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.

Circulating tricarboxylic acid cycle metabolite levels in citrin-deficient children with metabolic adaptation, with and without sodium pyruvate treatment.

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid ß-oxidation cycle between 22 citrin-deficient children (age, 3-13years) with normal liver functions and 37 healthy controls (age, 5-13years). TCA cycle analysis showed that basal plasma citrate and α-ketoglutarate levels were significantly higher in the affected than the control group (p<0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p<0.001). The plasma level of 3-OH-butyrate derived from fatty acid ß-oxidation was significantly higher in the affected group (p<0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p<0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development.

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.

Identical deletion at 14q13.3 including PAX9 and NKX2-1 in siblings from mosaicism of unaffected parent.

By screening patients with undiagnosed multiple congenital anomalies and intellectual disability using array-comparative genomic hybridization, we identified an 884 kb heterozygous microdeletion at 14q13.3 in two siblings presenting with oligodontia, hypothyroidism and persistent pulmonary hypertension of the newborn, resulting from their parental gonosomal mosaicism. Among the six genes included in the deletion, haploinsufficiency of PAX9 and NKX2-1 was probably associated with their phenotypes. These results highlighted a possibility of recurrence of pathogenic copy-number variants associated with parental mosaicism, which requires careful genetic counseling.

Differences in carrier frequency between mothers of Duchenne and Becker muscular dystrophy patients.

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked inherited muscular disorders caused by mutations in the dystrophin gene. Two-thirds of DMD cases are thought to be caused by inheritance from carrier mothers and this study aimed to clarify and compare the carrier frequency of mothers of DMD and BMD patients according to the mutation type. We included 139 DMD and 19 BMD mothers. Of these, 113 patients (99 DMD and 14 BMD) and 13 patients (12 DMD and 1 BMD) had deletions and duplications of one or more exons, respectively. Small mutations, including nonsense mutations, small deletions/insertions and splice site mutations, were identified in 32 patients (28 DMD and four BMD). The overall carrier frequency for BMD mothers was significantly higher than for DMD (89.5% vs 57.6%, P<0.05), probably as BMD patients can leave descendants. The carrier frequency tended to be lower in mothers of DMD patients with deletion mutations than with duplications and small mutations (53.5%, 66.7% and 67.9%, respectively). It was suggested that de novo mutations are more prevalent for deletions than other mutations. This is the first report to analyze the carrier frequency according to mutation type.

A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy.

Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease, is mostly caused by exon deletion mutations in the DMD gene. The reading frame rule explains that out-of-frame deletions lead to muscle dystrophin deficiency in DMD. In outliers to this rule, deletion junction sequences have never previously been explored as splicing modulators. In a Japanese case, we identified a single exon 45 deletion in the patient's DMD gene, indicating out-of-frame mutation. However, immunohistochemical examination disclosed weak dystrophin signals in his muscle. Reverse transcription-PCR amplification of DMD exons 42 to 47 revealed a major normally spliced product with exon 45 deletion and an additional in-frame product with deletion of both exons 44 and 45, indicating upstream exon 44 skipping. We considered the latter to underlie the observed dystrophin expression. Remarkably, the junction sequence cloned by PCR walking abolished the splicing enhancer activity of the upstream intron in a chimeric doublesex gene pre-mRNA in vitro splicing. Furthermore, antisense oligonucleotides directed against the junction site counteracted this effect. These indicated that the junction sequence was a splicing silencer that induced upstream exon 44 skipping. It was strongly suggested that creation of splicing regulator is a modifier of dystrophinopathy.

[Clinical manifestations of three neonates with family histories of Menkes disease].

