Differential metabolic secretion between muscular dystrophy mouse-derived spindle cell sarcomas and rhabdomyosarcomas drives tumor type development.

The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.

Longitudinal data of serum creatine kinase levels and motor, pulmonary, and cardiac functions in 337 patients with Duchenne muscular dystrophy.

INTRODUCTION/AIMS: Duchenne muscular dystrophy (DMD) presents with skeletal muscle weakness, followed by cardiorespiratory involvement. The need for longitudinal data regarding DMD that could serve as a control for determining treatment efficacy in clinical trials has increased notably. The present study examined the longitudinal data of Japanese DMD patients collectively and assessed individual patients with pathogenic variants eligible for exon-skipping therapy. METHODS: Patients with DMD who visited Kobe University Hospital between March 1991 and March 2019 were enrolled. Data between the patients' first visit until age 20 years were examined. RESULTS: Three hundred thirty-seven patients were included. Serum creatine kinase levels showed extremely high values until the age of 6 years and a rapid decline from ages 7-12 years. Both the median 10-m run/walk velocity and rise-from-floor velocity peaked at the age of 4 years and declined with age. The values for respiratory function declined from the age of 11 years. The median left ventricular ejection fraction was >60% until the age of 12 years and rapidly declined from ages 13-15 years. Examination of the relationship between pathogenic variants eligible for exon-skipping therapy and longitudinal data revealed no characteristic findings. DISCUSSION: We found that creatine kinase levels and motor, respiratory, and cardiac functions each exhibited various changes over time. These findings provide useful information about the longitudinal data of several outcome measures for patients with DMD not receiving corticosteroids. These data may serve as historical controls in comparing the natural history of DMD patients not on regular steroid use in appropriate clinical trials.

An adolescent case of ASXL3-related disorder with delayed onset of feeding difficulty.

BACKGROUND: ASXL3-related disorder, first described in 2013, is a genetic disorder with an autosomal dominant inheritance that is caused by a heterozygous loss-of-function variant in ASXL3. The most characteristic feature is neurodevelopmental delay with consistently limited speech. Feeding difficulty is a main symptom observed in infancy. However, no adolescent case has been reported. CASE PRESENTATION: A 14-year-old girl with ASXL3-related syndrome was referred to our hospital with subacute onset of emotional lability. Limbic encephalitis was ruled out by examination; however, the patient gradually showed a lack of interest in eating, with decreased diet volume. Consequently, she experienced significant weight loss. She experienced no symptoms of bulimia, or food allergy; therefore, avoidant/restrictive food intake disorder (ARFID) was clinically suspected. CONCLUSIONS: We reported the first case of ASXL3-related disorder with adolescent onset of feeding difficulty. ARFID was considered a cause of the feeding difficulty.

Spinal Muscular Atrophy: The Past, Present, and Future of Diagnosis and Treatment.

Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance. The first cases of SMA were reported by Werdnig in 1891. Although the phenotypic variation of SMA led to controversy regarding the clinical entity of the disease, the genetic homogeneity of SMA was proved in 1990. Five years later, in 1995, the gene responsible for SMA, SMN1, was identified. Genetic testing of SMN1 has enabled precise epidemiological studies, revealing that SMA occurs in 1 of 10,000 to 20,000 live births and that more than 95% of affected patients are homozygous for SMN1 deletion. In 2016, nusinersen was the first drug approved for treatment of SMA in the United States. Two other drugs were subsequently approved: onasemnogene abeparvovec and risdiplam. Clinical trials with these drugs targeting patients with pre-symptomatic SMA (those who were diagnosed by genetic testing but showed no symptoms) revealed that such patients could achieve the milestones of independent sitting and/or walking. Following the great success of these trials, population-based newborn screening programs for SMA (more precisely, SMN1-deleted SMA) have been increasingly implemented worldwide. Early detection by newborn screening and early treatment with new drugs are expected to soon become the standards in the field of SMA.

Identification of plexin D1 on circulating extracellular vesicles as a potential biomarker of polymyositis and dermatomyositis.

