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3.
Ann Clin Biochem ; 38(Pt 4): 371-5, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11471879

ABSTRACT

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.


Subject(s)
Cerebrospinal Fluid/chemistry , Hemolysis , Subarachnoid Hemorrhage/cerebrospinal fluid , Subarachnoid Hemorrhage/diagnosis , Artifacts , Erythrocytes , Humans , Oxyhemoglobins/cerebrospinal fluid , Reproducibility of Results , Specimen Handling/methods , Spectrophotometry/methods
4.
Prenat Diagn ; 19(3): 219-23, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10210119

ABSTRACT

Mid-trimester biochemical screening of 38 143 pregnancies in south-east Scotland revealed 127 cases (0.34 per cent) in which the maternal serum (MS) intact human chorionic gonadotrophin (hCG) concentration was > or = 4 multiples of the median in singleton pregnancies (MOM). Three were lost to follow-up but in 72 (58 per cent) complications developed or there were associated fetal abnormalities. This percentage was greatest at very high hCG concentrations, 92 per cent with hCG > or = 10 MOM (n = 12) compared with 48 per cent with hCG concentrations of 4-4.99 MOM (n=69). 22 cases had an MS alpha-fetoprotein > or = 2 MOM in addition to an MS hCG > or = 4 MOM, and in only 3 of these was the pregnancy uneventful; 86 per cent were associated with abnormalities or pregnancy complications.


Subject(s)
Chorionic Gonadotropin/blood , Mass Screening/methods , Pregnancy/blood , Chromosome Aberrations/diagnosis , Chromosome Disorders , Chromosomes, Human, Pair 16 , Female , Follow-Up Studies , Humans , Male , Maternal Age , Mosaicism , Pregnancy Complications/diagnosis , Pregnancy Trimester, Second , Pregnancy, High-Risk , Retrospective Studies , Scotland , Trisomy
5.
Ann Clin Biochem ; 35 ( Pt 2): 226-35, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9547893

ABSTRACT

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.


Subject(s)
Founder Effect , Hypercholesterolemia/genetics , Mutation , Polymerase Chain Reaction/methods , Base Sequence , Female , Haplotypes , Heterozygote , Humans , Hypercholesterolemia/drug therapy , Hypercholesterolemia/epidemiology , Male , Microsatellite Repeats , Molecular Sequence Data , Pedigree , Receptors, LDL/genetics , United Kingdom
6.
Clin Chem ; 43(9): 1630-4, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9299944

ABSTRACT

Familial defective apolipoprotein (apo) B-100 (FDB), a condition that may give rise to hypercholesterolemia, is caused by mutations around codon 3500 of the apo B gene. We have compared the ability of three molecular-scanning techniques, heteroduplex analysis, single-strand conformation polymorphism (SSCP) analysis, and denaturing gradient gel electrophoresis (DGGE), to detect these mutations in a cohort of 432 hypercholesterolemic individuals. Heteroduplex analysis and DGGE detected 11 individuals with apo B mutations, 9 of whom were heterozygous for apo B R3500Q and 2 who were heterozygous for apo B R3531C. Whereas DGGE was able to distinguish between these two mutations, heteroduplex analysis was technically simpler and gave a higher sample throughput. In contrast, SSCP analysis detected only 7 of the R3500Q and none of the R3531C heterozygotes and was the most complex of the three techniques. We believe heteroduplex analysis to be the method of choice for screening large numbers of samples for FDB.


Subject(s)
Apolipoproteins B/deficiency , Apolipoproteins B/genetics , Hypercholesterolemia/blood , Hypercholesterolemia/genetics , Polymorphism, Single-Stranded Conformational , Apolipoprotein B-100 , Apolipoproteins B/blood , Codon , DNA/blood , DNA/chemistry , DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel/methods , Heterozygote , Homozygote , Humans , Nucleic Acid Heteroduplexes , Point Mutation , Polymerase Chain Reaction/methods
7.
Atherosclerosis ; 129(2): 185-92, 1997 Mar 21.
Article in English | MEDLINE | ID: mdl-9105560

ABSTRACT

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.


Subject(s)
Apolipoproteins B/genetics , Hyperlipoproteinemia Type II/genetics , Lipoproteins, LDL/metabolism , Point Mutation , Adult , Aged , Apolipoprotein B-100 , Apolipoproteins B/metabolism , Codon/genetics , Ethnicity/genetics , Female , Gene Frequency , Genes, Dominant , Haplotypes/genetics , Humans , Hyperlipoproteinemia Type II/ethnology , Ligands , Male , Middle Aged , Nucleic Acid Hybridization , Pedigree , Polymorphism, Restriction Fragment Length , Receptors, LDL/genetics , Receptors, LDL/metabolism , United Kingdom/epidemiology
8.
Ann Clin Biochem ; 33 ( Pt 5): 443-50, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8888978

ABSTRACT

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.