OBJECTIVE: To examine indications for the early diagnosis of Menkes disease (MD). METHODS: We compared clinical examination results of 3 neonate males with family histories of MD who were at risk of being affected (1 infant was affected and 2 were unaffected). RESULTS: Kinky hair, a characteristic shown by MD patients, was observed just after birth in the affected infant; however, this characteristic was extremely mild and easy to miss. In the first month after birth, serum copper level declined over time in the affected infant,while it increased in the unaffected infants. Urine homovanillic acid/vanillylmandelic acid (HVA/VMA) ratio, which is typically high in MD patients, was observed to be 4.9-8.0 (cut-off, 4.0) in the affected infant. In the unaffected infants, the urine HVA/MVA ratio showed a high value of 11.0 during catecholamine administration, but this decreased to below the cut-off value when catecholamine was not administered. CONCLUSIONS: Variations in the serum copper level and urine HVA/VMA ratio over time were considered as useful indicators for the early diagnosis of MD.

Improvement in a post-stroke pediatric patient with hemiplegia: Use of a hand-arm bimanual intensive therapy with hybrid assistive limb.

BACKGROUND: Pediatric stroke is a rare medical condition that often leads to long-lasting motor and cognitive impairments. Although therapies for adults after a stroke are well described, treatments for motor deficits following a pediatric stroke are yet to be investigated. We report a case of pediatric stroke in the chronic phase, in which a combination of novel treatments resulted in a significant improvement in physical function. CASE REPORT: A seven-year-old girl with a left hemispheric cerebral infarction lost almost all right upper extremity motor function. Following onabotulinumtoxinA treatment, she underwent hand-arm bimanual intensive therapy augmented with a hybrid assistive limb for 90 h over 15 days. Evaluation after the training revealed significant improvements in physical function, daily activities, and occupational performance. CONCLUSIONS: This report highlights the importance of innovative combinations of techniques in the treatment of pediatric stroke.

Molecular characterization of an X(p21.2;q28) chromosomal inversion in a Duchenne muscular dystrophy patient with mental retardation reveals a novel long non-coding gene on Xq28.

Duchenne muscular dystrophy (DMD) is the most common inherited muscular disease and is characterized by progressive muscle wasting. DMD is caused by mutations in the dystrophin gene on Xp21.2. One-third of DMD cases are complicated by mental retardation, but the pathogenesis of this is unknown. We have identified an intrachromosomal inversion, inv(X)(p21.2;q28) in a DMD patient with mental retardation. We hypothesized that a gene responsible for the mental retardation in this patient would be disrupted by the inversion. We localized the inversion break point by analysis of dystrophin complementary DNA (cDNA) and fluorescence in situ hybridization. We used 5' and 3' rapid amplification of cDNA ends to extend the known transcripts, and reverse transcription-PCR to analyze tissue-specific expression. The patient's dystrophin cDNA was separated into two fragments between exons 18 and 19. Exon 19 was dislocated to the long arm of the X-chromosome. We identified a novel 109-bp sequence transcribed upstream of exon 19, and a 576-bp sequence including a poly(A) tract transcribed downstream of exon 18. Combining the two novel sequences, we identified a novel gene, named KUCG1, which comprises three exons spanning 50 kb on Xq28. The 685-bp transcript has no open-reading frame, classifying it as a long non-coding RNA. KUCG1 mRNA was identified in brain. We cloned a novel long non-coding gene from a chromosomal break point. It was supposed that this gene may have a role in causing mental retardation in the index case.

Childhood-onset hereditary pancreatitis with mutations in the CT gene and SPINK1 gene.

Hereditary pancreatitis (HP) is an autosomal-dominant gene disorder. The affected genes have been identified as the cationic trypsinogen (CT) gene and the serine protease inhibitor Kazal type 1 (SPINK1) gene. These gene abnormalities alone, however, do not necessarily regulate the onset or severity of pancreatitis, suggesting the involvement of other gene abnormalities and environmental factors. Reported herein is the case of a 9-year-old boy with early-onset HP due to mutations in the CT and SPINK1 genes. The patient had a p.R122H heterozygous mutation in the CT gene and a p.N34S heterozygous mutation in the SPINK1 gene. The father had heterozygous mutation of the SPINK1 gene, and the mother had heterozygous mutation of the CT gene, although neither had a prior history of pancreatitis. In this patient, early onset of HP was attributed to the presence of gene abnormalities in the CT and SPINK1 genes.

Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers.

BACKGROUND: Duchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors. RESULTS: Using a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group. CONCLUSIONS: The decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.

Early clinical signs and treatment of Menkes disease.

Menkes disease (MD) is an X-linked recessive disorder caused by mutations in ATP7A. Patients with MD exhibit severe neurological and connective tissue disorders due to copper deficiency and typically die before 3 years of age. Early treatment with copper injections during the neonatal period, before the occurrence of neurological symptoms, can alleviate neurological disturbances to some degree. We investigated whether early symptoms can help in the early diagnosis of MD. Abnormal hair growth, prolonged jaundice, and feeding difficulties were observed during the neonatal period in 20 of 69, 16 of 67, and 3 of 18 patients, respectively. Only three patients visited a physician during the neonatal period; MD diagnosis was not made at that point. The mean age at diagnosis was 8.7 months. Seven patients, who were diagnosed in the prenatal stage or soon after birth, as they had a family history of MD, received early treatment. No diagnosis was made based on early symptoms, highlighting the difficulty in diagnosing MD based on symptoms observed during the neonatal period. Patients who received early treatment lived longer than their elderly relatives with MD. Three patients could walk and did not have seizures. Therefore, effective newborn screening for MD should be prioritized.

Natural Course of Pachychoroid Pigment Epitheliopathy.

Purpose: To investigate the natural course of pachychoroid pigment epitheliopathy (PPE). Design: A retrospective cohort study. Subjects: From the Kyoto central serous chorioretinopathy (CSC) cohort consisting of 548 patients with CSC as of September 2020, we included consecutive unilateral patients with acute or chronic CSC between January 2013 and December 2016. Methods: All patients underwent complete ophthalmic examination, including multimodal imaging such as fundus autofluorescence, spectral-domain optical coherence tomography, and fluorescein angiography/indocyanine green angiography and/or optimal coherence tomography angiography. The fellow eyes of eyes diagnosed with CSC were screened for PPE, and their natural course was evaluated. We also evaluated the association of ARMS2 rs10490924, CFH rs800292, TNFRSF10A rs13278062, and GATA5 rs6061548 genotypes with the natural course. Main Outcome Measures: Incidence of CSC, pachychoroid neovasculopathy, and pachychoroid geographic atrophy (GA). Results: In total, 165 patients with unilateral CSC (mean age, 55.7 ± 12.6 years; female, 22.4%) were included from the Kyoto CSC cohort. Among them, 148 (89.7%) were diagnosed as having PPE in their non-CSC eye. Survival analysis revealed that 16.8% of PPE eyes developed CSC during the 6-year follow up, whereas non-PPE eyes did not. Although genetic factors did not have significant association with CSC development (P > 0.05, log-rank test), choroidal vascular hyperpermeability (CVH) and subfoveal choroidal thickness (SFCT) were significantly associated with CSC incidence (P = 0.001, log-rank test). Survival analysis showed that eyes without CVH and eyes with SFCT < 300 µm did not develop CSC during the 6-year follow-up. Pachychoroid neovasculopathy developed in only 1 eye with PPE during a follow-up of 46.4 months. Pachychoroid GA did not develop in any of the studied eyes. Conclusions: This study revealed a natural history of PPE in a relatively large Japanese cohort. Choroidal vascular hyperpermeability and SFCT were significant risk factors for the development of CSC in PPE eyes. Although the current results cannot be generalized for all eyes with PPE, these findings present an important clinical implication.

A patient with mitochondrial trifunctional protein deficiency due to the mutations in the HADHB gene showed recurrent myalgia since early childhood and was diagnosed in adolescence.

