Antisense oligonucleotide induced pseudoexon skipping and restoration of functional protein for Fukuyama muscular dystrophy caused by a deep-intronic variant.

Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3'-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2'-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, antisense oligonucleotide-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.One Sentence Summary Antisense oligonucleotide treatment restored normal FKTN protein production and functional O-mannosyl glycosylation of alpha-dystroglycan via pseudoexon skipping in patient-derived cells carrying the compound heterozygous deep-intronic variant of Fukuyama muscular dystrophy.

Breakpoints in complex chromosomal rearrangements correspond to transposase-accessible regions of DNA from mature sperm.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

Congenital hearing impairment associated with peripheral cochlear nerve dysmyelination in glycosylation-deficient muscular dystrophy.

Hearing loss (HL) is one of the most common sensory impairments and etiologically and genetically heterogeneous disorders in humans. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. Aberrant glycosylation of α-dystroglycan causes at least eighteen subtypes of MD, now categorized as MD-dystroglycanopathy (MD-DG), with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes and increasing evidence regarding their molecular pathogeneses, no comprehensive study has investigated sensorineural HL (SNHL) in MD-DG. Here, we found that two mouse models of MD-DG, Largemyd/myd and POMGnT1-KO mice, exhibited congenital, non-progressive, and mild-to-moderate SNHL in auditory brainstem response (ABR) accompanied by extended latency of wave I. Profoundly abnormal myelination was found at the peripheral segment of the cochlear nerve, which is rich in the glycosylated α-dystroglycan-laminin complex and demarcated by "the glial dome." In addition, patients with Fukuyama congenital MD, a type of MD-DG, also had latent SNHL with extended latency of wave I in ABR. Collectively, these findings indicate that hearing impairment associated with impaired Schwann cell-mediated myelination at the peripheral segment of the cochlear nerve is a notable symptom of MD-DG.

A case of a parthenogenetic 46,XX/46,XY chimera presenting ambiguous genitalia.

Sex-chromosome discordant chimerism (XX/XY chimerism) is a rare chromosomal disorder in humans. We report a boy with ambiguous genitalia and hypospadias, showing 46,XY[26]/46,XX[4] in peripheral blood cells. To clarify the mechanism of how this chimerism took place, we carried out whole-genome genotyping using a SNP array and microsatellite analysis. The B-allele frequency of the SNP array showed a mixture of three and five allele combinations, which excluded mosaicism but not chimerism, and suggested the fusion of two embryos or a shared parental haplotype between the two parental cells. All microsatellite markers showed a single maternal allele. From these results, we concluded that this XX/XY chimera is composed of two different paternal alleles and a single duplicated maternal genome. This XX/XY chimera likely arose from a diploid maternal cell that was formed via endoduplication of the maternal genome just before fertilization, being fertilized with both X and Y sperm.

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.

De novo variants in RHOBTB2, an atypical Rho GTPase gene, cause epileptic encephalopathy.

By whole exome sequencing, we identified three de novo RHOBTB2 variants in three patients with epileptic encephalopathies (EEs). Interestingly, all three patients showed acute encephalopathy (febrile status epilepticus), with magnetic resonance imaging revealing hemisphere swelling or reduced diffusion in various brain regions. RHOBTB2 encodes Rho-related BTB domain-containing protein 2, an atypical Rho GTPase that is a substrate-specific adaptor or itself is a substrate for the Cullin-3 (CUL3)-based ubiquitin ligase complex. Transient expression experiments in Neuro-2a cells revealed that mutant RHOBTB2 was more abundant than wild-type RHOBTB2. Coexpression of CUL3 with RHOBTB2 decreased the level of wild-type RHOBTB2 but not the level of any of the three mutants, indicating impaired CUL3 complex-dependent degradation of the three mutants. These data indicate that RHOBTB2 variants are a rare genetic cause of EEs, in which acute encephalopathy might be a characteristic feature, and that precise regulation of RHOBTB2 levels is essential for normal brain function.

Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease.

The pattern of X-chromosome inactivation (XCI) can affect the clinical severity of X-linked disorders in females. XCI pattern analysis has been conducted mainly by HUMARA assay, a polymerase chain reaction-based assay using a methylation-sensitive restriction enzyme. However, this assay examines the XCI ratio of the androgen receptor gene at the genomic DNA level and does not reflect the ratio of either targeted gene directly or at the mRNA level. Here, we report four females with Dent disease, and we clarified the correlation between XCI and female cases of Dent disease using not only HUMARA assay but also a novel analytical method by RNA sequencing. We constructed genetic analysis for 4 female cases showing high level of urinary low-molecular-weight proteinuria and their parents. Their XCI pattern was analyzed by both HUMARA assay and an ultra-deep targeted RNA sequencing of the CLCN5 gene using genomic DNA and mRNA extracted from both leukocytes and urine sediment. All four cases possessed pathogenic variants of the CLCN5 gene. XCI analysis revealed skewed XCI in only two cases, while the other two showed random XCI. All assay results of HUMARA and targeted RNA sequencing in both leukocytes and urinary sediment were clearly identical in all four cases. We developed a novel XCI analytical assay of ultra-deep targeted RNA sequencing and revealed that skewed XCI explains the mechanism of onset of female Dent disease in only half of such cases.

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome.

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins.

Current Situation of Treatment for Anaphylaxis in a Japanese Pediatric Emergency Center.

OBJECTIVE: Anaphylaxis is a systemic allergic reaction that sometimes requires prompt treatment with intramuscular adrenaline. The aim of the study was to investigate the current situation regarding anaphylaxis treatment in a representative pediatric primary emergency facility in Japan. METHODS: We retrospectively examined the medical records dating from April 2011 through March 2014 from Kobe Children's Primary Emergency Medical Center, where general pediatricians work on a part-time basis. Clinical characteristics and current treatments for patients with anaphylaxis who presented to the facility were investigated. Furthermore, we compared the clinical characteristics between anaphylaxis patients given intramuscular adrenaline and those not given it. RESULTS: During the study period, 217 patients were diagnosed with anaphylaxis. The median Sampson grade at the time of visit was 2, and 90 patients (41%) were grade 4 or higher. No patients received self-intramuscular injected adrenaline before arrival at our emergency medical center because none of the patients had been prescribed it. Further treatment during the visit was provided to 128 patients (59%), with only 17 (8%) receiving intramuscular adrenaline. Patients given intramuscular adrenaline had significantly lower peripheral saturation of oxygen at the visit (P = 0.025) and more frequent transfer to a referral hospital (P < 0.001) than those not given intramuscular adrenaline. CONCLUSIONS: Education for Japanese pediatric practitioners and patients is warranted, because no patients used self-intramuscular injected adrenaline as a prehospital treatment for anaphylaxis, and only severely affected patients who needed oxygen therapy or hospitalization received intramuscular adrenaline in a pediatric primary emergency setting.

Clinical factors associated with prehospital exacerbation of anaphylaxis in children.

BACKGROUND: The severity of anaphylaxis often varies with time. Because prehospital intervention and initial treatment at hospital are affected by changing symptoms, the aim of this study was to determine the clinical factors associated with prehospital remission and exacerbation in the course of anaphylaxis in children. METHODS: Data from medical records on anaphylactic children who were treated for 3 years at Kobe Children's Primary Emergency Medical Center were retrospectively analyzed. Severity of symptoms was evaluated using Sampson's grade (S-G). Patients with increased S-G at the hospital visit from disease onset (worsened group) were compared with those with decreased S-G at the visit (improved group). Uni- and multivariate analyses were performed to identify clinical differences between the groups, with P<0.05 considered statistically significant. RESULTS: Among 115 anaphylactic children who showed S-G changes from onset to hospital visit, 43 were assigned to the worsened group and 72 to the improved group. Univariate analysis showed no significant differences in age, sex, history of asthma, prehospital treatment, type of antigen, or period from symptom onset to hospital visit between the groups. However, the time from antigen exposure to symptom onset was significantly longer, and S-G at onset was significantly lower in the worsened group than in the improved group. Multivariate analysis identified time from antigen exposure to symptom onset (odds ratio: 3.89, P<0.01) and S-G at onset (odds ratio: 0.06, P<0.001) as independent predictors of exacerbation. CONCLUSIONS: Anaphylactic children with slower and milder symptoms at onset are more likely to show deterioration.

