Familial ligand-defective apolipoprotein B-100: detection, biochemical features and haplotype analysis of the R3531C mutation in the UK.

Familial ligand-defective apolipoprotein (apo) B-100 (FDB) is an autosomal codominant disorder which may give rise to hypercholesterolaemia. It is caused by the substitution of glutamine for arginine at codon 3500 of the apo B gene (apo B R3500Q), resulting in decreased binding of low density lipoprotein (LDL) to the LDL receptor. In order to search for other mutations in this region of the apo B gene, we have screened genomic DNA, obtained from 412 hypercholesterolaemic individuals, using heteroduplex analysis. Additional heteroduplex bands were observed following analysis of DNA from 11 individuals, nine of whom were heterozygous for apo B R3500Q. The two remaining individuals, both of Celtic origin, were shown by DNA sequencing to be heterozygous for a C-->T transition at nucleotide 10800 of the apo B gene, resulting in the substitution of cysteine for arginine at codon 3531 (apo B R3531C). Both had a strong family history of atherosclerosis and family studies revealed a further four individuals heterozygous for the mutation, three of whom were hypercholesterolaemic. Individuals heterozygous for apo B R3531C and R3500Q had mean +/- S.E.M. cholesterol concentrations of 7.82 +/- 0.68 and 8.53 +/- 0.31 mmol/l, respectively. These values were significantly higher than the value of 5.51 +/- 0.23 mmol/l observed in their unaffected relatives. These findings suggest that apo B R3531C is both less common in the UK and gives rise to a less severe form of hypercholesterolaemia than the classical 3500 mutation. In one of the families, the R3531C mutation occurred on a haplotype, compatible with that previously assigned to the mutation in a North American family also of Celtic origin. This is consistent with the mutation having been inherited from a common distant ancestor in individuals of Celtic origin.

Rare apolipoprotein E variant identified in a patient with type III hyperlipidaemia.

We report a rare apolipoprotein E variant in an Irish female with Type III hyperlipidaemia who has the phenotype E2E1 as determined by isoelectric focusing. Sequence analysis of the apolipoprotein E gene from the proband and from four other family members, using DNA amplified by the polymerase chain reaction, demonstrated the presence of a point mutation in the common epsilon 2 allele with a G-->A transition at nucleotide 3791. This was confirmed by digestion with the restriction endonuclease TaqI, which cuts at a new site within the apolipoprotein E gene, created by the base change. This mutation results in a substitution of aspartic acid for glycine at position 127 of the mature protein. We believe this to be the first description of this apolipoprotein E variant in a family from the British Isles. The mutation appears to be 'recessive' with respect to the expression of Type III hyperlipidaemia, although it may be somewhat more potent in this regard than the parent epsilon 2 allele. The Type III hyperlipidaemia is responsive to treatment with diet and gemfibrozil.

Serum gamma-glutamyltransferase isoenzymes in extrahepatic biliary obstruction.

The gamma-glutamyltransferase isoenzymes in the sera of patients with extrahepatic biliary obstruction have been studied, using electrophoretic, gel filtration, and ultracentrifugation techniques, and compared with those present in normal sera. Five isoenzymes were shown to exist in patients' sera, three of which were not demonstrated in normal sera. The observations are discussed in relation to the influence of biliary regurgitation and the possible solubilisation of membrane-bound enzymes. The results are compared with those of previous studies on alkaline phosphatase.

gamma-Glutamyl transferase isoenzymes in human bile.

The gamma-glutamyl transferase isoenzymes of bile were studied using electrophoretic, gel filtration, and ultracentrifugation techniques. In view of the known association of other biliary enzymes with lipids the effects of butanol extraction were investigated. The results show the presence of four isoenzymes of gamma-glutamyl transferase in bile, differing in electrophoretic mobilities, molecular size, and density. The correlation between the properties of biliary gamma-glutamyl transferase and of alkaline phosphatase is discussed.

Gamma-glutamyltransferases in bile and sera from patients with extrahepatic biliary obstruction.

Gamma-glutamyltransferase has been studied in bile and sera from patients with extrahepatic biliary obstruction. In both fluids gamma-glutamyltransferase activity is found largely in the high molecular mass fraction present in the void volume following G200 gel chromatography and which remains at the origin after 7% polyacrylamide gel electrophoresis. When bile, sera and liver microsomes are treated with deoxycholate, a different form of gamma-glutamyltransferase, with an approximate molecular mass of 157 000 estimated by gel chromatography and of 125 000 by gradient gel electrophoresis, is obtained. After treatment with papain, all three types of specimen gave rise to a form of gamma-glutamyltransferase with a molecular mass of 115 000 estimated by gel chromatography and of 98 000 by gradient gel electrophoresis. The possible relationship between these various forms of gamma-glutamyltransferase, and their relevance to the increases in activity in serum seen in extrahepatic biliary obstruction, are discussed.