Mitochondrial trifunctional protein (MTP) is a multienzyme complex involved in the metabolism of long-chain hydroxyacyl-CoA, a product of the fatty acid ß-oxidation cycle. MTP is an α4ß4 hetero-octomer encoded by two different genes: HADHA (OMIM 600890) and HADHB (OMIM 143450). MTP deficiency induces three different types of presentation: (1) a lethal phenotype with neonatal onset (severe); (2) a hepatic phenotype with infant onset (intermediate); and (3) a neuromyopathic phenotype with late-adolescent onset (mild). While acylcarnitine analysis has revealed increased levels of long-chain hydroxyacylcarnitine in blood when an MTP deficiency exists, the neuromyopathic type is usually asymptomatic and does not always result in an abnormality in acylcarnitine analysis results. We report here the case of a 13-year-old girl with recurrences of intermittent myalgia since her early childhood, for whom the disorder had not been definitely diagnosed. Since she was referred to our hospital because of rhabdomyolysis, we have repeatedly performed blood acylcarnitine analysis and found slight increases in long-chain 3-OH-acylcarnitine levels, on the basis of which we made a chemical diagnosis of MTP deficiency. Immunoblot analysis of skin fibroblasts revealed loss of α- and ß-subunits of MTP. In addition, analysis of the HADHB gene, which encodes long-chain 3-ketoacyl-CoA thiolase, one of the enzymes constituting MTP, identified compound heterozygous mutations of c.520C>T (p.R141C) and c.1331G>A (p.R411K). MTP deficiency is considered an extremely rare disorder, as only five cases (lethal phenotype, two patients; hepatic phenotype, two patients; and neuromyopathic phenotype, one patient) have thus far been reported in Japan. However, it is likely that the neuromyopathic phenotype of MTP deficiency has not yet been diagnosed among patients with recurrences of intermittent myalgia and rhabdomyolysis, as in our patient reported here.

Two closely spaced nonsense mutations in the DMD gene in a Malaysian family.

In Duchenne muscular dystrophy (DMD), identification of one nonsense mutation in the DMD gene has been considered an endpoint of genetic diagnosis. Here, we identified two closely spaced nonsense mutations in the DMD gene. In a Malaysian DMD patient two nonsense mutations (p.234S>X and p.249Q>X, respectively) were identified within exon 8. The proband's mother carried both mutations on one allele. Multiple mutations may explain the occasional discrepancies between genotype and phenotype in dystrophinopathy.

Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies.

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

A Japanese child with asymptomatic elevation of serum creatine kinase shows PTRF-CAVIN mutation matching with congenital generalized lipodystrophy type 4.

Congenital generalized lipodystrophy (CGL), characterized by generalized absence of adipose tissue, has heterogeneous causes. Recently, a novel type of CGL complicated by muscular dystrophy was categorized as CGL4 caused by PTRF-CAVIN deficiency. However, it is unknown whether CGL4 exhibits clinical abnormalities during the infantile period. Here, we describe the youngest Japanese case of CGL4-a Japanese girl with asymptomatic high serum creatine kinase (CK) levels at 3months old. She was referred to our hospital at 5months of age because of her elevated serum CK (2528IU/L). Generalized absence of adipose tissue was first recognized at 2years of age. Mutation analysis of genes known to be responsible for CGL1-3 failed to disclose any abnormalities. Instead, analysis of the PTRF-CAVIN gene encoding PTRF-CAVIN revealed compound heterozygous mutations, one allele contained an insertion (c.696_697insC) and the other allele harbored a novel nonsense mutation (c.512C>A). Our patient had low serum leptin and adiponectin levels and insulin resistance. Pathological studies on biopsied muscle disclosed mild dystrophic change and highly reduced expression of PTRF-CAVIN. It was concluded that our PTRF-CAVIN deficient patient showed not only CGL but also asymptomatic elevation of serum CK because of her mild muscle dystrophic change.

Low incidence of limb-girdle muscular dystrophy type 2C revealed by a mutation study in Japanese patients clinically diagnosed with DMD.

