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S Humphries

Publications and source records attributed to S Humphries.

At least 91 records · Page 5Linked to original sources
PubMed1991

Rapid screening for specific mutations in patients with a clinical diagnosis of familial hypercholesterolaemia.

We describe a rapid screening procedure to identify known DNA sequence changes in individuals diagnosed as having heterozygous familial hypercholesterolaemia (FH). The screening is made possible by combining a rapid DNA extraction protocol and small scale polymerase chain reaction DNA amplification, followed by oligonucleotide melting or restriction enzyme digestion. We have screened for two different mutations; firstly a mutation in the apolipoprotein B (apo B) gene that results in the substitution of glutamine (Gln) for arginine (Arg) at amino acid residue 3500 (apo B3500 mutation). Apo B is the principal component of the protein moiety of low density lipoprotein (LDL) and the mutation reduces the affinity for the LDL receptor (LDL-R). Secondly we have screened for a point mutation in the LDL-R gene itself that creates a new Pst I restriction enzyme site. This mutation in the LDL-R gene (LDL-R664 mutation) results in the substitution of leucine (Leu) for proline (Pro) at amino acid 664 and is known to slow processing of the LDL-R precursor to the mature form and to reduce the affinity of the receptor on the cell surface for LDL. In 77 unrelated patients with a clinical diagnosis of FH two out of 77 (2.6%) were positive for the apo B3500 mutation. Three (3.9%) were positive for the LDL-R664 mutation. Thus these two mutations might account for 5-6% of patients in the U.K. with a clinical diagnosis of FH (5000-6000 people).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Identification of the 664 proline to leucine mutation in the low density lipoprotein receptor in four unrelated patients with familial hypercholesterolaemia in the UK.

Mutations in the gene for the low density lipoprotein (LDL) receptor cause Familial Hypercholesterolaemia (FH). One such mutation, a cytosine to thymine change in the codon for amino acid 664, causes proline (CCG) to be replaced by leucine (CTG) at this position, and creates a Pst I site in exon 14 of the gene. This mutation, previously identified in an FH homozygote of Asian Indian origin, results in a receptor with a reduced binding affinity for LDL and in delayed processing of the precursor form of the protein in cultured cells. A total of 224 unrelated heterozygous and 4 homozygous FH patients from London was screened for this mutation using direct amplification of genomic DNA by the polymerase chain reaction (PCR) and restriction digestion of the PCR product. Four patients were identified who were heterozgous for this mutation and the C to T base change was confirmed by sequencing. Affected relatives of these patients were also found to have the mutation. The effect of the mutation on LDL-receptor function in lymphoblastoid cell lines obtained from two of these patients was similar to that observed in heterozygous relatives of the original proband (MM). Eight polymorphisms of the LDL receptor gene were used to determine the haplotype of the defective allele carried by the patients and the individual (MM) in whom the mutation was first discovered. Two different haplotypes were found, suggesting that the mutation, which occurs at a CpG 'hotspot', has arisen independently at least twice. The presence of the same single base change in the LDL-receptor gene in several unrelated patients has not previously been reported in a population which is not geographically or culturally isolated.

Adolescent↗
PubMed1990

Variation in the size of human apolipoprotein(a) is due to a hypervariable region in the gene.

We have investigated whether the size heterogeneity of the human apolipoprotein(a) [apo(a)] is due to differences in the number of plasminogen kringle 4-like repeat units present in the different alleles. Using the Southern blot hybridization technique and a DNA probe for the kringle 4 domain of plasminogen, we have observed that in 31 different individuals a 5.8-kb PvuII restriction fragment band varies widely in intensity relative to other bands. A strong correlation (r = 0.76, P less than 0.001) was found between apo(a) protein size and the variation in intensity of the detected restriction fragment band. We confirmed this correlation in a large family where the parents are heterozygous for the apo(a) protein size isoforms. The specificity of the 5.8-kb band was established by using an apo(a)-specific oligonucleotide. These correlations strongly suggest that the observed size heterogeneity in apo(a) protein is due to different numbers of copies of the kringle 4 sequence in the apo(a) glycoprotein gene.

Alleles↗
PubMed1990

XbaI polymorphism of the apolipoprotein B gene influences plasma lipid response to diet intervention.

