Amplification of 18 dystrophin gene exons in DMD/BMD patients: simultaneous resolution by capillary electrophoresis in sieving liquid polymers.

Duchenne (DMD) and Becker (BMD) muscular dystrophies are the two most common myopathies described so far. In the late 80s, Chamberlain et al. and Beggs et al. proposed two PCR assays allowing detection of over 98% DMD/BMD deletions. Since each of them is based on specific co-amplification of 9 dystrophin gene exons, a method attempting simultaneous analysis of DMD/BMD should offer unambiguous resolution and identification of 18 DNA fragments ranging in size from approximately 100 to 500 bp. We have developed a novel capillary electrophoresis method that allows simultaneous analysis of the two PCR sets with full diagnostic value. It consists of (a) an ultrastable inner capillary coating based on a novel acrylamide monomer (N-acryloyl amino ethoxy ethanol); (b) a very low viscosity (barely 70 mPa) sieving polymer solution, formed by short-chain (average mol wt of 230,000, 55,000 Mn) polyacrylamides; (c) substitution of four fragments in the classical multiplex reaction (181 and 535 bp in the Beggs, 416 and 459 bp in the Chamberlain) with four new fragments of different lengths (170, 313, 154 and 88 bp, respectively). These new conditions allow resolution and unambiguous identification of all 18 PCR-amplified fragments in a single electrophoretic run. The set of 18 fragments comprises the following: 88, 113, 139, 154, 170, 196, 202, 238, 268, 271, 313, 331, 357, 360, 388, 410, 506 and 547 bp.

Molecular characterization of 21-hydroxylase deficiency in 70 Italian families.

Seventy Italian families affected by 21-hydroxylase deficiency were studied in order to evaluate the distribution of mutations. The coding P450c21B gene, the highly homologous P450c21A pseudogene and the linked C4A, C4B and DRB genes, mapping within the major histocompatibility complex region, were studied by multiple restriction analysis and in vitro amplification. In the affected individuals, 21.4% of the chromosomes were found to carry either gene deletions or large and small gene conversions. Our findings, consistent with previous reports in other ethnic groups, provide further evidence for the genetic heterogeneity of the disease.

Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction. A multicenter study.

OBJECTIVE--To assess the efficiency, reliability, and ease of use of DNA diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) using the polymerase chain reaction (PCR). DESIGN--DNA from the patients was screened for deletion mutations using multiplex PCR, and the results were compared with those obtained by Southern blot analysis. The PCR multiplex reaction detects nine specific "hot-spot" exons in the dystrophin gene while the Southern analysis detects 66 specific dystrophin gene restriction fragments. The multiplex reaction requires 50-fold less DNA than Southern analysis and thus is considerably more sensitive. SETTING--Fourteen university-affiliated and private genetic disease diagnostic laboratories. PATIENTS--Male patients with clinical signs of DMD/BMD. Cases were selected for analysis randomly, without knowledge of whether a deletion was present within the dystrophin gene. MAIN OUTCOME MEASURES--The percentage of cases that were detectable by multiplex PCR in comparison with Southern analysis, the frequency, extent, and location of the detected deletion mutations. In some cases, duplication mutations were monitored. RESULTS--The accuracy of a single PCR multiplex amplification (nine exons) was compared with Southern analysis with 10 cDNA probes that cover the full length of the gene. The multiplex PCR analytic method detected 82% of those deletions detected by Southern analysis methods. In one of 745 analyses, the multiplex method suggested a single exon deletion, which was not confirmed by Southern analysis, representing a false-positive rate of 0.013%. CONCLUSIONS--Multiplex PCR represents a sensitive and accurate method for deletion detection of 46% of all cases of DMD/BMD. The method requires 1 day for analysis, is easy to perform, and does not use radioactive tracers. As such, multiplex PCR represents an efficient and rapid method for prenatal or postnatal diagnosis of DMD/BMD.

Double minute chromosomes and a homogeneously staining chromosome region in C3H10T1/2 murine cells transformed "in vitro" by proton radiation.

Four foci (type II or type III) of transformed cells, isolated from the murine line C3H10T1/2 after exposure to proton radiations, were expanded and cytogenetically examined. While the overall numerical chromosome distributions were similar, there were some differences between the various cell lines with regard to the presence and frequency of specific-marker chromosomes and to the colony-forming efficiency in soft-agarose medium. No association between any of these markers and the transformed phenotype could be established. However, in the line F4, derived from a type II focus, numerous double-minute chromosomes (DM) were observed after passage 22, and the phenomenon became more pronounced in the subclone C2. The finding of DMs in radiation-transformed cells is unusual. The DMs were observed in long-term subcultures, and in one of them they were partially replaced by a homogeneously staining chromosome region (HSR). DNAs from transformed cells of the line F4 and subclone C2 was digested with restriction enzymes and analyzed by Southern blotting with probes for seven oncogenes commonly amplified in cancer cells (c-myc, N-myc, N-ras, Ki-ras, Ha-ras, c-myb, c-abl) and with probes for the mouse MHC class I region. None of the regions tested was structurally altered or amplified in these transformed cells. The origin of the genetic material carried by DMs or homogeneously staining intrachromosomal regions (HSR) in cells of the line F4 and subclone C2, where it is believed to provide a selective advantage for in vitro growth, remains unknown.