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.

Service provision of complex mutation analysis: a technical and economic appraisal using dystrophin point mutation analysis as an example.

Duchenne muscular dystrophy (DMD) results from mutations in the dystrophin gene. One-third of cases arise from point mutations, which are heterogeneous and difficult to detect. The aims of this study of dystrophin point mutation analysis were to assess its technical feasibility in a routine diagnostic laboratory, and to estimate its costs and clinical benefits. The methods used were a laboratory based study using reverse transcription-polymerase chain reaction (RT-PCR) and a protein truncation test, and a mathematical model to estimate costs and clinical benefits. None of the cases analyzed had an identifiable dystrophin deletion or duplication. They were 12 males affected with DMD and two obligate female carriers; two female carriers of known dystrophin point mutations were also analyzed. Point mutations were detected in six out of 12 males, but in none of the female carriers. Assuming a sensitivity of 50% the model predicts significant clinical benefits of point mutation analysis over linkage analysis, including a reduction in the number of prenatal diagnoses (by 0.77 per family), terminations of pregnancy (by 0.18 per family), and terminations of unaffected fetuses (by 0.16 per family). The mean cost of point mutation analysis to prevent the termination of an unaffected fetus is 6220 US dollars.

Primary torsion dystonia due to the Tor1A GAG deletion in an Irish family.

BACKGROUND: Early, limb-onset primary torsion dystonia (PTD) is commonly due to a trinucleotide GAG deletion in the TOR1A (DYT1) gene on chromosome 9q34. The majority of carriers of this mutation conform to a characteristic phenotype that is similar in different ethnic populations. AIM: To describe the clinical features of affected members of a large Irish family with PTD due to the TOR1A deletion. METHODS: Fourteen consenting family members from three generations were examined according to a standardised protocol. RESULTS: Five affected individuals were identified. Two had a somewhat atypical phenotype with focal and segmental upper-limb dystonia without further progression. CONCLUSION: The authors describe the clinical features of PTD due to the TOR1A GAG deletion in an Irish family illustrating the presence of intrafamilial phenotypic variability.

Cross-modal links in endogenous spatial attention are mediated by common external locations: evidence from event-related brain potentials.

Recent behavioural and event-related potential (ERP) studies reported cross-modal links in spatial attention between vision, audition and touch. Such links could reflect differences in hemispheric-activation levels associated with spatial attention to one side, or more abstract spatial reference-frames mediating selectivity across modalities. To distinguish these hypotheses, ERPs were recorded to lateral tactile stimuli, plus visual (experiment 1) or auditory stimuli (experiment 2), while participants attended to the left or right hand to detect infrequent tactile targets, and ignored other modalities. In separate blocks, hands were either in a crossed or uncrossed posture. With uncrossed hands, visual stimuli on the tactually attended side elicited enhanced N1 and P2 components at occipital sites, and an enhanced negativity at midline electrodes, reflecting cross-modal links in spatial attention from touch to vision. Auditory stimuli at tactually attended locations elicited an enhanced negativity overlapping with the N1 component, reflecting cross-modal links from touch to audition. An analogous pattern of results arose for crossed hands, with tactile attention enhancing auditory or visual responses on the side where the attended hand now lay (i.e. in the opposite visual or auditory hemifield to that enhanced by attending the same hand when uncrossed). This suggests that cross-modal attentional links are not determined by hemispheric projections, but by common external locations. Unexpectedly, somatosensory ERPs were strongly affected by hand posture in both experiments, with attentional effects delayed and smaller for crossed hands. This may reflect the combined influence of anatomical and external spatial codes within the tactile modality, while cross-modal links depend only on the latter codes.

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.

The parental origin of de novo X-autosome translocations in females with Duchenne muscular dystrophy revealed by M27 beta methylation analysis.

The parental origin of 3 de novo X-autosome translocations in females with Duchenne Muscular Dystrophy (DMD) was studied by means of methylation analysis using the X-linked probe M27 beta. In all three the translocation was found to be paternal in origin. The parental origin of X-autosome translocations in females with and without DMD is compared with other structural abnormalities of the X and with autosomal translocations.

Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus.

The 37-amino acid peptide called amylin is a major component of the islet amyloid deposited in the pancreases of persons with type 2 diabetes mellitus. We report the isolation of a partial cDNA clone and a phage lambda genomic clone of the coding region of the amylin gene. The DNA sequence encodes a protein sequence identical to that of amylin isolated from the amyloid found in the diabetic pancreas and shows that amylin is likely to be synthesized as a precursor peptide, now named proamylin. We have demonstrated that the amylin gene is present on chromosome 12 and that it is probably transcribed in the islets of Langerhans. The sequences of the genes for amylin and the calcitonin gene-related peptides (CGRPs) show strong similarity, especially over their 5' coding regions, where both peptides have a conserved intramolecular disulfide bridge, and also over their 3' coding regions, where the presence of a glycine codon strongly suggests that the carboxyl-terminal residue of amylin, like that of CGRP, is amidated. To examine the functional relevance of these posttranslational modifications, the biological activity of amylin synthesized with or without the disulfide bridge and/or amidation was measured. It was found that both features are necessary for full biological activity, thereby confirming the functional importance of those regions of the molecule whose sequences are conserved at both protein and genetic levels.

Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q.

The localization to 19q of the gene causing myotonic dystrophy (DM) has been defined more precisely by refinement of the physical location of several linked markers. A somatic cell hybrid mapping panel from cells with t(1;19), t(12;19), and t(X;19) translocation products was constructed to define five different intervals across 19q. In addition, we have derived a series of cell hybrids by irradiation of a der(19)-only hybrid to further subdivide the cen-q13.1 region. Using an array of 36 cloned genes, anonymous DNAs, and enzyme markers, we have tested the location of the panel breakpoints and refined the regional assignment of several of these markers. All markers tightly linked to DM are localized mainly within 19q13.2, thus suggesting that the DM gene is also close to this region.

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy.

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.

Spontaneous internal jugular vein thrombosis and venous-stasis retinopathy.

A case is presented in-which the detection of venous-stasis retinopathy in one eye led to investigation of the carotid circulation. There were no neurologic symptoms of carotid insufficiency, and noninvasive tests failed to reveal significant carotid pathology. Digital subtraction angiography and carotid angiography demonstrated a carotid plaque of doubtful significance. At carotid endarterectomy, the venous-stasis retinopathy was found to be associated with venous thrombosis distant from the eye and in the internal jugular vein. This site is beyond the range over which isolated ocular vascular effects would be expected and was thought to be unrelated to the hemodynamically insignificant, nonulcerated carotid artery plaque. The possibility of this association being causal is discussed.

Clinical significance of hemorrhages in the optic disc.

A consecutive series of 8029 first visit optometric examinations revealed 20 patients having hemorrhages of the optic disc. Assessment of optic disc contour, intraocular pressure (IOP), and visual fields was undertaken at detection of the hemorrhage and at follow-up not less than 3 months later. The disc hemorrhage patients were assessed for the presence of systemic occlusive disease. The sensitivity of disc hemorrhage as a sign of glaucoma was 0.3 in the sample. There was some risk of visual field loss after detection of a hemorrhage in eyes having glaucoma (1 of 6 eyes); a similar risk was observed in non-glaucomatous eyes in which bleeding occurred (4 of 14 eyes). Hemorrhages occurred in 8 eyes that remained clinically normal during the course of follow-up. Notching of the neural rim of the disc was common but overall changes in contour of the disc were observed in only one patient. An association between disc hemorrhage and systemic disease could not be established.