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R G Worton

Publications and source records attributed to R G Worton.

At least 19 recordsLinked to original sources
PubMed1992

Partial gene duplication as a cause of human disease.

Tandem duplication of large regions of DNA, including duplication of whole genes, provides a substrate for genetic evolution. Tandem duplication of smaller regions involving parts of genes is now recognized as a contributor to the mutation spectrum that results in genetic disease. In this review, more than 30 unrelated partial gene duplications that have been implicated in the genesis of human genetic disease are presented and the pathogenic effects and frequency of such duplications are summarized. The mechanisms of duplication formation are analyzed with special emphasis on the molecular details of the nucleotide sequences at the duplication junctions. Evidence to date suggests that duplication may arise from either homologous (Alu-Alu) recombination or nonhomologous recombination, the latter possibly mediated by topoisomerases. For the dystrophin gene, in which most duplications have been identified, these recombination events are intrachromosomal, suggesting that unequal sister chromatid exchange is the major mechanism.

Biological Evolution↗
PubMed1992

Duchenne muscular dystrophy: gene and gene product; mechanism of mutation in the gene.

The X-linked gene responsible for Duchenne muscular dystrophy encodes dystrophin, a high-molecular-weight cytoskeletal protein. Studies in several laboratories have revealed deletion of one or more exons in 60% of affected boys; quantitative analysis in our laboratory has detected duplication of exons in another 6%. The severe Duchenne phenotype is associated with deletions or duplications that shift the reading frame of the message, whereas the milder Becker muscular dystrophy is associated with deletions or duplications that maintain the reading frame. Patients who have neither deletion nor duplication may have nonsense mutations, one of which has been detected by predicting the site of the mutation from the size of the truncated protein. Rare females with the disease have a translocation that disrupts the dystrophin gene on one X chromosome and causes non-random inactivation of the normal X, resulting in the expression of the disease. The high frequency of new mutation provides an opportunity to study the mechanism of chromosomal rearrangement that is characteristic of the disease. Our laboratory has focused on the translocations in females and on duplications in affected males. The X-autosome translocations of affected females are all de novo events that originated in the paternal set of chromosomes. Molecular characterization of the translocation junctions revealed reciprocal translocation with both deletion and addition of nucleotides at the junction, suggestive of a breakage and reunion mechanism. Duplications studied to date are all tandem in nature and sequence analysis of duplication junctions has revealed both homologous and non-homologous recombination. Marker segregation analysis has revealed that five out of five duplications originated in a single X chromosome of one of the maternal grandparents, suggesting that the recombination event is unequal sister chromatid exchange.

Chromosome Aberrations↗
PubMed1992

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.

Amino Acids↗
PubMed1992

The role of the skeletal muscle ryanodine receptor gene in malignant hyperthermia.

Malignant hyperthermia (MH) is an inherited, potentially lethal condition in which sustained muscle contracture with attendant hypermetabolism and hyperthermia is triggered in humans, heterozygous for the gene defect, by inhalational anaesthetics and skeletal muscle relaxants, and in pigs, homozygous for the defect, by stress. Because muscle contracture could result from a defective Ca2+ release channel, we have focussed our attention on the linkage of MH to defects in the gene (RYR1) encoding the skeletal muscle Ca2+ release channel. We have cloned and sequenced human RYR1 cDNA and found restriction fragment length polymorphisms (RFLPs) in the human gene. We also localized RYR1 to human chromosome 19q13.1. Studies of the cosegregation of MH with these RFLPs established RYR1/MH linkage on human chromosome 19q13.1 (lod score of 4.2; recombinant fraction 0.0). We then sequenced MH and normal porcine RYR1 cDNAs. Mutation of C1843 to T, leading to substitution of Cys for Arg615, was the sole amino acid change noted between MH and normal animals. Linkage of this mutation to MH was established in a study of 338 informative meioses (lod score of 102; recombinant fraction 0.0). We identified the corresponding mutation in 1 of 35 human MH families studied and found cosegregation of the mutation and MH. The combination of a high lod score with crossing of a species barrier supports the causal nature of this mutation. Future studies are aimed at finding the major human MH mutations and establishing assays for their accurate diagnosis.

Amino Acid Sequence↗
PubMed1992

Dystrophin in frameshift deletion patients with Becker muscular dystrophy.

In a previous study we identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. We have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases we detected slightly lower-molecular-weight dystrophin in 12%-15% abudance relative to the normal. By sequencing amplified mRNA we have found that exon 2 is spliced to exon 8, a splice that produces a frameshifted mRNA, and have found no evidence for alternative splicing that might be involved in restoration of dystrophin mRNA reading frame in the patients with a mild phenotype. Other transcriptional and posttranscriptional mechanisms such as cryptic promoter, ribosomal frameshifting, and reinitiation are suggested that might play some role in restoring the reading frame.

Adolescent↗
PubMed1991

Molecular analysis of X-autosome translocations in females with Duchenne muscular dystrophy.

