Search PubMed⌕ Search

Biomedical subjects

R G Elles

Publications and source records attributed to R G Elles.

11 recordsLinked to original sources
PubMed2004

Mutation scanning of the NF2 gene: an improved service based on meta-PCR/sequencing, dosage analysis, and loss of heterozygosity analysis.

We describe the development and implementation of a neurofibromatosis type 2 (NF2) mutation scanning service based on novel techniques. All 17 exons of the NF2 gene are amplified in four polymerase chain reaction (PCR) reactions, using the meta-PCR technique to link the NF2 exons into chimeric concatamers. The meta-PCR products are then scanned for point mutations by direct sequencing. A four-exon dosage assay is used to test for large deletion/duplication mutations. In certain cases when tumour studies are necessary, these techniques are also combined with loss of heterozygosity analysis with three highly polymorphic microsatellite markers located within or close to the NF2 gene. Over a period of 2 years, we have applied these techniques in a service setting to the analysis of 271 patient samples (245 lymphocyte DNA; 26 schwannoma DNA). Meta-PCR and sequencing identified 90 point mutations in the 271 blood and tumor samples, 48 of which have not been reported previously. Dosage analysis identified large deletions in 12 of the lymphocyte DNA samples. In addition, over 84% of mutations were identified in 23 schwannoma DNA samples in which complete analysis was possible. Adoption of this novel strategy has increased the overall mutation detection rate in familial NF2 cases to 88% and sporadic NF2 cases to 59%. It has also allowed us to decrease our reporting turnaround times, and because of a low overall failure rate, permitted the running of an efficient and cost-effective service.

Costs and Cost Analysis↗
PubMed1999

Meta-PCR: a novel method for creating chimeric DNA molecules and increasing the productivity of mutation scanning techniques.

Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Gene patenting.

Explore the source record for details and available documents.

Base Sequence↗
PubMed1994

Diagnosis of adult polycystic kidney disease by genetic markers and ultrasonographic imaging in a voluntary family register.

Diagnosis of autosomal dominant adult polycystic kidney disease (APKD) is possible by ultrasonographic scanning (USS) or by using DNA markers linked to the PKD1 locus. Ultrasonography is complicated by the age dependent penetrance of the gene and linkage studies are subject to recombination errors owing to meiotic crossing over and locus heterogeneity. This study draws on data collected from a voluntary family register of APKD over 10 years. Records of 150 families were examined, ultrasound reports were obtained from 242 people at 50% prior risk, and 37 families were typed for DNA markers. The fraction of APKD resulting from loci unlinked to PKD1 (designated PKD2 here) was calculated at 2.94% (upper confidence limit 8.62%). Some subjects who were negative on initial scan later gave a positive scan, but there was no example of a definite gene carrier aged over 30 giving a negative scan. In families large enough for linkage analysis, most people who were at 50% prior risk could be given a final risk below 5% or above 95%, by using combined ultrasound and DNA studies.

Adolescent↗
PubMed1990

Recombination or heterogeneity: is there a second locus for adult polycystic kidney disease?

Twenty-four families with adult onset polycystic kidney disease were typed for markers flanking the PKD1 locus on chromosome 16. The aggregated results gave a significant lod score in favour of linkage to PKD1. Within this group of families two showed unusual features: recombinations, including double recombinations, and, in one family, an unexpectedly high proportion of affected people. We consider the evidence that in these families the disease might result from a mutation at a different locus, PKD2, not linked to PKD1. We suggest that a useful test is to compare the relative numbers of meioses apparently non-recombinant and doubly recombinant for markers flanking the normal disease locus, ignoring meioses recombinant for only a single marker. Using this test, neither our two families nor the data published so far on other families provide compelling evidence for the existence of a second locus for adult polycystic kidney disease. For genetic counselling in families too small to give internal evidence for or against linkage, the extra uncertainty can be handled by using a higher recombination rate.

Adolescent↗
PubMed1987

Clinic experience of prenatal diagnosis of cystic fibrosis by use of linked DNA probes.