Fresh blood samples were collected from 103 North Karelians who had in 1981-84 participated in dietary intervention studies and analysis of the XbaI restriction fragment length polymorphism (RFLP) of apolipoprotein B (apoB) was carried out. Reanalysis of the original plasma lipid and apolipoprotein data indicated that while baseline concentrations did not differ significantly between genotypes, the response to a low-fat, low-cholesterol diet was influenced by apoB XbaI genotype: reductions in total and low density lipoprotein (LDL) cholesterol, apoB and high density lipoprotein (HDL) cholesterol were greater in subjects homo- or heterozygous for the presence of the XbaI cutting site (X1X2 or X2X2 genotype, designated X2+) as compared to those lacking the cutting site (X1X1 genotype, designated X2-). The corresponding average reductions induced by dietary intervention in X2+ and X2- subjects were: for total cholesterol 1.30 and 0.99 mmol/l (p = 0.036), for LDL cholesterol 1.04 and 0.78 mmol/l (p = 0.049), for apoB 18.3 and 8.1 mg/100 ml (p = 0.012) and for HDL cholesterol 0.26 and 0.17 mmol/l (p = 0.008).

Adult↗
PubMed1990

Association of DNA-haplotypes in the human LDL-receptor gene with normal serum cholesterol levels.

For the low density lipoprotein receptor (LDLR), many mutations have been characterized which identify this gene as one with an important role in lipid metabolism in patients with familial hypercholesterolemia (FH). Genetic heterogeneity at this locus raises the possibility that the LDLR may also contribute to variation in cholesterol levels in the normocholesterolemic population. We have determined genotypes at the LDLR locus using restriction fragment length polymorphisms (RFLPs) detected with the enzymes StuI, ApalI, PvuII and NcoI in 324 normocholesterolemic individuals from Germany. A significant association (p less than 0.01) was detected between the cutting site for the PvuII RFLP and lower cholesterol levels, and variation associated with this polymorphism explains 3% of the sample variance in cholesterol levels. In family studies we have determined four-RFLP haplotypes of 148 independent LDLR genes and have observed 9 haplotypes in the population. Three of these haplotypes containing the cutting site for PvuII are associated with a reduction in plasma LDL-cholesterol levels. Phylogenetic analysis indicates that these three haplotypes are related by evolutionary history, and this suggests that a single functionally important sequence change in the LDLR explains our observations. Our data confirm other reports and strongly suggest that the LDLR locus may be one of those genes involved in determining serum cholesterol levels in the normal population.

Adult↗
PubMed1990

Apolipoprotein B signal peptide insertion/deletion polymorphism is associated with Ag epitopes and involved in the determination of serum triglyceride levels.

We have investigated the insertion/deletion polymorphism in the signal peptide region of the apoB gene in 106 Finnish individuals from North Karelia. The relative frequency of the insertion allele in this sample was 0.73. Strong linkage disequilibrium was detected between this apoB insertion/deletion polymorphism and the Ag(c/g) epitope pair of apoB, while weak linkage disequilibrium was detected between the polymorphism and the four other reported Ag epitope pairs [(a1/d), (x/y), (h/i) and (t/z)], as well as the apoB PvuII and the XbaI RFLPs. Using one-way analysis of variance there was a statistically significant association (P less than 0.05) between the apoB insertion/deletion polymorphism and serum triglyceride levels in this sample. Individuals homozygous for the insertion allele had higher triglyceride levels than individuals homozygous for the deletion allele, while individuals heterozygous for the polymorphism had intermediate levels. These differences were reduced when individuals were consuming a low fat diet but were statistically significant when the individuals returned to their normal diet. It is possible that insertion or deletion of three hydrophobic amino acids (leu-ala-leu) from the signal peptide of apoB may have a direct effect on plasma triglyceride levels by altering the intracellular processing of apoB or apoB-containing lipoproteins in the liver or intestine.

Adult↗
PubMed1990

G to A substitution in the promoter region of the apolipoprotein AI gene is associated with elevated serum apolipoprotein AI and high density lipoprotein cholesterol concentrations.

