Mariko Yagi

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 patients number.

Intravenous Infusion of an Antisense Oligonucleotide Results in Exon Skipping in Muscle Dystrophin mRNA of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease that is characterized by muscle dystrophin deficiency. We report that intravenous (IV) infusion of an antisense oligonucleotide created an in-frame dystrophin mRNA from an out-of-frame DMD mutation (via exon skipping) which led to muscle dystrophin expression. A 10-year-old DMD patient possessing an out-of-frame, exon 20 deletion of the dystrophin gene received a 0.5 mg/kg IV infusion of an antisense 31-mer phosphorothioate oligonucleotide against the splicing enhancer sequence of exon 19. This antisense construct was administered at one-week intervals for 4 wk. No side effects attributable to infusion were observed. Exon 19 skipping appeared in a portion of the dystrophin mRNA in peripheral lymphocytes after the infusion. In a muscle biopsy one week after the final infusion, the novel in-frame mRNA lacking both exons 19 and 20 was identified and found to represent approximately 6% of the total reverse transcription PCR product. Dystrophin was identified histochemically in the sarcolemma of muscle cells after oligonucleotide treatment. These findings demonstrate that phosphorothioate oligonucleotides may be administered safely to children with DMD, and that a simple IV infusion is an effective delivery mechanism for oligonucleotides that lead to exon skipping in DMD skeletal muscles.

Oligonucleotides against a splicing enhancer sequence led to dystrophin production in muscle cells from a Duchenne muscular dystrophy patient

Yasuhiro Takeshima, Hiroko Wada, Mariko Yagi, Yukitoshi Ishikawa, Yuka Ishikawa, Ryoji Minami, Hajime Nakamura, Masafumi Matsuo* Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuoku, Kobe 650 0017, Japan Division of Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuoku, Kobe 650 0017, Japan Department of Pediatrics, National Yakumo Hospital, 128 Miyazonocho, Yakumocho, Hokkaido 049 3198, Japan

Genetic polymorphisms associated with adverse events and elimination of methotrexate in childhood acute lymphoblastic leukemia and malignant lymphoma

AbstractMethotrexate is administered in high doses to treat childhood acute lymphoblastic leukemia and malignant lymphoma. Hepatotoxicity and bone marrow suppression often limit its use, however. The objective of this study was to determine the genetic polymorphisms associated with the hepatotoxicity and elimination of methotrexate. Genetic polymorphisms of glutathione S-transferase (GST) genes including GSTT1 positive/null, GSTM1 positive/null, and GSTP1 A313G, and genes for reduced folate carrier 1 G80A (RFC1 G80A), methylenetetrahydrofolate reductase C677T (MTHFR C677T), and breast cancer resistant protein C421A (BCRP C421A) were determined for 26 patients by the polymerase chain reaction (PCR) method or by direct sequencing. A high frequency of hepatotoxicity (P = 0.035) was observed for patients with GSTM1 positive and RFC1 AA80, and serum concentrations of methotrexate 48 h after the start of infusion were higher for patients with the TT677 genotype of MTHFR (P = 0.028). In conclusion, GSTM1 positive/null and RFC1 G80A polymorphisms could be predictors for hepatotoxicity, and the MTHFR C677T polymorphism is associated with elimination of methotrexate.

Chemical treatment enhances skipping of a mutated exon in the dystrophin gene

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by a loss of the dystrophin protein. Control of dystrophin mRNA splicing to convert severe DMD to a milder phenotype is attracting much attention. Here we report a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene. Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein is produced in the patient cells. An analysis of the mRNA reveals that the mutation promotes exon skipping and restores the open reading frame of dystrophin. Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer. Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promotes the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increases the production of the dystrophin protein in the patients cells.

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.