96 families at risk of having a child with cystic fibrosis have been counselled about prenatal diagnosis by the use of linked DNA probes. Of the first 30 pregnancies 9 children have been born and confirmed to be free from CF, as predicted; 8 pregnancies were terminated (1 of these was found to be miscarrying at time of termination); 12 pregnancies, in which the fetuses are predicted to be unaffected, are in progress; and 1 has miscarried. No couple with a prenatal prediction of CF decided to continue with the pregnancy. 1 pregnancy was terminated because of a 50:50 chance of an affected fetus. In 1 twin pregnancy the affected fetus was selectively aborted, and the other baby was confirmed after birth to be unaffected, as predicted. In the other twin pregnancy the two chorionic plates could not be samples separately; the pregnancy was continued on the basis of normal findings for the lower plate, and both twins have turned out to be normal. Many couples at risk see early prenatal diagnosis as a way to help them have an unaffected child.

Abortion, Induced↗
PubMed1986

A register based system for gene tracking in Duchenne muscular dystrophy.

A total of 102 families with Duchenne muscular dystrophy has been studied with linked DNA polymorphisms as an aid to estimating carrier risks for female relatives. Early work using probes RC8, L1.28, and pXUT23 gave very little clinically useful information because of the high recombination rates between these probes and Duchenne muscular dystrophy and the low proportion of women who were heterozygous. Clinically useful results were obtained using probes 99-6, 754, and particularly pERT87. Examples are given of deductions which can be made using these probes. The importance of a genetic register is stressed as a tool for long term contact with the families and other professionals.

Female↗
PubMed1984

Clinical use of DNA markers linked to the gene for Duchenne muscular dystrophy.

Seventy families with Duchenne muscular dystrophy (DMD) known to the Institute of Child Health fall into three categories with respect to potential linkage analysis with the X chromosome DNA markers RC8 and L1.28 that bridge the DMD gene. Families in which there is at least one obligatory female heterozygote (n = 13). Here 'prediction' and 'exclusion' of DMD gene transmission may be possible, the accuracy being dependent on the closeness of the linkage of the DNA marker(s) to the DMD gene; an illustrative case is reported. Families in which there is a single affected boy, who also has one or more healthy brothers (n = 26). Given an informative restriction fragment length polymorphism (RFLP), the probability that the boy represents a new mutation can be reassessed; it is also possible to 'exclude' the DMD gene in a sister. Families with a single affected boy with no brother (n = 30). Here 'exclusion' of the DMD gene in a sister may be possible. Only in one family was there no possibility of useful linkage analysis. The linkage analysis required is described, and the need to check DMD families for informative RFLPs is stressed.

Base Sequence↗
PubMed1983

Absence of maternal contamination of chorionic villi used for fetal-gene analysis.

Chorionic villi can be obtained by direct transcervical aspiration at 9 to 10 weeks' gestation and used for analysis of fetal DNA. However, for the method to be reliable, there must be no detectable contamination by maternal DNA. To investigate the question of contamination, we compared the DNA of chorionic villi from five fetuses with that obtained from maternal lymphocytes, using the restriction endonuclease Taql and specific DNA probes for a pair of alleles on the X chromosome. The alleles yield fragments of different lengths when digested with Taql (length polymorphism), which can be demonstrated by electrophoresis and hybridization with the radioactive DNA probes. If the pattern obtained with the chorionic DNA is different from that obtained with the maternal DNA, contamination is not present. In two cases the fetal DNA of the chorionic villi was shown to be uncontaminated by maternal tissue. In one of these cases the mother was heterozygous and the fetus was homozygous; in the other the mother was homozygous and the fetus was heterozygous. In three other cases no definitive conclusions could be drawn, because the genotypes of the fetus and mother were identical. We conclude that chorionic villi at 9 to 10 weeks' gestation are a source of fetal DNA that can be used for gene analysis, with no detectable contamination by maternal DNA.

Alleles↗
PubMed1981

Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry.

A library of 50,000 recombinants representative of the human X chromosome has been constructed. Human X chromosomes were physically separated using a fluorescence-activated cell sorter. The DNA was purified from the chromosomes, digested to completion with the restriction enzyme EcoRI and cloned into the phage lambda gtWES.lambda B. The X-derived nature of the recombinants was confirmed by hybridization to rodent/human cell line DNA containing only the human X chromosome. Such libraries will be particularly useful for the investigation of genetic diseases such as Duchenne muscular dystrophy, where the basic defect has not been elucidated, and of neoplasia, where several specific chromosomal anomalies, particularly for the leukaemias, have been identified.

Bacteriophage lambda↗