The cloning of FRAXF: trinucleotide repeat expansion and methylation at a third fragile site in distal Xqter.

Three fragile sites, FRAXA, FRAXE and FRAXF lie in the Xq27-28 region of the human X chromosome. The expression of FRAXA is associated with the fragile X syndrome, the most prevalent form of inherited mental retardation whilst the expression of FRAXE is associated with a rarer and comparatively milder form of mental handicap. Both the FRAXA and FRAXE sites have been cloned and the fragile site expression found to be due to the expansion of analogous CGG/GCC trinucleotide repeat arrays. We describe here the cloning of the third fragile site, FRAXF, and demonstrate that it involves the expansion of a (GCCGTC)n(GCC)n compound array. PCR analyses across the repeat of normal individuals show that the number of triplets in the array ranges from 12-26 and the most common allele consists of 14 triplet units. Sequencing analyses show that 95% of normal individuals have three copies of the GCCGTC motif and in these individuals, the size variation observed by PCR is due to copy number alterations in the GCC array. In a cytogenetically positive male with developmental delay, the array is expanded by > 900 triplets and the adjacent CpG-rich region is methylated. The array is also expanded in cytogenetically positive carrier females from the family originally used to define the FRAXF site. We conclude that the expanded array corresponds to the FRAXF fragile site.

Holt-Oram syndrome is a genetically heterogeneous disease with one locus mapping to human chromosome 12q.

Holt-Oram syndrome (HOS) is an autosomal dominant condition affecting the heart and upper limbs. We have sought to identify the location of this gene using microsatellite DNA markers in a linkage study. Of seven families analysed, five show linkage between HOS and markers on chromosome 12q. But the two remaining families, phenotypically indistinguishable from the others, do not show this linkage. Analysis with the computer program HOMOG indicates that HOS is a heterogeneous disease. Our analysis places one HOS locus in a 21 cM interval in the distal region of chromosome 12q. The localization of a gene for HOS, reported here, represents an important step towards a better understanding of limb and cardiac development.

Regulation of expression of a Xenopus borealis embryonic/larval alpha 3 skeletal-actin gene.

We have isolated a genomic clone, related in sequence to the skeletal-actin gene sub-family. It is expressed in the skeletal muscle of embryos from the neurula stage onwards and in tadpoles, but not in adults. The equivalent Xenopus laevis gene is expressed as a major transcript in adult muscle, as well as at earlier stages. The intron/exon structure is typical of vertebrate skeletal-actin genes, as is the possession of multiple copies of three serum-response elements in the promoter of this gene. The Xenopus actin and beta-globin genes were fused in their second introns. This construct, which contained 2.4 kb of upstream sequence, was injected into fertilized eggs at the two-cell stage. It showed the normal pattern of tissue-specific transcription. Thus all of the information necessary for appropriate expression of this actin gene in the embryo is contained in the region that extends from a point 2.4 kb upstream of transcription initiation to the centre of the second exon. A series of enhancer constructs were made in which upstream regions of the actin gene were placed upstream of a X. laevis beta-globin gene. The region immediately adjacent to the promoter, containing the three serum-response elements, was able to drive muscle-specific expression, and there was also a general enhancement of transcription by regions further upstream.

Sequences of junction fragments in the deletion-prone region of the dystrophin gene.

The Duchenne muscular dystrophy locus is remarkable in that it shows a high mutation rate and the majority of mutations found are deletions. These deletions are generated as meiotic as well as mitotic events and occur preferentially in the central region of the gene. Nothing is known so far about the mechanisms involved. This paper reports the first sequencing of deletion junctions in the dystrophin gene. The data from a study of two patients with deletions in the central region of dystrophin show the breakpoints to lie in regions of introns in which stretches of dA-dT are seen. The relationship between these observations and possible mechanisms for the mutations is discussed.

Molecular analysis of muscular dystrophy.