To further an understanding of the mechanism of constitutional chromosomal rearrangement, the translocation breakpoints of two X-autosome translocations carried by females with Duchenne or Becker muscular dystrophy have been mapped, cloned and sequenced. Breakpoints were mapped to specific introns within the dystrophin gene and intron sequences spanning the two breakpoints were cloned and used as probes to identify DNA fragments containing the translocation junctions. The junction-containing fragments were cloned after amplification by inverse PCR or single-specific-primer PCR. Sequence through the junctions and the autosomal regions spanning the breakpoints identified the mechanism of rearrangement as non-homologous exchange with minor additions or deletions (0-8 nucleotides) at the breakpoints. Paternal origin of these X-autosome translocations, coupled with evidence for non-transmission of X-autosome translocations through male meiosis suggested that the translocations were the result of a post-meiotic rearrangement in spermiogenesis.

Adult↗
PubMed1991

Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination.

Three tandem duplications were previously identified in patients with Duchenne muscular dystrophy and were shown in each case to have a subset of dystrophin gene exons duplicated. The origin of these duplications was traced to the single X chromosome of the maternal grandfathers, suggesting that an intrachromosomal event (unequal sister chromatid exchange) was involved in the formation of these duplications. In the present study, a DNA segment containing the duplication junction and the normal DNA that corresponds to both ends of the duplicated region have been cloned. Subsequent mapping studies confirmed the tandem arrangement (head to tail) of these duplications and revealed their sizes to be 130 kb, approximately 300 kb, and 35-80 kb, respectively. Sequence analysis of the duplication junctions showed that one duplication was due to homologous recombination between two repetitive elements (Alu sequences) and the other two were due to recombination between unrelated nonhomologous sequences. In the latter cases, the preferred cleavage sites of the eukaryotic type I and II DNA topoisomerases were found at the junctions of these duplications, suggesting a possible role of these enzymes in the chromatid exchange events. This study provides the first insight into the molecular basis of gene duplications formed through unequal sister chromatid exchange in humans.

Base Sequence↗
PubMed1991

Dystrophin is tightly associated with the sarcolemma of mammalian skeletal muscle fibers.

Sarcolemmal vesicles with right-side-out configuration were prepared from normal fresh human and rabbit skeletal muscle bundles by incubation in 140 mM KCl solution containing collagenase. The vesicles were used to examine the association of dystrophin, the protein product of the Duchenne muscular dystrophy gene, with the sarcolemma. Western blot analysis, indirect immunofluorescence, and immunoperoxidase staining using specific antibodies raised against the N-terminal and the C-terminal domains show that dystrophin remains associated with the membrane of sarcolemmal vesicles. Indirect immunofluorescence microscopy using permeabilized and unpermeabilized vesicles indicated that both the N-terminus and the C-terminus of dystrophin are localized to the cytoplasmic surface of the sarcolemma. These results suggest that dystrophin has much stronger attachment to the surface membrane than it has to the internal domain of skeletal muscle fibers. Sarcolemmal vesicles thus represent a new system for studying the function of dystrophin and the molecular basis of its association with the sarcolemma.

Amino Acid Sequence↗
PubMed1991

A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia.

Malignant hyperthermia (MH) is a devastating, potentially lethal response to anesthetics that occurs in genetically predisposed individuals. The skeletal muscle ryanodine receptor (RYR1) gene has been linked to porcine and human MH. Furthermore, a Cys for Arg substitution tightly linked to, and potentially causative of, porcine MH has been identified in the ryanodine receptor. Analysis of 35 human families predisposed to malignant hyperthermia has revealed the presence, and cosegregation with phenotype, of the corresponding substitution in a single family. This substitution, by analogy to the findings in pig, may be causal for predisposition to MH in this family.

Adolescent↗
PubMed1991

Physical mapping at a potential X-linked retinitis pigmentosa locus (RP3) by pulsed-field gel electrophoresis.

A genetic locus (RP3) for X-linked retinitis pigmentosa (XLRP) has been assigned to Xp21 by genetic linkage studies and has been supported by two Xp21 male deletion patients with XLRP. RP3 appears to be the most centromeric of several positioned loci, including chronic granulomatous disease (CGD), McLeod phenotype (XK), and Duchenne muscular dystrophy (DMD). In one patient, BB, the X-chromosome deletion includes RP3 and extends to within the DMD locus. Using a DMD cDNA, the centromeric endpoint of this patient was cloned and used as a starting point for chromosome walking along a normal X chromosome. A single-copy probe, XH1.4, positioned near the centromeric junction but deleted in BB, was used along with a CGD cDNA probe to establish a refined long-range physical map. Both probes recognized a common SfiI fragment of 205 kb. As the CGD gene covers approximately 30-60 kb, the RP3 locus has been restricted to approximately 150-170 kb. A CpG island, potentially marking a new gene, was identified within the SfiI fragment at a position approximately 35 kb from the deletion endpoint in BB.

Centromere↗
PubMed1991

Point mutation in the human dystrophin gene: identification through western blot analysis.