Detection of apolipoprotein E polymorphisms using PCR/ASO probes and Southern transfer: application for routine use.

The detection of apolipoprotein E genotypes is of importance both for diagnostic and research purposes. We have previously used the polymerase chain reaction to amplify a specific region of the apolipoprotein E gene which, when used in conjunction with allele specific oligonucleotide probes, permits the detection of the six common apolipoprotein genotypes. In our present report we have modified the above procedure by immobilising the amplified DNA using Southern blotting. This enables the technique to be used in routine laboratories with minimal expenditure and little risk of cross-contamination between samples. Furthermore, it is inherently robust and may be rapidly performed.

Physical properties of gamma-glutamyltransferase in human serum.

Using gel chromatography on Sephacryl S300 we have separated serum gamma-glutamyltransferase into three fractions with estimated relative molecular masses of (a) greater than 1000 000, (b) 250 000-500 000 and (c) about 120 000. Similarly, serum leucine aminopeptidase has been separated into three fractions. We have studied, particularly, the gamma-glutamyltransferase fraction of intermediate relative molecular mass (250 000-500 000) in serum from patients with a number of liver diseases. We have shown, both by polyanion and immuno-precipitation, that it consists significantly of a complex between gamma-glutamyltransferase and high density lipoprotein. The physical properties of this fraction, namely its mass and charge, can be altered by incubating serum with either bile or bile salts.

The nature of gamma-glutamyltransferase and other hepatocyte plasma membrane enzymes in human bile.

High molecular mass gamma-glutamyltransferase in human bile is converted by physiological concentrations of the bile salts glycocholate and glycochenodeoxycholate to a form with a molecular mass estimated by gel chromatography to be 165,000. A molecule of intermediate molecular mass estimated to be 330,000 is obtained following treatment with the non-ionic detergent Triton X-100. When human bile is centrifuged at 150,000 X g, between 25-73% of gamma-glutamyltransferase activity is recovered in the supernatant. This proportion is increased following prior addition of bile salts to the bile. Analogous results are obtained for the high molecular mass enzymes alkaline phosphatase and leucine aminopeptidase, present in human bile, and also considered to originate from the hepatocyte plasma membrane. From these results it is suggested that these high molecular mass enzymes found in bile may, in part at least, represent artefacts following aggregation of the enzymes in aqueous media containing no bile salts.

In vitro studies upon the release of gamma-glutamyltransferase from human liver.

Samples of human liver have been incubated in different fluids for up to 48 h and the released gamma-glutamyltransferase studied by gel chromatography on Sephacryl S-300 and polyacrylamide gradient gel electrophoresis. When human liver is incubated in serum, most of the released enzyme is of high Mr (greater than 1,000,000). Incubation in hepatic bile, or in a solution of glycochenodeoxycholate, results in the release of enzyme that is hydrophobic in nature and which reaggregates to a varying extent after the removal of bile salts. In contrast, incubation in saline, or in a solution of human albumin results in the release of a hydrophilic low Mr (about 120,000) form of the enzyme. These observations are discussed in relation to possible mechanisms for the release of these multiple forms.

Apolipoprotein E phenotyping: a word of caution.

Using two different techniques, phenotyping and genotyping, we have studied allelic variation at amino acids 112 and 158 of the apolipoprotein E gene locus in 52 patients with insulin-dependent diabetes and in 58 non-diabetic controls. Phenotypes were determined by isoelectric focusing and immunoblotting of delipidated, neuraminidase-treated plasma. Genotypes were determined by using the polymerase chain reaction to amplify a 227 base pair fragment of the apolipoprotein E gene spanning both allelic sites. This was then digested with the restriction endonuclease CfoI and the alleles identified by polyacrylamide gel electrophoresis. Discrepancies between phenotype and genotype were observed in 16 (15%) of the individuals studied, 7 (13%) in the diabetics and 9 (17%) in the controls. From these results it is concluded that isoelectric focusing can lead to the erroneous assignment of apolipoprotein E phenotype even after pretreatment with neuraminidase. It is suggested that genotyping by DNA analysis is the method of choice in determining apolipoprotein E status.

Interference in spectrophotometric analysis of cerebrospinal fluid by haemolysis induced by transport through a pneumatic tube system.

The hypothesis that sending blood-stained cerebrospinal fluid (CSF) through a pneumatic tube causes in vitro haemolysis has been tested. Spectrophotometric scanning of CSF supernatants demonstrated a significantly greater absorbance at 415 nm in those CSF samples that had been sent through the tube system compared to those that had not (P=0.0034). It is concluded that passage of blood-stained CSF down a pneumatic tube system causes in vitro haemolysis, accompanied by the release of oxyhaemoglobin from the lysed cells into the surrounding CSF. In view of this observation, it is recommended that CSF samples requiring spectrophotometric analysis, as part of the investigation of subarachnoid haemorrhage, should not be transported via a pneumatic tube system.

A short review of techniques for the localisation of gamma-glutamyl transferase isoenzymes after electrophoresis.

