Search PubMed⌕ Search

Biomedical subjects

S Humphries

Publications and source records attributed to S Humphries.

At least 109 records · Page 6Linked to original sources
PubMed1988

The use of recombinant DNA techniques for the diagnosis of familial hypercholesterolaemia.

In the UK, about 5% of patients with familial hypercholesterolaemia have a detectable deletion or rearrangement of part of the LDL-receptor gene. This results in the detection of shorter or abnormal sized fragments of the LDL-receptor gene in a Southern blot hybridization. This can be used to follow the inheritance of the defective gene, and for diagnosis in the families of these individuals. In the families of the rest of the patients, diagnosis may be possible using linked restriction fragment length polymorphisms (RFLPs) detected with the LDL-receptor probe. There are now ten common RFLPs of the LDL-receptor gene, with variable sites in the 3' half of the gene. Over 80% of patients are heterozygous for at least one of these RFLPs, and therefore potentially informative for DNA diagnosis. For a foetus at risk of homozygous familial hypercholesterolaemia, antenatal diagnosis may also be possible using these methods. However, family studies require samples to be available from affected or unaffected relatives of the patient, and this limits the applicability of the tests. For some mutations, the base pair change causing the defect in the LDL-receptor itself creates or destroys a site for a restriction enzyme. Such 'mutation-specific' RFLPs could be used for population screening, but so far have only been reported for the familial hypercholesterolaemia mutation that is common in Lebanon. In the future it may be possible to develop mutation-specific oligonucleotide probes for the diagnosis of familial hypercholesterolaemia. These would be appropriate for population screening or screening patients with hyperlipidaemia. This information may be useful if different mutations require different therapeutic strategies.

DNA, Recombinant↗
PubMed1988

A cladistic analysis of phenotype associations with haplotypes inferred from restriction endonuclease mapping. II. The analysis of natural populations.

Genes that code for products involved in the physiology of a phenotype are logical candidates for explaining interindividual variation in that phenotype. We present a methodology for discovering associations between genetic variation at such candidate loci (assayed through restriction endonuclease mapping) with phenotypic variation at the population level. We confine our analyses to DNA regions in which recombination is very rare. In this case, the genetic variation at the candidate locus can be organized into a cladogram that represents the evolutionary relationships between the observed haplotypes. Any mutation causing a significant phenotypic effect should be imbedded within the same historical structure defined by the cladogram. We showed, in the first paper of this series, how to use the cladogram to define a nested analysis of variance (NANOVA) that was very efficient at detecting and localizing phenotypically important mutations. However, the NANOVA of haplotype effects could only be applied to populations of homozygous genotypes. In this paper, we apply the quantitative genetic concept of average excess to evaluate the phenotypic effect of a haplotype or group of haplotypes stratified and contrasted according to the nested design defined by the cladogram. We also show how a permutational procedure can be used to make statistical inferences about the nested average excess values in populations containing heterozygous as well as homozygous genotypes. We provide two worked examples that investigate associations between genetic variation at or near the Alcohol dehydrogenase (Adh) locus and Adh activity in Drosophila melanogaster, and associations between genetic variation at or near some apolipoprotein loci and various lipid phenotypes in a human population.

Alcohol Dehydrogenase↗
PubMed1988

Four DNA polymorphisms in the LDL receptor gene: their genetic relationship and use in the study of variation at the LDL receptor locus.

We have studied four different restriction fragment length polymorphisms (RFLPs) for the LDL receptor gene, detected using the restriction enzymes StuI, PvuII, ApaLI, and NcoI, in normal subjects and in patients with familial hypercholesterolaemia (FH) from London. Significant linkage disequilibrium was detected between all four RFLPs. Used together they give a polymorphism information content (PIC) of greater than 0.7 which makes them useful for studying the inheritance of the LDL receptor gene in more than 70% of families with FH. The NcoI and ApaLI RFLPs were found to be the most useful, giving a combined PIC value of 0.6. The allele frequencies of all four polymorphisms were compared in the normal and FH groups and the frequency of the rarer N2 allele of the NcoI RFLP was found to be significantly higher in the FH group. This suggests that a mutation has occurred on the rare NcoI N2 allele and that it may be making a significant contribution to the defects causing FH in this patient group. We have also used these RFLPs to look for evidence that variation at the LDL receptor gene locus contributes to the determination of cholesterol levels in the normal population. People with different RFLP genotypes do not have significantly different levels of serum total or LDL cholesterol. At present we have no evidence that variation at this locus may be determining cholesterol levels in the non-FH population.

Alleles↗
PubMed1988

Genetic evidence from two families that the apolipoprotein B gene is not involved in abetalipoproteinemia.

Abetalipoproteinemia (ABL) is a recessive disorder in which affected individuals have extremely low or undetectable levels of serum apo B-containing lipoproteins. Using restriction fragment length polymorphisms, we have studied two families, each with two children with classical ABL born of normal parents. In each of these families, the two affected children have inherited different apo B alleles from at least one parent, whereas the siblings would be anticipated to share common alleles if this disorder were due to an apo B gene mutation. This linkage study shows that in these families, the apo B gene is discordant with ABL and therefore the disorder is caused by a defect in another gene, which is important for the normal synthesis or secretion of apo B-containing lipoproteins from both the liver and intestine.

Abetalipoproteinemia↗
PubMed1987

Unequal crossing-over between two alu-repetitive DNA sequences in the low-density-lipoprotein-receptor gene. A possible mechanism for the defect in a patient with familial hypercholesterolaemia.

