutations, including Ile395Val and Leu520Argfs in family 1, Val95Met and Gly491Arg in family 2, and a dominant missense mutation (Gly456Arg) in family 3. The 3D-modeling of human TNSALP revealed three mutations (Val95Met, Ile395Val and Gly456Arg) at the homodimer interface. Severe collisions between the side chains were predicted for the Gly456Arg variant. Discussion: One of the characteristic findings of this present study was a high prevalence of coexisting asthma and a high level serum IgE level. These characteristics may account for the fragility of tracheal tissues and a predisposition to asthma in patients with mild HPP. The genotypes of the five mild HPP patients in our present study series included 1) compound heterozygous for severe and hypomorphic mutations, and 2) dominant-negative mutations. All of these mutations were at the homodimer interface, but only the dominant-negative mutation was predicted to cause a severe collision effect between the side chains. This may account for varying mechanisms leading to different effects on TNSALP function.

BACKGROUND: Several immunosuppressants have been used to treat children with steroid-dependent nephrotic syndrome (SDNS). Mizoribine (MZR) is an immunosuppressant used to maintain remission in children with SDNS, although its effectiveness for treating SDNS remains controversial. Therefore, in this study, we assessed the clinical factors associated with children having SDNS who were successfully treated with MZR. METHODS: A total of 47 children with SDNS who underwent MZR treatment were retrospectively evaluated. Clinical features including pharmacokinetics after MZR administration were compared between MZR responders and non-responders. RESULTS: The comparison of the two groups revealed no significant differences in age, body weight (BW), daily dose of MZR per BW, serum concentration 2 h after administration (C2), peak serum concentration (Cmax), and area under the concentration curve 0-4 h after administration (AUC0-4). C2/(single dose/BW), Cmax/(single dose/BW), and AUC0-4/(single dose/BW) were significantly higher in the MZR responders than in the non-responders (all p < 0.01). Receiver operating characteristic analysis revealed that the cutoff values of C2 (single dose/kg), Cmax/(single dose/BW), and AUC0-4/(single dose/BW) were 0.55, 0.58, and 1.37, respectively. CONCLUSIONS: MZR is a useful immunosuppressant for treating frequent-relapse NS in children who are susceptible to the drug. The efficacy of MZR may be associated with not only serum concentrations defined by the dosage or absorption efficiency through MZR transporters, but also the susceptibility defined by the expression level and performance of MZR transporters on the target cells.

Phosphoglucomutase 1 (PGM1) deficiency is a recently defined disease characterized by glycogenosis and a congenital glycosylation disorder caused by recessive mutations in the PGM1 gene. We report a case of a 12-year-old boy with first-cousin parents who was diagnosed with a PGM1 deficiency due to significantly decreased PGM1 activity in his muscle. However, Sanger sequencing revealed no pathogenic mutation in the PGM1 gene in this patient. As this case presented with a cleft palate in addition to hypoglycemia and elevated transaminases and creatine kinase, karyotyping was performed and identified homozygous inv(1)(p31.1p32.3). Based on the chromosomal location of the PGM1 gene at 1p31, we analyzed the breakpoint of the inversion. Fluorescence in situ hybridization (FISH) combined with long PCR analysis revealed that the inversion disrupts the PGM1 gene within intron 1. Since the initiation codon in the PGM1 gene is located within exon 1, we speculated that this inversion inactivates the PGM1 gene and was therefore responsible for the patient's phenotype. When standard molecular testing fails to reveal a mutation despite a positive clinical and biochemical diagnosis, the presence of a gross structural variant that requires karyotypic examination must be considered.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is an X-linked recessive disorder involving a defect in the urea cycle caused by OTC gene mutations. Although a total of 417 disease-causing mutations in OTC have been reported, structural abnormalities in this gene are rare. We here describe a female OTCD case caused by an exonic duplication of the OTC gene (exons 1-6). CASE PRESENTATION: A 23-year-old woman with late-onset OTCD diagnosed by biochemical testing was subjected to subsequent genetic testing. Sanger sequencing revealed no pathogenic mutation throughout the coding exons of the OTC gene, but multiplex ligation-dependent probe amplification (MLPA) revealed duplication of exons 1-6. Further genetic analyses revealed an inversion of duplicated exon 1 and a tandem duplication of exons 2-6. Each of the junctions of the inversion harbored a microhomology and non-templated microinsertion, respectively, suggesting a replication-based mechanism. The duplication was also of de novo origin but segregation analysis indicated that it took place in the paternal chromosome. CONCLUSION: We report the first OTCD case harboring an exonic duplication in the OTC gene. The functional defects caused by this anomaly were determined via structural analysis of its complex rearrangements.