The development of a sensitive method for the localisation of gamma-glutamyl transferase isoenzymes after electrophoresis on various media is described. Recommendations on final conditions are given for optimal concentrations of substrate, acceptor and buffer, and for pH of the reaction mixture. The reaction conditions are compared with other isoenzyme localisation techniques previously reported.

Familial defective apolipoprotein B-100: a study of patients from lipid clinics in Scotland and Wales.

Familial defective apolipoprotein (apo) B-100 (FDB) is an autosomal codominant disorder, which may be associated with hypercholesterolaemia. The defect is caused by the substitution of glutamine for arginine at amino acid residue 3500 of apo B-100. A total of 357 hypercholesterolaemic patients, 48 with a clinical diagnosis of familial hypercholesterolaemia attending lipid clinics in Scotland and Wales, were screened for the presence of FDB. Seven unrelated individuals, five of whom had a family history of coronary heart disease, and a further 11 first-degree relatives, were shown to be heterozygous for the mutation. Pedigree analysis demonstrated the mutation to be present on a single haplotype, suggesting that in Britain it is inherited from a common ancestor. Treatment of 11 heterozygous individuals with lipid-lowering medication showed falls in total and low density lipoprotein cholesterol ranging from 11.6 to 38.8% and 5.3 to 49.5%, respectively. In view of the condition's association with coronary heart disease and hypercholester-olaemia, it may be worthwhile identifying carriers attending lipid clinics, so that affected siblings can be offered cholesterol-lowering treatment where necessary.

Simplified detection of a mutation causing familial hypercholesterolaemia throughout Britain: evidence for an origin in a common distant ancestor.

Familial hypercholesterolaemia (FH) is an inherited autosomal codominant disorder caused by many different mutations in the low-density lipoprotein receptor (LDLR) gene. The one described most frequently in patients with FH from England, arises from a G-->A transition at the first nucleotide of codon 80, resulting in the substitution of lysine for glutamic acid at residue 80 of the mature protein, FH E80K. We describe a simple method to detect this mutation in genomic DNA using the polymerase chain reaction (PCR). A 69 base pair (bp) fragment of exon 3 of the LDLR gene is amplified using a mutagenic upstream PCR primer. This substitutes a T for an A residue in the amplified product, 2 bp upstream from the mutant site, generating a restriction site for the endonuclease Taq I, in normal, but not in mutant DNA. Following digestion of amplified DNA with Taq I, normal but not mutant DNA is cut into two fragments of 29 and 40 bp, which are readily identified by polyacrylamide gel electrophoresis. Using this method, 410 patients with clinically diagnosed FH, attending lipid clinics in Edinburgh (72), Newport (158), Walsall (30) and Southampton (150), were screened for the mutation. Five individuals tested positive as heterozygotes, one from Edinburgh, three from Newport and one from Southampton. This finding was confirmed by DNA sequence analysis. We conclude that FH due to this mutation occurs in individuals throughout Great Britain and that it can be detected accurately using this simple technique. DNA from these and other individuals previously identified to be heterozygous for FH E80K, was then studied using PCR of highly informative microsatellite markers flanking the LDLR gene. Sixteen of 17 apparently unrelated individuals heterozygous for FH E80K also were heterozygous for an identical size (239 nucleotide) allele, of polymorphic microsatellite D19S394, located approximately 250 kb away from the LDLR gene. This supports the hypothesis that FH E80K in these 16 individuals arose from a single ancestor less than 1000 years ago.

Measurement of plasma phenytoin by EMIT in the Monarch centrifugal analyzer: studies on the elimination of within-rotor drift.

We investigated the measurement of phenytoin in plasma by the EMIT method, in the Monarch centrifugal analyzer. When we used the standard protocol supplied by Instrumentation Laboratory, significant drift was observed across a full rotor loaded with 31 replicates of a single specimen. Although the cause remains obscure, the drift was eliminated by using a load-spin-reload-spin procedure. This makes it possible to analyze large batches of samples without excessive use of standards and reagents.

Analysis of apolipoprotein E genotypes by the Amplification Refractory Mutation System.

The Amplification Refractory Mutation System (ARMS) has been successfully applied to the detection of apolipoprotein (apo) E genotypes in human DNA extracted from peripheral blood. By using four allele-specific oligonucleotide primers and one common primer, one can identify the three common alleles of the apo E genetic polymorphism, epsilon 2, epsilon 3, and epsilon 4. The system amplifies two sequences of the apo E gene, one of 181 bp and the other 319 bp. These sequences are amplified when DNA containing a particular allele is incubated with its allele-specific oligonucleotide primer and a common primer. The method is simple, reliable, and nonisotopic and obviates the need for digestion with restriction endonucleases or for hybridization with allele-specific oligonucleotide probes. Genotyping DNA by this method overcomes the problem of post-translational modification of the apo E phenotype encountered with isoelectric focusing of the mature plasma apo E protein.