It is now possible to map almost any disease locus to a chromosomal region in the human genome by family studies with restriction fragment length polymorphisms. Duchenne and Becker muscular dystrophies have been shown to be localized within the same small region of Xp21 on the human X chromosome. Myotonic dystrophy has been localized to a region close to the centromere of chromosome 19. Technologies are now available to identify candidate genes for the diseases. Autosomal recessive muscular dystrophies are more difficult to study, but even these will be amenable to analysis in the very near future. The next decade should witness some exciting advances in the molecular analysis and clinical management of human muscular dystrophies.

Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies.

Fetal muscle cDNA clones covering at least 11.4 kb of the Duchenne muscular dystrophy (DMD) gene sequence were used to identify a deletion-prone region in DNA from DMD and Becker muscular dystrophy (BMD) patients. Of 36 BMD cases, 17 (47%) had deletions and all of the deletions began in the same intron of the gene. Of 107 DMD patients, 27 (25%) were deleted for this region, and 19 deletions originate in the same intron. Using a cDNA probe for an adjacent region of the gene, 32 new deletions were detected in DMD patients (total 44%). No new BMD deletions were detected. The DMD deletions were very heterogeneous. Thus two cDNA probes covering 2.4 kb could detect 53% of these deletions. Considering the whole locus, DMD and BMD are caused by a deletion of the gene sequence in at least 67% of cases.

Cytoskeletal actin gene families of Xenopus borealis and Xenopus laevis.

We have sequenced the coding and leader regions, as well as part of the 3' untranslated region, of a Xenopus borealis type 1 cytoskeletal actin gene [defined according to the arrangement of acidic residues at the N-terminus; Vandekerckhove et al. (1981) J Mol Biol 152:413-426]. The encoded amino acid sequence is the same as the avian and mammalian beta (type 1) cytoskeletal actins, except for an isoleucine at position 10 (as found in the mammalian gamma cytoskeletal actins), and an extra amino acid, alanine, after the N-terminal methionine. Five introns were found, in the same positions as those of the rat and chicken beta-actin genes. The 5' and 3' untranslated regions resemble those of the human gamma (type 8) cytoskeletal actin gene more closely than the mammalian beta genes. Primer extension showed that this type 1 gene is transcribed in ovary and tadpole. Sequencing of primer extension products demonstrated two additional mRNA species in X. borealis, encoding type 7 and 8 isoforms. This contrasts with the closely related species Xenopus laevis, where type 4, 5, and 8 isoforms have been found. The type 7 isoform has not previously been found in any other species. The mRNAs of the X. borealis type 1 and 8 and X. laevis type 5 and 8 isoforms contain highly homologous leaders. The X. borealis type 7 mRNA has no leader homology with the other mRNA species and, unlike them, has no extra N-terminal alanine codon. The evolutionary implications of these data are discussed.

Duchenne and Becker muscular dystrophy mutations: analysis using 2.6 kb of muscle cDNA from the 5' end of the gene.

We have isolated overlapping human fetal muscle cDNAs encompassing 2.6kb which are localised very close to the 5' end of the Duchenne muscular dystrophy (DMD) gene. Using DNA from patients with deletions of previously reported genomic probes, we have mapped the exons across the region. Investigation of deletions in both DMD and Becker muscular dystrophy (BMD) patients shows the deletions to be present in 10% of cases and heterogeneous.

Effective strategy for prenatal prediction of Duchenne and Becker muscular dystrophy.

Deletions in the gene sequence for Duchenne (DMD) and Becker (BMD) muscular dystrophy were detected in affected males with four cDNA probes, Cf56a, Cf23a, Ca1A, and Cf27. Most of the deletions were seen with only one of the probes. Cf23a detected all BMD deletions seen with Cf56a and some that were not. The same markers also detected restriction fragment length polymorphisms for those cases where deletions were not evident. The probes were also used successfully for prenatal diagnosis in two families each with two DMD affected males. In DMD families successive application of probes Cf56a, Ca1A, and Cf27 will give a 70% chance of detecting the mutation. BMD families should first be screened with the Cf23a probe.

