Detection of a nonsense mutation in the dystrophin gene by multiple SSCP.

A combination of multiplex PCR with the single strand conformation polymorphism (SSCP) technique was employed to screen for point mutations in the human dystrophin gene. Co-amplification of 11 exons from genomic DNA of Duchenne and Becker muscular dystrophy (DMD/BMD) patients with no deletion or duplication was performed and the samples subjected to multiple SSCP analysis. We report the case of a nonsense mutation in a Duchenne patient identified by this approach. The mutation introduces a termination codon within exon 8 of the dystrophin gene. It is predicted to cause a very premature translational termination accounting for the severe phenotype observed. The patient inherited this mutation from his mother. In addition the analysis revealed 5 polymorphisms useful for internal control.     

Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene

Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping.     

A novel insertional mutation of a single base in exon 12 of the dystrophin gene

A new point mutation in exon 12 of the dystrophin gene was identified in a DMD patient using multiple SSCP analysis, which allows the simultaneous study of several exons. The mutation is an A insertion at position 1580 of the cDNA sequence, leading to a stop codon in the translational reading frame. This mutation was not observed in a sample of 70 DMD patients.     

Microarray-based mutation detection in the dystrophin gene

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans>2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6 to 10% of males with either DMD or BMD. The remaining 30 to 35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, multiplex ligation-dependent probe amplification (MLPA), detection of virtually all mutations-SSCP (DOVAM-S), and single condition amplification/internal primer sequencing (SCAIP); however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution comparative genomic hybridization (CGH) microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin.     

Characterization of two nonsense mutations in the human dystrophin gene.

Forty Duchenne muscular dystrophy patients from the province of Moravia in the Czech Republic, who were previously found negative for large deletions in the dystrophin gene, were tested for the presence of point mutations in selected exons. Besides several intron and exon polymorphisms, two cases of nonsense mutations were detected in exon 70, thus causing the loss of the C-terminal domain of dystrophin. One of these, the mutation, S3365X, is newly reported here while the other, R3381X, has been described previously. These mutations, only 16 bp distant from each other, have a very different impact on the mental abilities of the corresponding patients.     

Double missense mutation in exon 41 of the human dystrophin gene detected by double strand conformation analysis

Development of late-onset Becker muscular dystrophy is reported in a patient whose two healthy brothers showed high serum creatine kinase level. No cases of neuromuscular disorders had been previously reported in this family. The analysis of the dystrophin gene showed that the three brothers had A → C transversion at nucleotide 6092 in exon 41, a missense mutation which converts lysine into glutamine. The symptomatic patient showed an additional mutation in the same exon, a T → C transition at nucleotide 6119, converting a phenylalanine to leucine. The possible pathogenic role of this mutation is discussed. Am. J. Med. Genet. 80:99–102, 1998. © 1998 Wiley-Liss, Inc.     

Family with Pelizaeus-Merzbacher disease/X-linked spastic paraplegia and a nonsense mutation in exon 6 of the proteolipid protein gene

We report on a C-to-T transition in exon 6 of the PLP gene in a male with Pelizaeus-Merzbacher disease/X-linked spastic paraplegia. The transition changes a glutamine at amino acid residue 233 to a termination codon. This premature stop codon probably results in a truncated protein that is not functional. Six other relatives were analyzed for the mutation and two female carriers were identified. Autopsy data on one male are presented. Am. J. Med. Genet. 71:357–360, 1997. © 1997 Wiley-Liss, Inc.     

A point mutation in the human CD45 gene associated with defective splicing of exon A

CD45 is a receptor-type protein tyrosine phosphatase involved in the regulation of lymphocyte activation. Different CD45 isoforms are generated by alternative splicing of three variable exons (A, B and C). The pattern of CD45 splicing depends upon cell type and state of activation. CD45RA isoforms (containing exon A-encoded sequences) can usually be found on a subset of resting T cells, but not on activated T cells. We have recently described a variant pattern of CD45RA expression which is characterized by continuous expression of CD45RA molecules on activated and memory T cells. Here, we demonstrate that this phenotype is associated with heterozygosity for a point mutation at nucleotide position 77 of exon A, leading to a C → G transition. This mutation does not change the protein sequence of the CD45RA isoform. We conclude that position 77 is part of a motif necessary for splicing of exon A, which supports the hypothesis that sequences within exons have significant effects on alternative splicing. The mutation of this motif might prevent binding of a transacting splice factor. In the heterozygous state, this mutation is not associated with impaired T cell reactivity. Functional consequences of the homozygous state remain to be elucidated.     

