Tomoko Lee

Utility of transmural myocardial strain profile for prediction of early left ventricular dysfunction in patients with Duchenne muscular dystrophy.

Myocardial damage in Duchenne muscular dystrophy (DMD) has lethal outcomes, making early detection of myocardial changes in patients with DMD vital, because early treatment can help prevent the development of myocardial fibrosis. The aim of the present study was, therefore, to test the hypothesis that transmural strain profile (TMSP) analysis can predict future left ventricular (LV) dysfunction in patients with DMD with preserved ejection fraction. We studied 82 consecutive patients with DMD without LV wall motion abnormality, with an ejection fraction of 60 ± 5% (all ≥55%) and age 11 ± 3 years. Echocardiography was performed at baseline and 1 year of follow-up. TMSP in the posterior wall was evaluated from the mid-LV short-axis view. A normal TMSP pattern (1 peak in the endocardium, group 1) was seen in 44 patients, and TMSP with a notch (2 peaks in the endocardium, group 2) in the remaining 38 (46%). Wall motion abnormality in the posterior wall was observed in 16 patients (42%) in group 2 at 1 year of follow-up but in none of the patients in group 1 (42% vs 0%; p <0.001). Importantly, multivariate analysis showed that only TMSP with a notch (odds ratios 1.524, p <0.001) was an independent determinant of the presence of LV posterior wall motion abnormality at 1 year of follow-up. In conclusion, subclinical LV dysfunction can be detected by evaluation of TMSP in patients with DMD who do not have wall motion abnormalities by conventional echocardiography. TMSP with a notch proved effective for evaluating subtle early changes in patients with DMD and might be useful for predicting LV dysfunction.

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 prostaglandin D2 metabolite is elevated in the urine of Duchenne muscular dystrophy patients and increases further from 8 years old

BACKGROUND Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by muscle dystrophin deficiency. Downstream of the primary dystrophin deficiency is not well elucidated. Here, the hypothesis that prostaglandin D2 (PGD2)-mediated inflammation is involved in the pathology of DMD was examined by measuring tetranor PGDM, a major PGD2 metabolite, in urine of DMD patients. METHODS We measured tetranor PGDM in urine using LC-MS/MS. First morning urine samples were collected from genetically confirmed DMD patients and age-matched healthy controls aged 4 to 15y. RESULTS The urinary tetranor PGDM concentration was 3.08±0.15 and 6.90±0.35ng/mg creatinine (mean±SE) in 79 control and 191 DMD samples, respectively. The mean concentration was approximately 2.2-times higher in DMD patients than in controls (p<0.05). Remarkably, urinary tetranor PGDM concentrations in DMD patients showed chronological changes: it stayed nearly 1.5 times higher than in controls until 7y but surged at the age of 8y to a significantly higher concentration. CONCLUSION Urinary tetranor PGDM concentrations were shown to be increased in DMD patients and became higher with advancing age. It was indicated that PGD2-mediated inflammation plays a role in the pathology of DMD.

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.

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

BackgroundDuchenne 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.ResultsUsing 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.ConclusionsThe 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.

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 β-oxidation cycle. MTP is an α4β4 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 β-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.

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.

Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in the other. Examination of splicing regulatory factors revealed that intron 40 had the highest guanine–cytosine content of all examined introns in a 30-nt segment at its 3′ end. Further studies are needed to clarify the biological role of intron 40-retained dystrophin mRNA.

Validation of ambiguous MLPA results by targeted next-generation sequencing discloses a nonsense mutation in the DMD gene

BACKGROUND Duchenne muscular dystrophy (DMD) is the most common inherited muscular disease and caused by mutations in the DMD gene on the X-chromosome. Multiplex ligation-dependent probe amplification (MLPA) is recognized as a convenient and reliable technique to detect exon deletion/duplication mutations in the DMD gene. Here, we applied targeted semi-conductor next-generation sequencing to clarify the cause of ambiguous MLPA results. METHODS Targeted semi-conductor next-generation sequencing was carried out using the Inherited Disease Panel (IDP) on the Ion Torrent Personal Genome Machine (PGM). RESULTS MLPA analysis disclosed unclassifiable relative peak ratio of exon 18 in a DMD boy. His female cousin was indicated to have exon 18 deletion in one allele. To validate these incompatible results, targeted next-generation sequencing was conducted. A nucleotide change, C.2227 C>T creating a premature stop codon, was in exon 18. Concomitantly, both C and T nucleotides were identified in his cousins genome. Ambiguous values of the relative peak ratio in MLPA were considered due to the one nucleotide mismatch between the genomic sequence and the probe used in MLPA. CONCLUSION Analysis using IDP on PGM disclosed a nonsense mutation in the DMD gene as a cause of ambiguous results of MLPA.

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.