Deep-intronic variant of fukutin is the most prevalent point mutation of Fukuyama congenital muscular dystrophy in Japan.

Fukuyama congenital muscular dystrophy (FCMD), which is caused by mutations in the fukutin gene, is the second most common form of childhood muscular dystrophy in Japan. The founder haplotype is the most prevalent in the chromosomes of Japanese FCMD patients, and corresponds to an SVA retrotransposal insertion in the 3'-untranslated region of fukutin. Although other mutations have been reported, the mutation corresponding to the second most prevalent haplotype in Japanese FCMD patients remained unknown. Recently a deep-intronic point mutation c.647+2084G>T was identified in Korean patients with congenital muscular dystrophy. Here, we performed mutational analysis of 10 patients with the second most prevalent haplotype and found that all of them were compound-heterozygous for the SVA insertion and this c.647+2084G>T mutation. The fukutin mRNA of these patients contained a pseudoexon between exon 5 and exon 6, which was consistent with the previous Korean study. As expected, the mutated fukutin protein was smaller than the normal protein, reflecting the truncation of fukutin due to a premature stop codon. Immunostaining analysis showed a decrease in the signal for the glycosylated form of α-dystroglycan. These findings indicated that this mutation is the second most prevalent loss-of-function mutation in Japanese FCMD patients.

Corrigendum: Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

This corrects the article DOI: 10.1038/jhg.2017.48.

Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

Adams-Oliver syndrome (AOS, OMIM; 100300) is a rare genetic disease characterized by aplasia cutis congenita, terminal transverse limb defects and cutis marmorata with vascular anomalies such as congenital heart defects. The etiology of this syndrome has remained largely unknown but defective Notch signaling during vascular formation has been suggested. Here we describe a sporadic Japanese newborn case with clinically diagnosed AOS. Trio whole-exome sequencing identified a de novo, novel, heterozygous missense mutation in the Delta-like 4 ligand gene (DLL4 c.572G>A, p.Arg191His) in the patient. DLL4 functions as a requisite ligand for NOTCH1 receptor, which is essential for vascular formation. Amino acid substitution of Arg191 to His was predicted by molecular models to interfere with direct binding between DLL4 and NOTCH1. DLL4 has recently been identified as a causative gene of an autosomal dominant type of AOS with milder symptoms. The case described here showed gradual recovery from skull defects after birth and no psychomotor developmental delay has been observed. This is the second report of an AOS case with DLL4 mutation, and the phenotypic characteristics between the two cases are compared and discussed.

Cryptic exon activation in SLC12A3 in Gitelman syndrome.

Gitelman syndrome (GS) is an autosomal recessive renal tubulopathy characterized by hypokalemic metabolic alkalosis with hypocalciuria and hypomagnesemia. GS clinical symptoms range from mild weakness to muscular cramps, paralysis or even sudden death as a result of cardiac arrhythmia. GS is caused by loss-of-function mutations in the solute carrier family 12 member 3 (SLC12A3) gene, but molecular mechanisms underlying such a wide range of symptoms are poorly understood. Here we report cryptic exon activation in SLC12A3 intron 12 in a clinically asymptomatic GS, resulting from an intronic mutation c.1669+297 T>G that created a new acceptor splice site. The cryptic exon was sandwiched between the L3 transposon upstream and a mammalian interspersed repeat downstream, possibly contributing to inclusion of the cryptic exon in mature transcripts. The mutation was identified by targeted next-generation sequencing of candidate genes in GS patients with missing pathogenic SLC12A3 alleles. Taken together, this work illustrates the power of next-generation sequencing to identify causal mutations in intronic regions in asymptomatic individuals at risk of developing potentially fatal disease complications, improving clinical management of these cases.

Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.

Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Rare renal ciliopathies in non-consanguineous families that were identified by targeted resequencing.

BACKGROUND: Nephronophthisis-related ciliopathies (NPHP-RC) are a frequent cause of renal failure for children and adolescents. Although diagnosing these diseases clinically is difficult, a comprehensive genetic screening approach of targeted resequencing can uncover the genetic background in this complicated family of diseases. METHODS: We studied three Japanese female patients with renal insufficiency from non-consanguineous parents. A renal biopsy for clinical reasons was not performed. Therefore, we did not know the diagnosis of these patients from a clinical aspect. We performed comprehensive genetic analysis using the TruSight One Sequencing Panel next generation sequencing technique. RESULTS: We identified three different rare NPHP-RC variants in the following genes: SDCCAG8, MKKS, and WDR35. Patient 1 with SDCCAG8 homozygous deletions showed no ciliopathy-specific extrarenal manifestations, such as retinitis pigmentosa or polydactyly prior to genetic analysis. Patient 2 with a MKKS splice site homozygous mutation and a subsequent 39-amino acid deletion in the substrate-binding apical domain, had clinical symptoms of Bardet-Biedl syndrome. She and her deceased elder brother had severe renal insufficiency soon after birth. Patient 3 with a compound heterozygous WDR35 mutation had ocular coloboma and intellectual disability. CONCLUSIONS: Our results suggest that a comprehensive genetic screening system using target resequencing is useful and non-invasive for the diagnosis of patients with an unknown cause of pediatric end-stage renal disease.

Female X-linked Alport syndrome with somatic mosaicism.

BACKGROUND: X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although males with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and possess somatic mosaic variants of the type IV collagen α5 gene (COL4A5), with severity depending on variant frequencies. In females, somatic mosaic variants are rarely reported in XLAS, and it is not clear what determines severity. METHODS: Two females with somatic mosaic mutations in COL4A5 with variant frequencies of 17.9 and 22.1% were detected using the next-generation sequencing. One patient only had hematuria. The other, however, had moderate proteinuria, which is a severe phenotype for a female XLAS patient of her age. The molecular mechanisms for the severe phenotype were investigated by examining variant frequencies in urinary sediment cells and X chromosome inactivation patterns, and by looking for modifier variants in podocyte-related genes using the next-generation sequencing. RESULTS: The severe phenotype patient had a variant frequency of 36.6% in urinary sediment cells, which is not markedly high, nor did she show skewed X chromosome inactivation. However, she did have the heterozygous variant in COL4A3, which can affect severity. CONCLUSION: Factors determining severity in female XLAS patients remain unclear. One studied patient with the somatic variant in COL4A5 showed a severe phenotype without skewed X chromosome inactivation, which might be derived from digenic variants in COL4A3 and COL4A5. Further studies are required to determine molecular mechanisms behind female XLAS resulting in the severe phenotype.

Evaluation of BiliCare™ transcutaneous bilirubin device in Japanese newborns.