OBJECTIVES: We aimed to identify disease-specific surface proteins on extracellular vesicles (EVs) as novel serum biomarkers of PM/DM. METHODS: We performed liquid chromatography-tandem mass spectrometry (LC/MS) on purified EVs from sera of 10 PM/DM patients, 23 patients with other autoimmune diseases and 10 healthy controls (HCs). We identified membrane proteins preferentially present in EVs of PM/DM patients by bioinformatics and biostatistical analyses. We developed an EV sandwich ELISA for directly detecting serum EVs expressing disease-specific membrane proteins and evaluated their clinical utility using sera from 54 PM/DM, 24 RA, 20 SLE, 13 SSc and 25 Duchenne and Becker types of muscular dystrophy (DMD/BMD) patients and 36 HCs. RESULTS: LC/MS analysis identified 1220 proteins in serum EVs. Of these, plexin D1 was enriched in those from PM/DM patients relative to HCs or patients without PM/DM. Using a specific EV sandwich ELISA, we found that levels of plexin D1+ EVs in serum were significantly greater in PM/DM patients than in HCs or RA, SLE or DMD/BMD patients. Serum levels of plexin D1+ EVs were greater in those PM/DM patients with muscle pain or weakness. Serum levels of plexin D1+ EVs were significantly correlated with levels of aldolase (rs = 0.481), white blood cells (rs = 0.381), neutrophils (rs = 0.450) and platelets (rs = 0.408) in PM/DM patients. Finally, serum levels of plexin D1+ EVs decreased significantly in patients with PM/DM in clinical remission after treatment. CONCLUSION: We identified levels of circulating plexin D1+ EVs as a novel serum biomarker for PM/DM.

Magneto-optical diffractive deep neural network.

We propose a magneto-optical diffractive deep neural network (MO-D2NN). We simulated several MO-D2NNs, each of which consists of five hidden layers made of a magnetic material that contains 100 × 100 magnetic domains with a domain width of 1 µm and an interlayer distance of 0.7 mm. The networks demonstrate a classification accuracy of > 90% for the MNIST dataset when light intensity is used as the classification measure. Moreover, an accuracy of > 80% is obtained even for a small Faraday rotation angle of π/100 rad when the angle of polarization is used as the classification measure. The MO-D2NN allows the hidden layers to be rewritten, which is not possible with previous implementations of D2NNs.

Detecting pathogenic deep intronic variants in Gitelman syndrome.

Gitelman syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by loss of function in the SLC12A3 gene (NM_000339.2), which encodes the natrium chloride cotransporter. The detection of homozygous or compound heterozygous SLC12A3 variants is expected in GS, but 18%-40% of patients with clinical GS carry only one mutant allele. Previous reports identified some pathogenic deep intronic variants in SLC12A3. Here, we report the screening of SLC12A3 deep intronic variants in 13 patients with suspected GS carrying one mutated SLC12A3 allele. Variant screening used the HaloPlex Target Enrichment System Kit capturing whole introns and the promotor region of SLC12A3, followed by SureCall variant analysis. Rare intronic variants (<1% frequency) were identified, and pathogenicity evaluated by the minigene system. Deep intronic variant screening detected seven rare SLC12A3 variants from six patients. Only one variant showed pathogenicity in the minigene system (c.602-16G>A, intron 4) through activation of a cryptic acceptor site. No variants were detected in the promotor region. Deep intronic screening identified only one pathogenic variant in patients with suspected GS carrying monoallelic SLC12A3 variants. Our results suggest that deep intronic variants partially explain the cause of monoallelic variants in patients with GS.

Serum cardiac troponin I is a candidate biomarker for cardiomyopathy in Duchenne and Becker muscular dystrophies.