We have previously identified a patient with familial hypercholesterolaemia (FH), where the defect appears to be caused by a deletion in the 3' region of the low-density lipoprotein (LDL)-receptor gene. We have now isolated the LDL-receptor gene from the patient and have studied the defect at the DNA level. Restriction mapping and sequence analysis demonstrate that a 4-kb DNA deletion has occurred between two alu-repetitive sequences that are in the same orientation, one in intron 12 and the other in intron 14. This deletion eliminates exons 13 and 14, and changes the reading frame of the resulting spliced mRNA such that a stop codon is created in the following exon. Immuno- and ligand-blot analysis using cultured fibroblasts from this patient revealed the normal gene product, but failed to detect any smaller receptor protein. This implies that the truncated receptor protein that is synthesised is rapidly degraded. We suggest that in this patient the deletion is caused by an unequal crossing-over event that occurred between two homologous chromosomes at meiosis.

Chromosome Deletion↗
PubMed1987

Identification of deletions in the human low density lipoprotein receptor gene.

DNA samples from 70 unrelated UK patients with heterozygous familial hypercholesterolaemia were screened by Southern blot hybridisation with a 5' fragment of the human low density lipoprotein (LDL) receptor cDNA. In the majority of cases, the restriction fragment pattern of the LDL receptor gene was indistinguishable from that observed in normal subjects. However, three patients were found to have a deletion of approximately 1 kb in the central portion of the gene. Mapping experiments indicated that in two patients a similar deletion has occurred that includes all or part of exon 5, and in the third patient a deletion has occurred that includes exon 7. Taking into account our previously described patient with a deletion in the 3' part of the gene, this means that in four out of 70 UK patients with familial hypercholesterolaemia (6%), the defect is caused by a detectable deletion of part of the coding portion of the low density lipoprotein receptor gene.

Chromosome Deletion↗
PubMed1985

Allelic variation adjacent to the human insulin and apolipoprotein C-II genes in different ethnic groups.

We have used DNA probes for the human insulin gene and apolipoprotein C-II (apo C-II) gene to determine the extent of allelic variation in different ethnic groups. The distribution of an apo C-II DNA polymorphism revealed by the restriction endonuclease Taq I showed no significant variation amongst racial groups; in contrast, an insulin gene-related DNA polymorphism showed marked variability. In Japanese, Chinese, and Asian Indian groups there was an increased frequency of homozygosity for the class 1 allele compared to Caucasian groups (P less than 0.001, P less than 0.01, and P less than 0.05, respectively). In Caucasian, Japanese, Chinese, and Asian Indian groups no class 2 allele was observed; but in the Negroid populations (African and West Indian) the class 2 allele frequencies were 0.23 and 0.25 respectively. Possible reasons for this variation in allele distribution are considered in relation to disease associations.

Alleles↗
PubMed1985

Construction and partial characterization of a human liver cDNA library.

Total messenger RNA (mRNA) was isolated from adult human liver and copied to give complementary DNA (cDNA) with reverse transcriptase. Double stranded cDNA was cloned by GC tailing into the PstI site of the plasmid pAT153; approximately 2000 recombinant clones were isolated. The sensitivity of the bacterial cells to ampicillin was used as a marker to study the stability of the library during consecutive overnight growth in mixed liquid culture. A significant change in the composition of the library was observed after three overnight cultures. A heterologous rat albumin cDNA recombinant and two specific oligonucleotides were used to screen the library, and clones containing sequences of the human mitochondrial rRNA- and the alpha-1 antitrypsin-genes were isolated.

Albumins↗
PubMed1984

Polymorphisms in the 5'-flanking region of the insulin gene and non-insulin-dependent diabetes.

DNA insertions in the 5'-flanking region of the insulin gene have been studied by Southern-blot-hybridization techniques using a cloned gene probe in 159 unrelated Caucasians. Subjects homozygous for a large DNA insertion at this locus were found to have a higher risk of concurrence of non-insulin-dependent diabetes (NIDD) than controls (P less than 0.01). Analysis of a single large NIDD pedigree (n = 67 in four generations; 11 diabetic subjects) showed no linkage between the size of DNA inserts and diabetes (Lod score = -5.7), suggesting a separate diabetogenic locus (or loci) in this family.

DNA Transposable Elements↗
PubMed1984

The implications of genetic variation in human pathology.

Each human cell contains enough DNA to code for several million proteins. As a result of the technical advances which are broadly described as 'genetic engineering', each of these sequences can be isolated and purified in large amounts, free from those surrounding it in the genome. The sequences are not identical from person to person, nor even for each of a pair of chromosomes for a single person, but contain many variations. These include single base changes in both coding and non-coding regions, deletions, insertions and rearrangements. Using these changes as markers, the molecular geneticist can compare the structure and expression of normal and mutated human gene sequences and follow their inheritance in families.

Cloning, Molecular↗
PubMed1983

Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen.

cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen.

Adult↗
PubMed1983

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.

Adult↗
PubMed1980

The isolation of cloned cDNA sequences which are differentially expressed in human lymphocytes and fibroblasts.

Poly(A)+ RNA populations derived from normal lymphocytes and fibroblasts have been compared by hybridising each RNA to cDNA derived from the other RNA population. This indicated that approximately 75% of the sequences were common to both, and that these were present at different concentrations in the two cell types. The two RNA populations were further compared by hybridising them to a cDNA recombinant library derived from lymphocyte poly(A)+ RNA. This allowed the identification of clones containing sequences which are abundant in lymphocyte poly(A)+ RNA but absent or rare in fibroblast poly(A)+ RNA. A direct estimation of the abundance of five of these sequences in lymphocyte cDNA demonstrated that clones can be detected by such a procedure if they represent 0.2% or greater of the original cDNA population.

Animals↗