Duchenne muscular dystrophy-like phenotype in an LGMD2I patient with novel FKRP gene variants.

A 32-year-old man initially received a diagnosis of Duchenne muscular dystrophy (DMD). Genetic analysis revealed two novel heterozygous FKRP variants: c.169G>A (p.Glu57Lys) and c.692G>A (p.Trp231*). These results indicated that the patient had limb-girdle muscular dystrophy type 2I (LGMD2I) caused by recessive FKRP variants. Patients with LGMD2I and DMD have many overlapping phenotypes. LGMD2I should be considered in patients who have a DMD phenotype but not a DMD pathogenic variant.

Comparison of Causative Variant Prioritization Tools Using Next-generation Sequencing Data in Japanese Patients with Mendelian Disorders.

BACKGROUND: During the investigation of causative variants of Mendelian disorders using next-generation sequencing, the enormous number of possible candidates makes the detection process complex, and the use of multidimensional methods is required. Although the utility of several variant prioritization tools has been reported, their effectiveness in Japanese patients remains largely unknown. METHODS: We selected 5 free variant prioritization tools (PhenIX, hiPHIVE, Phen-Gen, eXtasy-order statistics, and eXtasy-combined max) and assessed their effectiveness in Japanese patient populations. To compare these tools, we conducted 2 studies: one based on simulated data of 100 diseases and another based on the exome data of 20 in-house patients with Mendelian disorders. To this end we selected 100 pathogenic variants from the "Database of Pathogenic Variants (DPV)" and created 100 variant call format (VCF) files that each had pathogenic variants based on reference human genome data from the 1000 Genomes Project. The later "in-house" study used exome data from 20 Japanese patients with Mendelian disorders. In both studies, we utilized 1-5 terms of "Human Phenotype Ontology" as clinical information. RESULTS: In our analysis based on simulated disease data, the detection rate of the top 10 causative variants was 91% for hiPHIVE, and 88% for PhenIX, based on 100 sets of simulated disease VCF data. Also, both software packages detected 82% of the top 1 causative variants. When we used data from our in-house patients instead, we found that these two programs (PhenIX and hiPHIVE) produced higher detection rates than the other three systems in our study. The detection rate of the top 1 causative variant was 71.4% for PhenIX, 65.0% for hiPHIVE. CONCLUSION: The rates of detecting causative variants in two Exomizer software packages, hiPHIVE and PhenIX, were higher than for the other three software systems we analyzed, with respect to Japanese patients.

Recurrent Erythema Nodosum in a Child with a SHOC2 Gene Mutation.

We report the case of a 6-year-old male who developed recurrent erythema nodosum (EN) at the age of 3 years. The patient exhibited hypertelorism, low-set ears, micrognathia, moderate intellectual disability, thin long fingers, loose anagen hair, and prominent palmoplantar wrinkles. A heterozygous single nucleotide variant in the SHOC2 gene (c.4 A > G, p.S2G) was identified. Patients with a SHOC2 mutation exhibit a unique combination of ectodermal abnormalities including darkly pigmented skin and loose anagen hair. This report is the first to describe EN in a patient with SHOC2 mutation, and to examine the patient's hair using scanning electron microscopy. We hypothesize that the RAS/MAPK pathway is associated with the pathogenesis of cutaneous lesions in patients with SHOC2 mutations via autoinflammation and disturbance of epithelial stem cells.

Pharmacoresistant epileptic eyelid twitching in a child with a mutation in SYNGAP1.

SYNGAP1 gene mutation has been associated with epilepsy which is often drug resistant, with seizure types including eyelid myoclonia. However, detailed descriptions, including ictal video-EEG, have not been reported. We report the case of a 4-year-old boy who developed recurrent epileptic eyelid twitching at 1 year and 5 months of age. Seizures gradually increased in frequency to more than 50 times per day and manifested with upward eye deviation, motion arrest, loss of consciousness, and eyelid twitching lasting for five seconds. Ictal EEG showed rhythmic, generalized slow or spike-and-wave complex activity with posterior predominance. Moderate psychomotor developmental delay and unsteady gait were also noted. Neuroimaging results were normal. Seizures were refractory to carbamazepine and levetiracetam but were reduced in frequency by ethosuximide and lamotrigine administration. Genetic analysis identified a c.3583-6 G>A mutation in the SYNGAP1 gene. SYNGAP1 gene analysis should be considered for intellectually disabled patients with early-onset drug resistant eyelid twitching and photosensitivity. Further clinical research on SYNGAP1 function may be necessary to treat epilepsy of this aetiology. [Published with video sequence on www.epilepticdisorders.com].