BACKGROUND: Non-invasive transcutaneous bilirubin (TcB) monitoring has been widely used to screen for hyperbilirubinemia. TcB measured using the recently developed BiliCare™ system, however, has not been fully evaluated. METHODS: One hundred and seven TcB measurements were obtained from 82 Japanese newborns ≥35 weeks' gestational age within 2 weeks after birth. Measurements were taken at the scaphoid fossa, conchal cavity, and lobe of the ear using BiliCare. BiliCare TcB were compared with total serum bilirubin (TB) and TcB obtained using another bilirubinometer (JM-105™). RESULTS: Transcutaneous bilirubin measured at all three sites significantly correlated with TB (r = 0.91, 0.93, and 0.93 at the scaphoid fossa, conchal cavity, and lobe, respectively). The mean differences were 0.1, -0.3, and 3.6 at the scaphoid fossa, conchal cavity, and lobe, respectively. BiliCare TcB at the scaphoid fossa significantly correlated with that using the JM-105 (r = 0.91). The mean difference was 0.0. BiliCare, however, produced a significantly higher and lower TcB than the JM-105 for TB <7 and ≥15 mg/dL, respectively. CONCLUSIONS: Transcutaneous bilirubin measurements taken at the scaphoid fossa or conchal cavity using BiliCare were more reliable than those at the earlobe. BiliCare TcB differed from those of the JM-105, for TB <7 or ≥15 mg/dL.

Changes in the numbers of patients with acute gastroenteritis after voluntary introduction of the rotavirus vaccine in a Japanese children's primary emergency medical center.

BACKGROUND: Acute gastroenteritis (AGE) is a major reason for presentation to pediatric primary emergency medical centers. Because rotavirus vaccines were introduced in November 2011 for voluntary vaccination in Japan, we analyzed the changes in the numbers of AGE patients. METHODS: The number and proportion of patients visiting Kobe children's primary emergency medical center from January 2011 to February 2015 due to AGE, out of all visiting children, were investigated retrospectively. The rotavirus and norovirus epidemic periods were defined as the periods from March to June and from November to February, respectively, based on their disease prevalence. RESULTS: In patients ≤2 years of age, the numbers and proportions of patients with AGE were significantly decreased from 2464/14098 (17%) in 2011 to 1888/12321 (15%) in 2014 (p < 0.01). In patients ≤2 and 3-5 years of age, significant decreases in AGE patients between 2011 and 2014 were observed during the rotavirus season (from 20% [1090/5329] to 14% [642/4482] in patients aged ≤2 years and from 23% [704/3047] to 20% [572/2807] in patients aged 3-5 years, p < 0.01 and p < 0.05, respectively), but not during the norovirus season (from 19% [834/4436] to 19% [797/4160] in patients aged ≤2 years and from 20% [679/3334] to 25% [710/2852] in patients aged 3-5 years). CONCLUSIONS: The estimated rotavirus vaccine coverage in our area increased from 1% in 2011 to 49% in 2014; this coverage may have resulted in a reduction in AGE patients, both directly and indirectly, in our Japanese children's primary emergency medical center.

Next-generation sequencing discloses a nonsense mutation in the dystrophin gene from long preserved dried umbilical cord and low-level somatic mosaicism in the proband mother.

Duchene muscular dystrophy (DMD) is a progressive muscle wasting disease, caused by mutations in the dystrophin (DMD) on the X chromosome. One-third of patients are estimated to have de novo mutations. To provide in-depth genetic counseling, the comprehensive identification of mutations is mandatory. However, many DMD patients did not undergo genetic diagnosis because detailed genetic diagnosis was not available or their mutational types were difficult to identify. Here we report the genetic testing of a sporadic DMD boy, who died >20 years previously. Dried umbilical cord preserved for 38 years was the only available source of genomic DNA. Although the genomic DNA was severely degraded, multiplex ligation-dependent probe amplification analysis was performed but no gross mutations found. Sanger sequencing was attempted but not conclusive. Next-generation sequencing (NGS) was performed by controlling the tagmentation during library preparation. A nonsense mutation in DMD (p.Arg2095*) was clearly identified in the proband. Consequently, the identical mutation was detected as an 11% mosaic mutation from his healthy mother. Finally, the proband's sister was diagnosed as a non-carrier of the mutation. Thus using NGS we have identified a pathogenic DMD mutation from degraded DNA and low-level somatic mosaicism, which would have been overlooked using Sanger sequencing.