Using antibodies directed against the amino-terminus of dystrophin, we identified a truncated protein in a Duchenne muscular dystrophy patient. Antibodies directed against the carboxy-terminus failed to identify any cross-reactive material, a result consistent with premature termination of dystrophin translation. The estimated molecular mass of 126 kDa predicted the approximate location of the mutation in the mRNA and in the gene. Sequencing of cloned PCR products from patient muscle cDNA revealed a nonsense mutation, which was confirmed by direct sequencing of amplified patient genomic DNA. The mutation, a G to T transversion, at position 3714 changes a glutamic acid codon to an Amber stop codon. Translation of mRNA containing this mutation would be expected to result in a truncated protein with a molecular mass of 133 kDa, in close agreement with the 126 kDa estimated by Western blot analysis. This is the first reported case of a point mutation in this very large human gene.

Adolescent↗
PubMed1991

Linkage analysis in X-linked ocular albinism.

We studied the linkage of X-linked Nettleship-Falls ocular albinism (OA1) to Xp22.1-Xp22.3 RFLPs at 12 loci in five families, including one in which OA1 cosegregates with a deletion of steroid sulfatase (STS). We found evidence for tight linkage of OA1 to the Xp22.3 loci DXS143, STS, and DXS452. DXS452, a newly described polymorphism detected by the probe E25B1.8, is part of the sequence family "DXS278" (pCRI-S232), but represents a single genetic locus. Every female in this study was heterozygous for the DXS452 RFLP. Thus, this marker will be extremely useful for family studies and genetic counseling. Analysis of individual recombinations suggests that OA1 maps between DXS143 and DXS85. Multipoint linkage analysis was consistent with this localization but was not statistically significant. These data suggest that OA1 lies proximal to the deletion in a previously described family with OA1 and STS deletion, but maps within the Xp22.3-Xp22.2 region.

Albinism, Ocular↗
PubMed1991

Immunogold labelling of dystrophin in human muscle, using an antibody to the last 17 amino acids of the C-terminus.

Immunolabelling with a 10 nm gold probe was used to localize dystrophin at the ultrastructural level in human skeletal muscle. The primary antibody was raised against a synthetic peptide containing the last 17 amino acids at the C-terminus of dystrophin. Using this antibody, labelling was almost entirely confined to a narrow band enclosing 40 nm either side of the plasma membrane and including the membrane itself. Histograms of the position of the gold probe relative to the plasma membrane showed modes lying over the membrane itself or the extracellular face of the membrane. One interpretation of these results is that the C-terminus of dystrophin is inserted in the plasma membrane alongside the glycoproteins with which it is tightly associated. Histograms of the distances between gold probes displayed modes at approximately 120 nm in both transverse and longitudinal sections suggesting that dystrophin forms a lattice-like network adjacent to the plasma membrane.

Amino Acid Sequence↗
PubMed1991

A comparison of the caffeine halothane muscle contracture test with the molecular genetic diagnosis of malignant hyperthermia.

Malignant hyperthermia (MH) is currently diagnosed by the caffeine-halothane contracture (CHC) test. In a previous study, this test was used to establish linkage between the human gene for MH susceptibility and the ryanodine receptor (RYR) gene. The current study extends the genetic linkage analysis to a large French-Canadian kindred. In this family, genetic linkage between RYR and MH genes was not demonstrable using the currently recommended limits of normal for the CHC test in the identification of MH-susceptible individuals. With CHC test threshold limits below those currently recommended, however, complete linkage between the RYR and MH genes was seen. Comparisons of CHC test results with genetic linkage studies will increase the diagnostic accuracy of both tests as well as generate new insights into the biology of MH.

Alleles↗
PubMed1991

Molecular genetic approaches to the study of individual risk in alcoholism.

Genetic studies of alcoholics, their families and controls have given credence to the idea that genetic influences in alcoholism exist, and set the stage for efforts to identify alcoholism-susceptibility genes (Devor and Cloninger, 1989). My purpose is not to review the genetics of alcoholism, but rather to review the genetic approaches that have been successful in identifying the genes responsible for genetic conditions such as muscular dystrophy and cystic fibrosis. In these disorders our current knowledge of the basic biochemical defect was derived directly from the cloning of the gene that is defective in the disorder. The cloned gene provides DNA probes for carrier identification and prenatal diagnosis, while knowledge of the basic defect allows new and direct investigation of potential therapeutic strategies. The genetic approach is much less definitive when it comes to the study of polygenic or multifactorial disorders such as schizophrenia or Alzheimer's disease. In the case of alcoholism the problem is exacerbated not only by environmental factors but also by phenotypic and genetic heterogeneity. The lack of a clear inheritance pattern means that plausible modes of inheritance must be invoked and tested on families with multiple affected members. Direct segregation analysis may not be possible and the less informative analysis of sib-pairs may be the method of choice. Ultimately, however, it should be possible to identify and clone those genes that play a major role in determining susceptibility to alcoholism. Once cloned, the protein products can be identified, and study of their function should lead to new understanding of the complex biological processes involved in this disorder.

Alcoholism↗