The presence of oxidized phosphatidylserine on Fas-mediated apoptotic cell surface.

A growing body of evidence suggests that phosphatidylserine (PS) oxidation is linked with its transmembrane migration from the inner to the outer leaflet of the plasma membrane during apoptosis. However, there is no direct evidence for the presence of oxidized PS (PSox) on the surface of cells undergoing apoptosis. The present study was performed to detect PSox externalized to the cell surface after Fas engagement in Jurkat cells. Treatment of Jurkat cells with anti-Fas antibody induced caspase-3 activation, chromatin condensation, PS externalization, generation of reactive oxygen species, intracellular glutathione depletion, disruption of mitochondrial transmembrane potential and release of cytochrome c from mitochondria. To determine externalized PS and phosphatidylethanolamine (PE), Jurkat cells were treated with anti-Fas antibody and then labeled with membrane-impermeable fluorescamine, a probe for visualizing lipids that contain primary amino groups. Their total lipids were extracted and subjected to two-dimensional high-performance thin-layer chromatography (HPTLC). The HPTLC plate was sprayed with N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride to detect phospholipid hydroperoxides. PSox was present in small amounts within but not on the surface of normal cells. Treatment with anti-Fas antibody increased PSox within the cells and caused PSox to appear on the cell surface. In contrast, PE on the surface of Fas-ligated cells was not oxidized. Thus, the present study demonstrates for the first time the presence of PSox both within and on the surface of apoptotic cells.

Geranylgeranylacetone protects against acetaminophen-induced hepatotoxicity by inducing heat shock protein 70.

Geranylgeranylacetone (GGA), an anti-ulcer drug, has been reported to induce heat shock protein (HSP) 70 in several animal organs. The present study was performed to determine whether GGA protects mouse liver against acetaminophen (APAP)-induced injury and whether it has potential as a therapeutic agent for APAP overdose. Hepatic damage was induced by single oral administration of APAP (500 mg/kg). GGA at 400 mg/kg was given orally 4 or 8h before, or 0.5h after APAP administration. Treatment of mice with GGA 4h before or 0.5h after APAP administration suppressed increases in transaminase activities and ammonia content in blood as well as hepatic necrosis. Such GGA treatment significantly increased hepatic HSP70 accumulation after APAP administration. Furthermore, GGA inhibited increases in hepatic lipid peroxide content and hepatic myeloperoxidase activity after APAP administration. In contrast, GGA neither inhibited hepatic cytochrome P450 2E1 activity nor suppressed hepatic glutathione depletion after APAP administration. The protective effect of GGA treatment 4h before APAP on hepatotoxicity induced by APAP was completely inhibited with quercetin, known as an HSP inhibitor. In conclusion, GGA has been identified as a new antidote to APAP injury, acting by induction of HSP70. The potential of GGA as a therapeutic tool is strongly supported by its ability to inhibit hepatic injury even when administered after ingestion of APAP.

Clinical Diagnosis of Mendelian Disorders Using a Comprehensive Gene-Targeted Panel Test for Next-Generation Sequencing.

BACKGROUND: Genetic diagnoses provide beneficial information to patients and families. However, traditional genetic diagnoses are often difficult even for experienced clinicians and require recognition of characteristic patterns of signs or symptoms to guide targeted genetic testing for the confirmation of diagnoses. Next-generation sequencing (NGS) is a powerful genetic diagnostic tool. However, whole-genome and whole-exome sequencing (WES) are expensive, and the interpretation of results is difficult. Hence, target gene capture sequencing of gene panels has recently been applied to genetic diagnoses. Herein, we demonstrate that targeted sequencing approaches using gene panel testing are highly efficient for the diagnosis of Mendelian disorders. METHODS: NGS using TruSight one gene panel was performed in 17 families and 20 patients, and we developed a bioinformatic pipeline at our institution for detecting mutations. RESULTS: We detected causative mutations in 6 of 17 (35%) families. In particular, 11 (65%) families had syndromic diagnosis and 6 (35%) had no syndromic diagnosis before NGS testing. The number of positive diagnoses was 5 of 11 (45%) in the syndromic group and were 1 of 6 (17%) among patients of the no syndromic diagnosis group. CONCLUSION: Diagnostic yields in the present study were higher than in previous reports of genetic and chromosomal tests and WES. The present comprehensive gene-targeted panel test is a powerful diagnostic tool for Mendelian disorders.