We have determined the sequence of 250 bases 5' of the transcriptional start site of the apolipoprotein AI gene in a human individual with high serum concentrations of apo AI. One of the alleles contained a G to A substitution at position -75, between the CACAT sequence and the TAAATA box, creating a tandem repeat, CAGGGC-CA*GGGC. The substitution destroys an MspI cutting site, and the polymerase chain reaction and MspI digestion were used to identify the presence of the A or G base. The frequency of the A substitution in 96 healthy men from Bristol was 0.11 and this was increased to 0.25 in men with serum apo AI concentrations greater than 180 mg/dl. Men with the A allele had significantly higher serum concentrations of apo AI, high density lipoprotein (HDL) cholesterol and HDL2 than those with the G allele. In this sample, variation associated with the G to A substitution accounted for 6% and 4.6% of the total variance in apo AI and HDL cholesterol concentrations, respectively. Although there is as yet no functional proof, it is possible that the A substitution may be having a direct positive effect on the rate of apo AI gene transcription and thus be associated with increased apo AI and HDL cholesterol concentrations because of increased production of apo AI protein from the liver or intestine.

Alleles↗
PubMed1989

Four DNA polymorphisms in the LDL-receptor gene and their use in diagnosis of familial hypercholesterolemia.

To examine the potential usefulness of restriction fragment length polymorphisms (RFLPs) for diagnosis of familial hypercholesterolemia (FH), we determined the genotype of FH patients and their relatives for the Apa1I, NcoI, PvuII and StuI RFLP of the LDL-receptor gene in a sample of German patients attending the Lipid Clinic in Munich. There was no significant difference in the relative allele frequency between the group of FH patients and controls for any of the four polymorphisms. Using linkage analysis, we could determine the four-RFLP haplotypes of 39 defective and 90 normal LDL-receptor genes in 38 FH families. In our sample, defective LDL-receptor genes occur on 6 different chromosomes determined by the four RFLPs. This suggests that at least 6 different genetic defects may cause FH in this sample. RFLPs of the LDL-receptor gene cannot be used to detect FH in individuals; however, appropriate diagnosis can be carried out in more than 90% of families using linkage analysis and these RFLPs.

Gene Frequency↗
PubMed1989

The gene for the Lp(a)-specific glycoprotein is closely linked to the gene for plasminogen on chromosome 6.

We have studied the segregation of the Lp(a) glycoprotein phenotypes and of the plasminogen (PLG) polymorphism in three two-generation families. The inheritance of the Lp(a) gene was followed using the Lp(a) glycoprotein size polymorphism and that of the plasminogen gene, using protein and DNA polymorphisms. In the three families studied, no recombination was observed in 18 meioses. The lod score for linkage between the Lp(a) glycoprotein locus and the plasminogen locus in these families is greater than 5.0 at a recombination fraction of theta = 0. Our results show that the structural gene for the Lp(a) glycoprotein is closely linked to the gene for plasminogen on chromosome 6.

Chromosomes, Human, Pair 6↗
PubMed1989

A radiographic investigation into bone resorption of mandibular alveolar bone in elderly edentulous adults.

Clinical experience suggests that severe alveolar bone resorption can limit the success of complete denture wearing. The loss of bone mineral with age occurs throughout the skeleton, which contributes to the high incidence of bone fractures later in life. Women are more commonly affected by some of these fractures. The decline in serum oestrogen concentration following the menopause has been implicated in the aetiology of these fractures. We have found that age is important in determining the bone resorption observed in females but not in males.

Aged↗
PubMed1989

Control of plasma fibrinogen levels.

It is clear that the control of plasma fibrinogen levels is complex, involving not only many environmental factors such as alcohol intake, smoking habit, age, obesity and the acute phase response, but also genetic factors as shown by the association of the Bcl I RFLP of the beta-fibrinogen gene with plasma fibrinogen levels. The advent of recombinant DNA technology has made the dissection of the different factors controlling plasma fibrinogen levels a valid proposition, and great progress is already being made. The goals of this research are twofold. First, it may be possible to develop DNA tests to identify individuals who, on the basis of their genotype, are at high risk of ischaemic heart disease. Once identified, the subsequent risk of these individuals can be reduced by modifying life-style or by drug therapy to reduce other known risk factors such as cholesterol levels. Second, once the mechanisms controlling fibrinogen concentration are better understood at the molecular level, it may be possible to develop directed therapeutic strategies that will reduce fibrinogen synthesis in a specific manner, an approach that is not possible at present. In the future, such pharmacological agents may have as wide an impact on reducing ischaemic heart disease as cholesterol-lowering drugs do today.