INTRODUCTION/AIMS: Serum cardiac troponin I (cTnI), its relation to cardiomyopathy, and the contribution of the ACTN3 genotype to serum levels of cTnI in Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) remain unknown. In this study we aimed to reveal the characteristics of cTnI, assess whether cTnI is a biomarker for cardiomyopathy in these dystrophinopathies, and evaluate the contribution of the ACTN3 genotype to the serum levels of cTnI in DMD patients. METHODS: Serum cTnI values obtained from 127 DMD and 47 BMD patients were analyzed retrospectively. The relationship between cTnI and echocardiography data or the ACTN3 XX genotype was assessed. RESULTS: The cTnI levels and proportion of patients with abnormal cTnI levels were significantly higher among DMD patients than BMD, especially in the second decade of life. In DMD, the cTnI level reached a maximum at 13 years, and left ventricular ejection fraction (LVEF) became abnormal approximately 1 year subsequently. In BMD, the cTnI level peaked at the age of 14 years, and LVEF became abnormal 3 years later. Decreased LVEF was observed after cTnI elevation in both populations. cTnI levels by age in DMD patients with the ACTN3 XX genotype tended to increase significantly and early. DISCUSSION: Myocardial injury indicated by cTnI elevation was more common and severe in DMD patients. cTnI elevation preceding cardiac dysfunction may represent an early phase of cardiomyopathy progression and may be a biomarker for early detection of cardiomyopathy in these dystrophinopathies. The ACTN3 XX genotype may be a risk factor for early myocardial injury.

Assessment of catabolic state in infants with the use of urinary titin N-fragment.

BACKGROUND: Urinary titin N-fragment levels have been used to assess the catabolic state, and we used this biomarker to evaluate the catabolic state of infants. METHODS: We retrospectively measured urinary titin N-fragment levels of urinary samples. The primary outcome was its changes according to postmenstrual age. The secondary outcomes included differences between gestational age, longitudinal change after birth, influence on growth, and relationship with blood tests. RESULTS: This study included 219 patients with 414 measurements. Urinary titin N-fragment exponentially declined with postmenstrual age. These values were 12.5 (7.1-19.6), 8.1 (5.1-13.0), 12.8 (6.0-21.3), 26.4 (16.4-52.0), and 81.9 (63.3-106.4) pmol/mg creatinine in full, late, moderate, very, and extremely preterm infants, respectively (p < 0.01). After birth, urinary levels of titin N-fragment exponentially declined, and the maximum level within a week was associated with the time to return to birth weight in preterm infants (ρ = 0.39, p < 0.01). This was correlated with creatine kinase in full-term infants (ρ = 0.58, p < 0.01) and with blood urea nitrogen in preterm infants (ρ = 0.50, p < 0.01). CONCLUSIONS: The catabolic state was increased during the early course of the postmenstrual age and early preterm infants. IMPACT: Catabolic state in infants, especially in preterm infants, was expected to be increased, but no study has clearly verified this. In this retrospective study of 219 patients with 414 urinary titin measurements, the catabolic state was exponentially elevated during the early postmenstrual age. The use of the urinary titin N-fragment clarified catabolic state was prominently increased in very and extremely preterm infants.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Long spin coherence length and bulk-like spin-orbit torque in ferrimagnetic multilayers.

Spintronics relies on magnetization switching through current-induced spin torques. However, because spin transfer torque for ferromagnets is a surface torque, a large switching current is required for a thick, thermally stable ferromagnetic cell, and this remains a fundamental obstacle for high-density non-volatile applications with ferromagnets. Here, we report a long spin coherence length and associated bulk-like torque characteristics in an antiferromagnetically coupled ferrimagnetic multilayer. We find that a transverse spin current can pass through >10-nm-thick ferrimagnetic Co/Tb multilayers, whereas it is entirely absorbed by a 1-nm-thick ferromagnetic Co/Ni multilayer. We also find that the switching efficiency of Co/Tb multilayers partially reflects a bulk-like torque characteristic, as it increases with ferrimagnet thickness up to 8 nm and then decreases, in clear contrast to the 1/thickness dependence of ferromagnetic Co/Ni multilayers. Our results on antiferromagnetically coupled systems will invigorate research towards the development of energy-efficient spintronics.

