Intronic breakpoint definition and transcription analysis in DMD/BMD patients with deletion/duplication at the 5' mutation hot spot of the dystrophin gene.

Dystrophin mutations occurring at the 5' end of the gene frequently behave as exceptions to the "frame rule," their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5' dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3-7, 3-6 and duplication of exons 2-4. Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3-7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3-7 deletion. Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2-4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5' dystrophin mutations, including recoding mechanisms and unusual splicing choices.

Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis.

BACKGROUND: Prenatal diagnosis for chromosome abnormality is routinely undertaken by full karyotype analysis of chromosomes from cultured cells; pregnant women must wait on average 13-14 days for their results. Autosomal trisomies, which account for around 80% of significant abnormalities, can be detected by quantitative fluorescence (QF) PCR. We report on the development and implementation of this technique as the first such routine service within a diagnostic department of the UK National Health Service (NHS). METHODS: We designed a "one-tube test" comprising four primer pairs for polymorphic tetranucleotide repeat sequences on chromosome 21, four primer pairs for sequences on chromosome 18, three primer pairs for sequences on chromosome 13, and one primer pair to identify the sex chromosomes. All prenatal samples received by our NHS diagnostic department between April, 2000, and April, 2001, were tested. After DNA extraction, PCR amplification was done and the products separated on a capillary-based genetic analyser; the results were interpreted with dedicated software. Follow-up karyotype analysis was done on all samples. FINDINGS: 1148 amniotic fluid samples, 188 chorionic villus samples, and 37 fetal tissue samples were tested; the amplification failure rate was zero with our current protocol. QF-PCR results were obtained and reported on 1314 (98%) of the prenatal samples; the remaining 22 (2%) were uninformative because of maternal-cell contamination. One case of mosaicism in a chorionic villus sample, and two cases indicating somatic expansion of a tetranucleotide repeat were found. No false positive or false negative results were obtained. The mean reporting time for the last 4 months of data collection was 1.25 working days. INTERPRETATION: QF-PCR aneuploidy testing is an efficient and accurate technique for the detection of autosomal trisomies in prenatal samples. Implementation of this service has led to the rapid diagnosis of abnormalities and early reassurance for women with normal results.

Identification and quantification of somatic mosaicism for a point mutation in a Duchenne muscular dystrophy family.

Relatively few point mutations have been found in the dystrophin gene and of these only two have been associated with mosaicism. A single base insertion has been identified and quantified in a mother of two sons affected with Duchenne muscular dystrophy. It has been determined that she is a somatic mosaic with the mutation present in 25% of lymphocyte DNA. Further tissue lineages have been tested and the time at which the mutation arose was determined to be before the cellular differentiation into the bilaminar disc at approximately eight days after fertilisation. We suggest that the incidence of mosaicism for dystrophin point mutations may be higher than current data suggest.

A small intraexonic deletion within the dystrophin gene suggests a possible mechanism of mutagenesis.

A case of Duchenne muscular dystrophy is described with an unusual mutation consisting of a 17-bp deletion within exon 47 of the dystrophin gene. The sequences on either side of the deletion have a high degree of intrastrand base complementarity. It is hypothesised that the mechanism generating the deletion may have been the formation of hairpin loop structure in a single strand of DNA followed by enzymatic degradation at unpaired regions within the loop.

Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis.

We have developed a semiautomated approach to amplify 25 exons of the dystrophin gene using two fluorescent multiplex PCR assays which detect over 98% of reported deletions and 90% of duplications causing Duchenne/Becker muscular dystrophy. The 5' multiplex detects 11 exons from the proximal deletion hotspot of the gene while the 3' multiplex detects 14 exons from the central deletion hotspot. The PCR products are accurately sized and quantified by a fluorescent DNA sequencer after only 18 cycles of amplification. The amount of product amplified from each exon in a multiplex is divided by that from each of the other exons, and this ratio is compared with those from control samples to obtain a series of dosage quotients (DQ), from which the copy number of each exon is determined. No overlap was observed between the DQ values obtained from single and double copy loci. The assays can be used to screen both affected males and at risk female relatives for a mutation. The method has been evaluated as a female carrier test by conducting a blind trial on 150 coded samples. Sixty-three deletion carriers, two duplication carriers, and 84 normal female controls were all correctly identified, showing that carrier diagnosis is possible even in families where the nature of the mutation is unknown. Additionally the analysis showed a non-pathogenic duplication involving the muscle specific promoter and exon 1. Together these two multiplex assays detect over 70% of all mutations in the dystrophin gene, greatly simplifying and partly automating molecular diagnosis in Duchenne and Becker muscular dystrophy.

Rapid molecular method for prenatal detection of Down's syndrome.

We have evaluated a rapid method that allows prenatal detection of Down's syndrome in less than 24 hours. DNA from uncultured amniotic fluid, fetal blood, and tissue samples was amplified with the small tandem repeat (STR) marker D21S11. Quantitative analysis of fluorescent STR products with evaluation of their sizes provided clear evidence for trisomy 21. Whilst most normal samples showed two amplification peaks of equal size, Down's syndrome samples were characterised by either three STR peaks or two peaks with a ratio of 2:1. Co-amplification with a non-polymorphic sequence allowed analysis of samples that were homozygous for the 21-derived STRs.

Direct diagnosis of carriers of point mutations in Duchenne muscular dystrophy.

In about one-third of patients with Duchenne/Becker muscular dystrophy, the causative mutation does not involve gross alterations in the structure of the dystrophin gene. Prenatal diagnosis and carrier detection for such families is therefore dependent on the indirect method of linkage analysis with polymorphic DNA markers, a method that is subject to error. The identification of point mutations in the dystrophin gene in six affected males enabled us to develop direct assays, based on the polymerase chain reaction, that are specific for each of the mutations. In all six cases, the assays allowed us to offer families accurate carrier testing and prenatal diagnosis.

A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridisation shows mistypings by both methods.

Existing reactions for the multiplex PCR amplification of exons in the dystrophin gene have been modified to produce two multiplex reactions which separately cover the 5' and 3' major deletion 'hotspots' in the gene, and together detect approximately 98% of all deletions detectable by Southern cDNA hybridisation. A comparative study of 148 patients showed mistypings in both the cDNA hybridisation data (4%) and the PCR analysis (1.2%). We suggest means of circumventing the underlying problems in order to avoid mistyping and subsequent misdiagnosis, and conclude that, with appropriate precautions, multiplex PCR amplification can be the method of choice for detecting deletions in the dystrophin gene.