The detection of large deletions or duplications in genomic DNA.

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications.

Is gene deletion in eukaryotes sequence-dependent? A study of nine deletion junctions and nineteen other deletion breakpoints in intron 7 of the human dystrophin gene.

Although large deletions comprise 65% of the mutations that underlie most cases of Duchenne and Becker muscular dystrophies, the DNA sequence characteristics of the deletions and the molecular processes leading to their formation are largely unknown. Intron 7 of the human dystrophin gene is unusually large (110 kb) and a substantial number of deletions have been identified with endpoints within this intron. The distribution of 28 deletion endpoints was mapped to local sequence elements by PCR. The break points were distributed among unique sequence, LINE-1, Alu, MIR, MER and microsatellite sequences with frequencies expected from the frequency of those sequences in the intron. Thus, deletions in this intron are not associated primarily with any one of those sequences in the intron. Nine deletion junctions were amplified and sequenced. Eight were deletions between DNA sequences with minimal homology (0-4 bp) and are therefore unlikely to be products of homologous recombination. In the ninth case, a complex rearrangement was found to be consistent with unequal recombinational exchange between two Alu sequences coupled with a duplication. We have hypothesized that a paucity of matrix attachment regions in this very large intron expanded by the insertion of many mobile elements might provoke a chromatin structure that stimulates deletions (McNaughton et al., 1997, Genomics 40, 294-304). The data presented here are consistent with that idea and demonstrate that the deletion sequences are not usually produced by homologous DNA misalignments.

Pulsed field gel electrophoresis for detection of gene rearrangements in duchenne muscular dystrophy.

In diseases with a high new mutation rate, such as Duchenne and Becker muscular dystrophy (DMD, BMD), linkage analysis often produces highly unsatisfactory results for carrier diagnosis compared to methods that rely on the direct detection of the mutation. The size of the dystrophin gene and the nature of mutations at this locus that give rise to DMD/BMD make pulsed field gel electrophoresis (PFGE) an appropriate and powerful technique for detection of mutations and hence accurate carrier diagnosis in these diseases.

Molecular characterization of further dystrophin gene microsatellites.

Microsatellites of the dystrophin gene have been used extensively in the genetic analysis of Duchenne and Becker muscular dystrophy families. The microsatellites that have been reported to date are clustered within disparate regions of the dystrophin gene, specifically at the 5'-end and in the central rod-domain. YACs encompassing the gene were screened for further microsatellites to improve the density of available genetic markers. Four microsatellites were localized to defined regions of the dystrophin gene by the analysis of patient DNA samples, somatic cell hybrids and YACs. In addition, varying combinations of microsatellite loci were amplified in multiplex PCRs, which complement those loci that have been studied to date.

Mapping of X chromosome translocation breakpoints in females with Duchenne muscular dystrophy with respect to exons of the dystrophin gene.

There are rare female patients who suffer from Duchenne or Becker muscular dystrophy because they carry an X;autosome translocation with a breakpoint in the dystrophin gene. We have defined the positions of seven of these breakpoints with respect to exon-containing HindIII fragments detected by dystrophin cDNA. One breakpoint lies between exon-containing HindIII fragments 7 and 8, five breakpoints between exon-containing HindIII fragments 31 to 41, and one lies close to exon-containing-HindIII fragment 50. The distribution of these and of a further seven translocation breakpoints whose positions are known is compared with that reported for deletions and duplications in affected males.

Assignment of apolipoprotein H (APOH: beta-2-glycoprotein I) to human chromosome 17q23----qter; determination of the major expression site.

Human apolipoprotein H (APOH) is associated with lipoprotein present in plasma. It has been shown that APOH has structural similarities with the regulation of complement activation (RCA) protein superfamily and is involved in phospholipid binding interactions on platelets and as an autoantigen in complex with anionic phospholipids. Nevertheless, additional functional studies are necessary to establish the physiological role of APOH. By hybridizing a cDNA probe for APOH to a panel of somatic cell hybrids, we show that the structural locus for this protein maps to 17q23----qter and is therefore not part of the RCA cluster on chromosome 1. The site of biosynthesis for APOH was established by Northern blot analysis. Hybridization of the APOH cDNA probe to total liver RNA identified a transcript of approximately 1.5 kb.