The ACTN3 577XX Null Genotype Is Associated with Low Left Ventricular Dilation-Free Survival Rate in Patients with Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease caused by mutations in the DMD gene. Dilated cardiomyopathy is the leading cause of death in DMD; therefore, further understanding of this complication is essential to reduce morbidity and mortality. METHODS: A common null variant (R577X) in the ACTN3 gene, which encodes α-actinin-3, has been studied in association with muscle function in healthy individuals; however it has not yet been examined in relationship to the cardiac phenotype in DMD. In this study, we determined the ACTN3 genotype in 163 patients with DMD and examined the correlation between ACTN3 genotypes and echocardiographic findings in 77 of the 163 patients. RESULTS: The genotypes 577RR(RR), 577RX(RX) and 577XX(XX) were identified in 13 (17%), 44 (57%) and 20 (26%) of 77 patients, respectively. We estimated cardiac involvement-free survival rate analyses using Kaplan-Meier curves. Remarkably, the left ventricular dilation (> 55 mm)-free survival rate was significantly lower in patients with the XX null genotype (P < 0.01). The XX null genotype showed a higher risk for LV dilation (hazard ratio 9.04). CONCLUSIONS: This study revealed that the ACTN3 XX null genotype was associated with a lower left ventricular dilation-free survival rate in patients with DMD. These results suggest that the ACTN3 genotype should be determined at the time of diagnosis of DMD to improve patients' cardiac outcomes.

Acute rhabdomyolysis following viral infection with coxsackie A4 in a 50-day-old infant with Fukuyama congenital muscular dystrophy.

BACKGROUND: Fukuyama congenital muscular dystrophy (FCMD), which is characterized by generalized muscle weakness, hypotonia, and motor delay during early infancy, gradually progresses with advanced age. Although acute rhabdomyolysis following infection in patients with FCMD has occasionally been reported, no studies have investigated rhabdomyolysis following viral infection in FCMD patients during early infancy. CASE REPORT: We report the case of a 50-day-old girl with no apparent symptoms of muscular dystrophy who developed severe acute rhabdomyolysis caused by viral infection, resulting in quadriplegia and respiratory failure therefore requiring mechanical ventilation. Brain magnetic resonance imaging incidentally showed the typical characteristics of FCMD, and FCMD was confirmed by genetic analysis, which revealed a 3-kb retrotransposon insertion in one allele of the fukutin gene and a deep intronic splicing variant in intron 5 in another allele. The virus etiology was confirmed to be Coxsackie A4. CONCLUSION: We report a severe case of acute rhabdomyolysis with the earliest onset of symptoms due to the Coxsackie A4 virus in a patient with FCMD. The present findings indicate that physicians should consider FCMD with viral infection a differential diagnosis if the patient presents with acute rhabdomyolysis following a fever.

Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients.

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3' terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3'-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5' end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.

Identification of the shortest splice variant of Dp71, together with five known variants, in glioblastoma cells.

BACKGROUND: Dystrophin Dp71 mRNA is produced from the most distal alternative promoter of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 is characterized by a wide variety of splice variants. In addition to being associated with cognitive disturbance in patients with DMD, Dp71 may also play a role in tumorigenesis. This study analyzed Dp71 transcripts in glioblastoma, the most common and most lethal type of cerebral malignancy. METHODS: Dp71 mRNA in the U-251 glioblastoma cell line was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). The amplified products were subcloned and sequenced. RESULTS: RT-PCR amplification of the 5' end of the Dp71 transcript yielded a product of expected size, indicating transcription from the Dp71 promoter in glioblastoma. Amplification of full-length Dp71, from exon G1 to DMD exon 79, yielded a product of expected size, as well as a faint, smaller sized band. Sequencing of 17 clones revealed six different alternatively spliced variants, with only one clone being of full-length Dp71 containing all 18 exons. Ten clones lacked exon 78 (Dp71b), indicating that Dp71b was a major type of Dp71 in glioblastoma. In addition, three clones lacked both exons 71 and 78 (Dp71ab), one clone lacked exons 71, 73 and 78 (Dp71ab △73), one clone lacked exons 71-74 and 78 (Dp71bc), and one clone lacked exons 68-76 and 78 (Dp71b△68-76). This novel transcript was the shortest Dp71 variant, with a predicted stop codon in exon 77 and was predicted to produce a 24.8 kDa protein, consisting of 216 amino acids including 15 amino acids from exon 77. This novel product was classified as Dp71g because of its unique C-terminal amino acid sequence. CONCLUSIONS: Six splice variants of Dp71 were identified in glioblastoma cells, with Dp71b being the most abundant. Deletion of exon 78 was an apparent default splicing pathway in glioblastoma, being observed in 16 of 17 clones. Glioblastoma cells contained the shortest Dp71 transcript (Dp71b△68-76) identified to date, with a unique C-terminal amino acid sequence. These findings suggest the need to assess the function of Dp71 variants in glioblastoma.

