Yo Okizuka

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.

Small Mutations Detected by Multiplex Ligation–Dependent Probe Amplification of the Dystrophin Gene

Currently, multiplex ligation-dependent probe amplification (MLPA) has been recognized as the most powerful and convenient method to identify exon deletions or duplications in the dystrophin gene, the mutation of which causes Duchenne and Becker muscular dystrophies (DMD/BMD). The mutation diagnosis is easily done by assessing the amounts amplified by MLPA (loss, single, or double). However, an ambiguous amount of amplified product has never been reported. When 77 Japanese DMD/BMD patients were examined by MLPA from MRC-Holland (Amsterdam, The Netherlands), deletions/duplications in the dystrophin gene were identified in 64.8%. Ten male patients showed loss of a single exon by MLPA, but one of them was found to have not an exon deletion, but a four-nucleotide deletion (c.3347-3350delAGAA) within the exon. Remarkably, two patients showed ambiguous amounts of product with less than half of that of a single copy, making the genetic diagnosis impossible. In one patient, a novel single-nucleotide change (c.4303G>T) leading to a nonsense mutation was identified. In another patient, a novel five-nucleotide deletion (c.4536-4540delGAGTG) was identified. It was considered that these two mutations partially disturbed MLPA amplification, resulting in ambiguous amplification. These results show that MLPA can serve as a tool for screening small mutations, as well as for detecting exon deletions or duplications.

Effect of CPS14217C>A genotype on valproic‐acid‐induced hyperammonemia

Background:  In order to clarify the factors causing hyperammonemia and to predict occurrences during treatment with valproic acid (VPA), we investigated the effect of the genetic polymorphism of carbamoyl‐phosphate synthase 1 (CPS14217C>A) on susceptibility of hyperammonemia, together with the effect of coadministration of other anticonvulsants.

Tandem duplications of two separate fragments of the dystrophin gene in a patient with Duchenne muscular dystrophy

AbstractMutations in the dystrophin gene result in the most common inherited muscle disease, Duchenne muscular dystrophy (DMD). Duplications spanning one or more exons have been found to be the second most common disease-causing mutation in the dystrophin gene. Although the duplicated exons are commonly thought to be arranged in tandem, rare noncontiguous exon duplications have been disclosed without clarifying their location or orientation. Here we present the first report that details the exact locations and orientations of noncontiguous duplications in the dystrophin gene. Multiplex ligation-dependent probe amplification analysis of the dystrophin gene of a Japanese boy with DMD revealed that his genomic DNA contained duplications of exons from two separate fragments of the gene: one from exon 45 to exon 48 and the other from exon 55 to exon 63. To clarify the locations and orientations of the duplicated exons, reverse transcription-nested PCR analysis of dystrophin mRNA was conducted. Interestingly, the extra copies of exons 45–48 and exons 55–63 were found to be properly oriented between exons 48 and 49 and exons 63 and 64, respectively. These results indicated that two tandem duplication events occurred in the dystrophin gene of this patient and should contribute to the understanding of the duplication mechanisms that contribute to the development of DMD.

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.

Two Japanese infants with congenital generalized lipodystrophy due to BSCL2 mutations

Background:  Congenital generalized lipodystrophy (CGL), Berardinelli‐Seip syndrome, is a rare autosomal recessive disorder characterized by the generalized absence of adipose tissue at birth, severe insulin resistance early in life, hypertriglyceridemia, hepatomegaly, and the development of diabetes mellitus during puberty. Recently, two genes, BSCL2 and AGPAT2, were identified as causative genes for CGL. It has been reported that patients with BSCL mutations present with more severe clinical findings than those with AGPAT2 mutations. However, the clinical course of CGL caused by BSCL2 mutations in infancy has not been fully elucidated.

In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G→A mutations in introns of the dystrophin gene

Background: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene. Objective: To identify elements determining alternative splicing pathways in intron +1G→A mutations of the dystrophin gene. Results: We found that exon 25 is spliced out in the +1G→A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G→A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G→A mutation of intron 25. Conclusion: It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G→A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.

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

BackgroundLimb-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.MethodsWe 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.ResultsIn 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 γ-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 γ-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.ConclusionsTo the best of our knowledge, this is the first study to demonstrate a low incidence of LGMD2C in the Japanese population.

Insertion of the IL1RAPL1 gene into the duplication junction of the dystrophin gene

Duplications of one or more exons of the dystrophin gene are the second most common mutation in dystrophinopathies. Even though duplications are suggested to occur with greater complexity than thought earlier, they have been considered an intragenic event. Here, we report the insertion of a part of the IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) gene into the duplication junction site. When the actual exon junction was examined in 15 duplication mutations in the dystrophin gene by analyzing dystrophin mRNA, one patient was found to have an unknown 621 bp insertion at the junction of duplication of exons from 56 to 62. Unexpectedly, the inserted sequence was found completely identical to sequences of exons 3–5 of the IL1RAPL1 gene that is nearly 100 kb distal from the dystrophin gene. Accordingly, the insertion of IL1RAPL1 exons 3–5 between dystrophin exons 62 and 56 was confirmed at the genomic sequence level. One junction between the IL1RAPL1 intron 5 and dystrophin intron 55 was localized within an Alu sequence. These results showed that a fragment of the IL1RAPL1 gene was inserted into the duplication junction of the dystrophin gene in the same direction as the dystrophin gene. This suggests the novel possibility of co-occurrence of complex genomic rearrangements in dystrophinopathy.

Treatment of preterm infants with West syndrome: Differences due to etiology

This study was conducted with a particular focus on preterm infants with West syndrome (WS) to evaluate differences in the first responses to oral medication based on etiology.