A synonymous mutation in SPINK5 exon 11 causes Netherton syndrome by altering exonic splicing regulatory elements

Netherton syndrome (NS) is a rare, life-threatening ichthyosiform syndrome caused by recessive loss-of-function mutations in SPINK5 gene encoding lymphoepithelial Kazal-type-related inhibitor (LEKTI), a serine protease inhibitor expressed in the most differentiated epidermal layers and crucial for skin barrier function. We report the functional characterization of a previously unrecognized synonymous variant, c.891C>T (p.Cys297Cys), identified in the SPINK5 exon 11 of an NS patient. We demonstrated that the c.891C>T mutation is associated with abnormal pre-mRNA splicing and residual LEKTI expression in the patient's keratinocytes. Subsequent minigene splicing assays and in silico predictions confirmed the direct role of the synonymous mutation in inhibiting exon 11 inclusion by a mechanism that involves the activity of exonic regulatory sequences, namely splicing enhancer and silencer. However, this deleterious effect was not complete and a residual amount of normal mRNA and LEKTI protein could be detected, correlating with the relatively mild patient's phenotype. Our study represents the first identification of a disease-causing SPINK5 mutation that alters splicing without affecting canonical splice sites.     

McArdle's disease: a nonsense mutation in exon 1 of the muscle glycogen phosphorylase gene explains some but not all cases

McArdle's disease is an inherited disease that results froma lack of functional muscle glycogen phosphorylase. We reporthere the identification of a C to T transition in exon 1 ofthe muscle phosphorylase gene found in all patients studied.This base pair mutation results in the substitution of a stopcodon (TGA) for the codon (CGA) for Arg49 in the mature protein,and generates a novel restriction site for Niaill. Of sixteenMcArdle's patients, ten are homozygous for this mutation; theremainder are heterozygous. Additional unidentified mutationsmust lead to the McArdle's phenotype in the latter group ofpatients.     

A nonsense mutation in the fibrillin-1 gene of a Marfan syndrome patient induces NMD and disrupts an exonic splicing enhancer

A nonsense mutation in the fibrillin-1 (FBN1) gene of a Marfan syndrome (MFS) patient induces in-frame exon skipping of FBN1 exon 51. We present evidence, based on both in vivo and in vitro experiments, that the skipping of this exon is due to the disruption of an SC35-dependent splicing enhancer within exon 51. In addition, this nonsense mutation induces nonsense-mediated decay (NMD), which degrades the normally spliced mRNA in the patient's cells. In contrast to NMD, skipping of FBN1 exon 51 does not require translation.     

Characterization of a complex Duchenne muscular dystrophy-causing dystrophin gene inversion and restoration of the reading frame by induced exon skipping

Out of three mutations in the dystrophin gene that cause Duchenne muscular dystrophy (DMD), the most common, serious childhood muscle wasting disease, two are genomic deletions of one or more exons that disrupt the reading frame. Specific removal of an exon flanking a genomic deletion using antisense oligonucleotide intervention during pre-RNA processing can restore the reading frame and could potentially reduce disease severity. We describe a rare dystrophin gene rearrangement; inversion of approximately 28 kb, flanked by a 10-bp duplication and an 11-kb deletion, which led to the omission of exons 49 and 50 from the mature mRNA and the variable inclusion of several pseudoexons. In vitro transfection of cultured patient cells with antisense oligonucleotides directed at exon 51 induced efficient removal of that exon, as well as one of the more commonly included pseudoexons, suggesting closely coordinated splicing of these exons. Surprisingly, several antisense oligonucleotides (AOs) directed at this pseudoexon had no detectable effect on the splicing pattern, while all AOs directed at the other predominant pseudoexon efficiently excised that target. Antisense oligomers targeting dystrophin exon 51 for removal are currently undergoing clinical trials. Despite the unique nature of the dystrophin gene rearrangement described here, a personalized multiexon skipping treatment is applicable and includes one compound entering clinical trials for DMD.     

Left-sided CHILD syndrome caused by a nonsense mutation in the NSDHL gene

Congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome is a rare X-linked dominant malformation syndrome characterized by unilaterally distributed ichthyosiform nevi, often sharply delimited at the midline, and ipsilateral limb defects. At least two-thirds of cases demonstrate involvement of the right side. Mutations in an essential enzyme of cholesterol biosynthesis, NAD(P)H steroid dehydrogenase-like [NSDHL], have been reported in five unrelated patients with right-sided CHILD syndrome and in a sixth patient with bilaterally, symmetric nevi and mild skeletal anomalies, but not with CHILD syndrome as originally defined. Although all of the molecularly diagnosed cases with the CHILD phenotype to date have had right-sided disease, we report here a novel nonsense mutation (E151X) of NSDHL in an infant with left-sided CHILD syndrome. This result demonstrates that both right- and left-sided CHILD syndrome can be caused by mutations in the same gene.