BACKGROUND: Limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive muscle dystrophy that resembles Duchenne muscular dystrophy (DMD). Although DMD is known to affect one in every 3500 males regardless of race, a widespread founder mutation causing LGMD2C has been described in North Africa. However, the incidence of LGMD2C in Japanese has been unknown because the genetic background remains uncharacterized in many patients clinically diagnosed with DMD. METHODS: We enrolled 324 patients referred to the Kobe University Hospital with suspected DMD. Mutations in the dystrophin or the SGCG genes were analyzed using not only genomic DNA but also cDNA. RESULTS: In 322 of the 324 patients, responsible mutations in the dystrophin were successfully revealed, confirming DMD diagnosis. The remaining two patients had normal dystrophin expression but absence of gamma-sarcoglycan in skeletal muscle. Mutation analysis of the SGCG gene revealed homozygous deletion of exon 6 in one patient, while the other had a novel single nucleotide insertion in exon 7 in one allele and deletion of exon 6 in the other allele. These mutations created a stop codon that led to a gamma-sarcoglycan deficiency, and we therefore diagnosed these two patients as having LGMD2C. Thus, the relative incidence of LGMD2C among Japanese DMD-like patients can be calculated as 1 in 161 patients suspected to have DMD (2 of 324 patients = 0.6%). Taking into consideration the DMD incidence for the overall population (1/3,500 males), the incidence of LGMD2C can be estimated as 1 per 560,000 or 1.8 per million. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate a low incidence of LGMD2C in the Japanese population.

Contemporary retrotransposition of a novel non-coding gene induces exon-skipping in dystrophin mRNA.

Non-autonomous retrotransposon-mediated mobilizations of the Alu family are known pathogenic mechanisms of human disease. Here, we report a pathogenic, contemporary, non-autonomous retrotransmobilization of part of a novel non-coding gene into the dystrophin gene. In a Japanese Duchenne muscular dystrophy patient, a 330-bp-long de novo insertion was identified in exon 67 of dystrophin. The insertion induced exon 67-skipping in the dystrophin mRNA, creating a premature stop codon. The sequence of the insertion had certain characteristics of retrotransposons: an antisense polyadenylation signal accompanied by a poly(T) sequence and a target site duplication. The insertion site matched the consensus recognition sequence for the L1 endonuclease, indicating a retrotransposon-mediated event, although the inserted sequence did not match any known retrotransposons. The origin of the inserted sequence was mapped to a gene-poor region of chromosome 11. The inserted fragment was expressed in multiple human tissue RNAs, indicating that it is a novel transcript. The full length of the transcript was cloned and showed no meaningful protein coding ability.

Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center.

Recent developments in molecular therapies for Duchenne muscular dystrophy (DMD) demand accurate genetic diagnosis, because therapies are mutation specific. The KUCG (Kobe University Clinical Genetics) database for DMD and Becker muscular dystrophy is a hospital-based database comprising 442 cases. Using a combination of complementary DNA (cDNA) and chromosome analysis in addition to conventional genomic DNA-based method, mutation detection was successfully accomplished in all cases, and the largest mutation database of Japanese dystrophinopathy was established. Among 442 cases, deletions and duplications encompassing one or more exons were identified in 270 (61%) and 38 (9%) cases, respectively. Nucleotide changes leading to nonsense mutations or disrupting a splice site were identified in 69 (16%) or 24 (5%) cases, respectively. Small deletion/insertion mutations were identified in 34 (8%) cases. Remarkably, two retrotransposon insertion events were also identified. Dystrophin cDNA analysis successfully revealed novel transcripts with a pseudoexon created by a single-nucleotide change deep within an intron in four cases. X-chromosome abnormalities were identified in two cases. The reading frame rule was upheld for 93% of deletion and 66% of duplication mutation cases. For the application of molecular therapies, induction of exon skipping was deemed the first priority for dystrophinopathy treatment. At one Japanese referral center, the hospital-based mutation database of the dystrophin gene was for the first time established with the highest levels of quality and patient's number.