Potential roles of hepatic heat shock protein 25 and 70i in protection of mice against acetaminophen-induced liver injury.

The aim of the present study was to assess the contribution of the level of expression of heat shock protein 25 (HSP25), 60 (HSP60), 70 (HSC70) and 70i (HSP70i) in mouse livers after a lethal dose of acetaminophen (APAP) to their survival. We examined changes in survival ratio, plasma APAP level and alanine aminotransferase (ALT) activity, and hepatic reduced glutathione (GSH), HSP25, HSP60, HSC70 and HSP70i levels following treatment of mice with APAP (500 mg/kg, p.o.). The plasma APAP level increased rapidly, and reached a maximum 0.5 h after APAP treatment. Hepatic GSH decreased rapidly, and was almost completely depleted 1 h after APAP treatment. Plasma ALT activity, an index of liver injury, significantly increased from 3 h onwards after APAP treatment. The survival ratios 9 h, 24 h and 48 h after APAP treatment were 96%, 38% and 36%, respectively. We found a remarkable difference in the patterns of hepatic HSP25 and HSP70i induction in mice that survived after APAP treatment. HSP70i levels increased from 1 h onwards after APAP treatment in a time-dependent manner, and reached a maximum at 9 h. In contrast, HSP25 could be detected just 24 h after APAP treatment, and maximal accumulation was observed at 48 h. Other HSPs examined were unchanged. Notably, the survival ratio dropped by only 2% after HSP25 expression. Recently, a novel role for HSP25 as an anti-inflammatory factor was suggested. We have already shown that 48-h treatment with APAP induces severe centrilobular necrosis with inflammatory cell infiltration in mouse livers. Taken together, the level of expression of hepatic HSP25 may be a crucial determinant of the fate of mice exposed to APAP insult.

Endogenously generated hydrogen peroxide is required for execution of melphalan-induced apoptosis as well as oxidation and externalization of phosphatidylserine.

Hydrogen peroxide (H(2)O(2)) is generated endogenously during execution of both intrinsic as well as extrinsic apoptotic programs suggesting that it may function as a secondary messenger in apoptotic pathways. In the present study, we investigated the role of endogenously generated H(2)O(2) by using two cell lines-HL-60 cells and its subclone, H(2)O(2) resistant HP100 cells overexpressing catalase (CAT). With the exception of CAT, we found no differences in the expression of other primary antioxidant enzymes (Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase) or apoptosis-related proteins (Bcl-2 and Bax) in HP100 cells as compared with the parental HL-60 cells. Production of H(2)O(2) was readily detectable as early as 1 h after melphalan (Mel) exposure of HL-60 cells but not HP-100 cells. Biomarkers of apoptosis, such as release of cytochrome c, disruption of mitochondrial transmembrane potential, caspase-3 activation, and chromatin condensation, became apparent much later, 3 h and onward after Mel treatment of HL-60 cells. The emergence of essentially all biomarkers of apoptosis was dramatically delayed in HP100 cells as compared with HL-60 cells. A relatively minor phospholipid species, phosphatidylserine (PS), was markedly oxidized 3 h after Mel treatment in HL-60 cells (but not in HP100 cells) where it was significantly inhibited by exogenously added CAT. The two most abundant classes of membrane phospholipids, phosphatidylcholine and phosphatidyletanolamine, did not undergo any significant oxidation. PS oxidation took place 3 h after exposure of HL-60 cells to Mel and paralleled the appearance of cytochrome c in the cytosol. Neither cytochrome c release nor PS oxidation occurred in Mel-treated HP100 cells, indicating that both endogenous H(2)O(2) and cytochrome c were essential for selective PS oxidation detected in HL-60 cells. Mel-induced PS oxidation was also associated with externalization of PS on the surface of HL-60 cells. Given that 3-amino-1,2,4-triazole, a CAT inhibitor, suppressed the resistance of HP100 cells to apoptosis, production of reactive oxygen species, PS oxidation, and PS externalization induced by Mel, the results from the present study suggest that H(2)O(2) is critical for triggering the Mel-induced apoptotic program as well as PS oxidation and externalization.