A case of probable Vogt-Koyanagi-Harada disease in a 3-year-old girl.

BACKGROUND: Vogt-Koyanagi-Harada (VKH) disease is a T-cell-mediated autoimmune disorder characterized by bilateral granulomatous panuveitis with various systemic manifestations. Although VKH disease rarely occurs in the pediatric population, the clinical course tends to be aggressive, and the visual prognosis is worse than that in adult patients due to severe ocular complications secondary to recurrent inflammation. CASE PRESENTATION: A 3-year-old girl with probable VKH was referred to Kobe University Hospital. She had severe bilateral panuveitis with posterior synechiae of the iris, marked optic disk swelling, and serous retinal detachment in both eyes, and her best corrected visual acuities (BCVAs) were 20/200 OD and 20/125 OS. A third course of therapy was administered because serous retinal detachment remained after two courses of therapy. She was treated with three courses of high-dose intravenous corticosteroid therapy, followed by slow tapering of oral corticosteroids. Her BCVAs recovered to 20/16 OU, and relapse of ocular inflammation and side effect of treatment were not observed during the 1.5-year follow-up period. CONCLUSIONS: We experienced a pediatric patient with probable VKH disease who was treated with three courses of high-dose intravenous corticosteroid therapy. With the favorable clinical course in our patient, initial treatment with repeated high-dose intravenous corticosteroid therapy might be beneficial in pediatric VKH disease.

Renal insufficiency mimicking glutaric acidemia type 1 on newborn screening.

BACKGROUND: Glutaryl carnitine (C5DC) in dried blood spots is used as a biomarker for glutaric aciduria type 1 (GA-1) screening. C5DC, however, is the only screening marker for this condition, and various pathological conditions may interfere with C5DC metabolism. Recently, C5DC elevation has been reported in cases of renal insufficiency. METHOD: Five patients who were positive for GA-1 on newborn screening with tandem mass spectrometry between September 2012 and March 2015 at Kobe University Hospital were enrolled in this study. RESULTS: GA-1 was not confirmed on urinary organic acids analysis in any of the patients. C5DC decreased immediately in four patients, but one patient, who had high C5DC for at least 4 months, was diagnosed with bilateral renal hypoplasia. CONCLUSION: In the case of persistently elevated C5DC, renal insufficiency should be considered as a differential diagnosis.

Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System.

BACKGROUND: Dystrophin Dp71 is one of the isoforms produced by the DMD gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle. METHODS: Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMDmdx mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively. RESULTS: Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression. CONCLUSIONS: Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.

A de novo 50-bp GNAS Intragenic Duplication in a Patient with Pseudohypoparathyroidism Type 1a.

Germline intragenic mutations in the GNAS locus result in pseudohypoparathyroidism type 1a (PHP1a) and related conditions. Nearly half of the previously reported GNAS intragenic mutations were structural variants, including 3 tandem duplications of 12-25 bp. However, the precise mutation spectrum and the genomic basis of GNAS structural variants remain to be clarified. Here, we report a de novo 50-bp tandem duplication in GNAS (c.723_772dup50, p.Glu259Leufs*29) identified in a patient with typical clinical features of PHP1a. The mutant transcript was predicted to undergo mRNA decay or encode a nonfunctional protein. The 2 breakpoints of the duplication shared a 1-bp microhomology but were not associated with long homology or nucleotide stretches. We also examined the breakpoint structures of 3 previously reported GNAS duplications and found that 1 had a structure similar to that of our case, while the remaining 2 had blunt-ended breakpoints without microhomologies. In silico analyses revealed that the GNAS-flanking region was not enriched with repeats, palindromes, noncanonical DNA motifs, or GC content. This study expands the mutation spectrum of GNAS and provides the first indication that GNAS intragenic structural variants are induced by multiple processes, including nonhomologous end-joining and/or microhomology-mediated break-induced replication, independently of known rearrangement-inducing DNA features.

Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G>A mutation identified by NGS.

Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.