Splicing analysis disclosed a determinant single nucleotide for exon skipping caused by a novel intraexonic four-nucleotide deletion in the dystrophin gene

Background: Mutations in exonic splicing enhancer sequences are known to cause splicing errors. Although exonic splicing enhancers have been identified as a stretch of purine-rich sequences, it has been difficult to precisely pinpoint the determinant nucleotides in these sequences. This article reports that a 4-bp deletion in exon 38 of the dystrophin gene induced complete exon 38 skipping in vivo. Moreover, the third nucleotide of the deletion was shown to be determinant for the exonic splicing enhancer activity in in vivo splicing analysis of hybrid minigenes encoding mutant exons. Method: Genomic DNA analysis of a 2-year-old boy with a raised level of serum creatine kinase yielded a 4-bp deletion 11 bp upstream of the 3′ end of exon 38 of the dystrophin gene (c. 5434–5437del TTCA), disrupting a predicted SC35-binding site. Result: Interestingly, his dystrophin mRNA was shown to completely lack exon 38 (exon 38− transcript). As the exon 38− transcript coded for a truncated dystrophin protein, this exon skipping was determined to be a modifying factor of his phenotype. In an in vivo splicing assay, a hybrid minigene encoding exon 38 with the 4-bp deletion was shown to induce complete exon 38 skipping, confirming the deleted region as a splicing enhancer sequence. Site-directed mutagenesis of the deleted sequence showed that the complete exon 38 skipping was caused by mutation of the third nucleotide position of the deletion (C5436), whereas mutations at the other three nucleotide positions induced partial exon skipping. Conclusion: Our results underline the potential of understanding the regulation of exonic splicing enhancer sequences and exon skipping therapy for treatment of Duchenne’s muscular dystrophy.

Intraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle

Antisense oligodeoxynucleotide against the splicing enhancer sequence (SES) in exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame in the cultured Duchenne muscular dystrophy (DMD) myocytes with the deletion of exon 20. Transfection of the antisense oligodeoxynucleotide, therefore, has been proposed as a promising means for therapeutic modification of dystrophin mRNA of DMD, a fatal disorder caused by defects in the dystrophin gene. A systemic delivery method targeting the large number of diseased muscles remains to be established for clinical application of antisense oligodeoxynucleotide. In this study, we investigated capability of oligodeoxynucleotide transfer into the skeletal muscles of mdx mouse, a mouse model of DMD. Thirty-one mer phosphorothioate oligodeoxynucleotide complementary to the SES of dystrophin exon 19 was intraperitoneally administered to mdx mice without any carrier. Histochemical study disclosed that fluorescence-labeled oligodeoxynucleotide appeared in the nuclei of femoral skeletal muscle cell at the second day after injection of 20 mg/kg BW oligodeoxynucleotide, and still visible at 14th day. Reverse transcription (RT)-PCR analysis of dystrophin transcript in these cells disclosed that a proportion of it showed skipping of exon 19 from second to seventh day after injection. These results showed that the intraperitoneally administered oligodeoxynucleotide could be transfected to nucleus of mdx skeletal muscle without any carrier and was able to induce exon skipping in vivo.

Dystrophin Nonsense Mutations Can Generate Alternative Rescue Transcripts in Lymphocytes

Secondary alterations in splicing have been reported to produce semi‐functional mRNA from several nonsense mutations in the dystrophin gene. Disruptions of exonic splicing enhancers by single nucleotide changes are thought to underlie such alterations. The precise frequencies of such nonsense mutation‐dependent splicing alterations, however, remain unknown. Here we analyzed the splicing patterns of dystrophin mRNA in lymphocytes from 38 patients with dystrophinopathies due to nonsense mutations in the dystrophin gene. In seven of the cases (18%), we observed partial skipping of the nonsense‐encoding exon. Two of the seven cases, however, exhibited complex activation of a nonsense mutation‐created splice site, which resulted in the generation of novel transcripts. Examination of cis‐regulatory splicing elements through calculation of splicing probability scores and identification of potential splicing enhancer or silencer sequences failed to disclose a single cause for exon skipping. Remarkably, individual differences in splicing patterns were observed for cells from patients with identical nonsense mutations (C.5899C>T). Although five cases produced semi‐functional dystrophin mRNAs, only one of these exhibited a mild clinical course. These results provide important insights about targets for exon skipping induced by candidate antisense oligonucleotides and for ribosomal read‐through of nonsense mutations.

Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Here, we report a novel mechanism for the occurrence of DMD in females. In a Vietnamese DMD girl, conventional PCR amplification analysis disclosed a deletion of exons 12–19 of the dystrophin gene on Xp21.2, with a karyotype of 46, XY. Furthermore, a novel mutation in the androgen-receptor gene on Xq11.2-q12 was identified in this girl, which led to male pseudohermaphroditism. Co-occurrence of mutations of these two genes constitutes a novel mechanism underlying female DMD.