Deletions of fetal and adult muscle cDNA in Duchenne and Becker muscular dystrophy patients.

We have isolated a cDNA molecule from a human adult muscle cDNA library which is deleted in several Duchenne muscular dystrophy patients. Patient deletions have been used to map the exons across the Xp21 region of the short arm of the X chromosome. We demonstrate that a very mildly affected 61 year old patient is deleted for at least nine exons of the adult cDNA. We find no evidence for differential exon usage between adult and fetal muscle in this region of the gene. There must therefore be less essential domains of the protein structure which can be removed without complete loss of function. The sequence of 2.0 kb of the adult cDNA shows no homology to any previously described protein listed in the data banks although sequence comparison at the amino acid level suggests that the protein has a structure not dissimilar to rod structures of cytoskeletal proteins such as lamin and myosin. There are single nucleotide differences in the DNA sequence between the adult and fetal cDNAs which result in amino acid changes but none that would be predicted to change the structure of the protein dramatically.

Preferential deletion of exons in Duchenne and Becker muscular dystrophies.

Duchenne and Becker muscular dystrophy (DMD and BMD) genes are located in Xp21 on the short arm of the X chromosome. DMD patients display a much more severe clinical course than BMD patients, and yet about 10% of cases of each have been reported to have deletions for parts of the gene. Using a complementary DNA subclone of the DMD gene we have screened 66 DMD and BMD patients who had not previously shown deletions with the probes then available. Fifteen patients have a deletion of this part of the gene, indicating a higher deletion frequency in this region (22%). Exons were deleted in five severely affected DMD patients and in ten BMD patients. Significantly, most of these deletions begin in the same region of the cDNA, which implies that there is a common mechanism for the generation of many of these mutations. An apparently identical deletion in one family gave classical BMD in two brothers (presenting in their teens) and only very mild muscle weakness in their 86-year-old great-great-uncle. Taking these data together with data using the probes previously published, we are able to detect deletions directly in 40% of our families requiring antenatal diagnosis or carrier detection.

The mapping of a cDNA from the human X-linked Duchenne muscular dystrophy gene to the mouse X chromosome.

The recent discovery of sequences at the site of the Duchenne muscular dystrophy (DMD) gene in humans has opened up the possibility of a detailed molecular analysis of the genes in humans and in related mammalian species. Until relatively recently, there was no obvious mouse model of this genetic disease for the development of therapeutic strategies. The identification of a mouse X-linked mutant showing muscular dystrophy, mdx, has provided a candidate mouse genetic homologue to the DMD locus; the relatively mild pathological features of mdx suggest it may have more in common with mutations of the Becker muscular dystrophy type at the same human locus, however. But the close genetic linkage of mdx to G6PD and Hprt on the mouse X chromosome, coupled with its comparatively mild pathology, have suggested that the mdx mutation may instead correspond to Emery Dreifuss muscular dystrophy which itself is closely linked to DNA markers at Xq28-qter in the region of G6PD on the human X chromosome. Using an interspecific mouse domesticus/spretus cross, segregating for a variety of markers on the mouse X chromosome, we have positioned on the mouse X chromosome sequences homologous to a DMD cDNA clone. These sequences map provocatively close to the mdx mutation and unexpectedly distant from sparse fur, spf, the mouse homologue of OTC (ornithine transcarbamylase) which is closely linked to DMD on the human X chromosome.

Tissue-specific expression of actin genes injected into Xenopus embryos.

We have isolated a complete Xenopus borealis cardiac actin gene, which is normally expressed in the myotomes and heart of the embryo and tadpole. After injection into the zygote, this cloned gene becomes distributed throughout the embryo, but it is expressed almost wholly in the myotomes. The same wide distribution of injected DNA but spatially restricted pattern of expression is found with a fusion between the first two actin gene exons and the last exon of a mouse beta-globin gene. By contrast, a histone-globin fusion gene is expressed fairly uniformly in all regions. We discuss the special advantages of using Xenopus in studies of tissue-specific gene expression from injected, cloned genes in early development.