Base Sequence↗
PubMed1989

A study of familial hypercholesterolaemia in Iceland using RFLPs.

We have studied 17 unrelated families from Iceland who have familial hypercholesterolaemia (FH), using three different restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene. In one family FH was caused by a 2 kb deletion in the LDL receptor gene in the region of exons 9 to 10. The PvuII (intron 15), NcoI (exon 18), and ApaLI (intron 15) RFLPs were used to determine the haplotypes associated with the defective LDL receptor gene in Iceland. Genotypes were determined in 77 subjects from these 17 families, both FH and non-FH. A rare new NcoI RFLP was detected in three subjects. Among the patients, at least four different haplotypes were observed indicating that FH in Iceland is caused by at least four different mutations and is a heterogeneous disease, even in the small, geographically isolated population of Iceland.

Adult↗
PubMed1989

Dynamics of alpha-tubulin deacetylation in intact neurons.

The majority of the alpha-tubulin in cultured neurons is acetylated (Black and Keyser, 1987). The present studies examine the relationships of the acetylation and deacetylation reactions to tubulin assembly and disassembly in intact neurons. Extraction assays which separate assembled and unassembled tubulin pools reveal that greater than or equal to 99% of the total acetylated, as well as newly acetylated, tubulin is cytoskeletal associated. Treatment of neurons with depolymerizing drugs results in a progressive decrease in the levels of total tubulin in polymer and a corresponding increase in the levels of soluble tubulin. These drugs also cause a progressive decrease in the levels of acetylated alpha-tubulin in polymer that closely parallels in rate and extent that of total alpha-tubulin. However, there is no corresponding increase in soluble acetylated tubulin. Because the total levels of alpha-tubulin remain unchanged during drug treatment, the decrease in levels of acetylated alpha-tubulin during depolymerization must reflect its rapid conversion to nonacetylated alpha-tubulin. These findings suggest alpha-tubulin is acetylated in the polymeric form and that deacetylation is closely coupled to depolymerization. The close coupling between alpha-tubulin deacetylation and depolymerization provided a means of estimating the rate at which subunits cycle off microtubules in intact neurons. Acetate turnover on tubulin in intact neurons was determined both by pulse-chase protocols with 3H-acetate and by measuring the loss of acetylated subunits (using quantitative immunoblotting) under conditions of net microtubule depolymerization induced by colchicine. Both methods yielded similar results. Acetate turnover occurred biphasically; 30-50% of the acetate on tubulin turns over with a t1/2 of 1.5-2 hr, and the remaining half or more turns over with a t1/2 of 5-10 hr. We suggest that these kinetically distinguishable pools of acetylated alpha-tubulin reflect distinct pools of acetylated microtubules that differ in their average rates of subunit turnover.

Acetates↗
PubMed1989

The molecular basis of truncated forms of apolipoprotein B in a kindred with compound heterozygous hypobetalipoproteinemia.

Krul et al. (1) have identified two truncated species of apolipoprotein B-100 in a kindred with familial hypobetalipoproteinemia. Five family members were identified who produce either one or both of two truncated apolipoprotein B-100 proteins estimated to be 40% and 90% the amino-terminal end of apolipoprotein B-100. Low density lipoprotein with the apolipoprotein B-90 binds more strongly to the low density lipoprotein-receptor on cultured fibroblasts. In this present study, we have identified the DNA mutations leading to these truncated apolipoprotein B-100 variants in this kindred. Sequencing of amplified DNA from the proband revealed that deletions of one or two nucleotide bases produced frameshift mutations and generated premature stop codons in both cases. Apolipoprotein B-40 (Val1829----Cys-TERM) is the result of a dinucleotide (TG) deletion in exon 26 that generates a stop codon at position 1830 and produces a protein with a predicted molecular mass of 207.14 kDa. The other truncated apolipoprotein B Glu4034----Arg-Gln-Leu-Leu-Ala-Cys-TERM) is due to a single nucleotide (G) deletion in exon 29. This results in a protein with 4039 amino acids and a predicted molecular mass of 457.6 kDa that is now designated apolipoprotein B-89. Mechanisms by which the removal of the last 497 amino acids might increase the binding of the apoB-89 to the LDL-receptor are discussed.

Apolipoproteins B↗