Subject(s)
Apolipoproteins B/deficiency , Hypercholesterolemia/blood , Adult , Aged , Aged, 80 and over , Apolipoprotein B-100 , Apolipoproteins B/genetics , Electrophoresis, Polyacrylamide Gel , Female , Humans , Hypercholesterolemia/epidemiology , Hypercholesterolemia/genetics , Male , Middle Aged , Pedigree , Restriction Mapping , Scotland/epidemiology , Wales/epidemiology
10.
Eur Respir J ; 6(9): 1282-6, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8287944

ABSTRACT

Total plasma lactate dehydrogenase (LDH) activity may be elevated in cryptogenic fibrosing alveolitis (CFA) and extrinsic allergic alveolitis (EAA), and may be a useful monitor of disease progress. In a retrospective, primary referral centre study, we compared LDH at presentation, prior to bronchoalveolar lavage BAL, and after treatment and follow-up with changes in pulmonary function, in patients with CFA, EAA and pulmonary sarcoidosis. Plasma levels of LDH at presentation in CFA (n = 47) and EAA (n = 10) were significantly higher than in patients with sarcoidosis (n = 36). LDH activity decreased in patients with improving lung function (EAA, p = 0.008; CFA, p = 0.02), whereas it increased in CFA patients with deteriorating lung function (p = 0.015). Total plasma LDH is a simple, though nonspecific test, which appears to reflect changes of disease activity in patients with CFA and EAA.


Subject(s)
Alveolitis, Extrinsic Allergic/diagnosis , Biomarkers/blood , L-Lactate Dehydrogenase/blood , Pulmonary Fibrosis/diagnosis , Adult , Aged , Aged, 80 and over , Alveolitis, Extrinsic Allergic/physiopathology , Follow-Up Studies , Humans , Lung/physiopathology , Lung Volume Measurements , Male , Middle Aged , Pulmonary Fibrosis/physiopathology , Retrospective Studies , Sarcoidosis, Pulmonary/diagnosis , Sarcoidosis, Pulmonary/physiopathology
12.
Atherosclerosis ; 99(2): 261-71, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8503953

ABSTRACT

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.


Subject(s)
Apolipoproteins E/genetics , Hyperlipidemias/genetics , Adult , Base Sequence , DNA/genetics , Female , Genotype , Humans , Hyperlipidemias/metabolism , Immunoblotting , Isoelectric Focusing , Molecular Sequence Data , Mutation , Pedigree , Phenotype
14.
Ann Clin Biochem ; 28 ( Pt 6): 599-605, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1776811

ABSTRACT

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.


Subject(s)
Apolipoproteins E/genetics , Diabetes Mellitus, Type 1/genetics , Alleles , Female , Genotype , Humans , Male , Phenotype
15.
Clin Chem ; 37(11): 1983-7, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1934475

ABSTRACT

We report a method for the diagnosis of familial defective apolipoprotein (apo) B-100, using the Amplification Refractory Mutation System (ARMS) and either whole blood or extracted DNA in the polymerase chain reaction. Normal and mutant alleles are identified by using two allele-specific oligonucleotide primers, each with the same common primer, to amplify a 187-bp fragment of the apo B-100 gene. Fragment amplification occurs only when the allele-specific primer matches the nucleotide sequence of the template DNA. The amplification product is detected by agarose gel electrophoresis, followed by staining with ethidium bromide. The technique is simple, reliable, and robust. It avoids the use of radiation or hybridization with allele-specific oligonucleotide probes, and is well suited for use in the routine clinical chemistry department.


Subject(s)
Apolipoproteins B/genetics , Lipid Metabolism, Inborn Errors/diagnosis , Polymerase Chain Reaction , Apolipoprotein B-100 , Base Sequence , Diagnosis, Differential , Dimethyl Sulfoxide , Heterozygote , Humans , Hyperlipoproteinemia Type II/diagnosis , Lipid Metabolism, Inborn Errors/genetics , Molecular Sequence Data , Mutation , Temperature
17.
Clin Chem ; 37(2): 241-4, 1991 Feb.
Article in English | MEDLINE | ID: mdl-1993332

ABSTRACT

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.


Subject(s)
Apolipoproteins E/genetics , DNA/genetics , Alleles , Apolipoproteins E/analysis , Base Sequence , Electrophoresis, Agar Gel , Gene Amplification , Genotype , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Genetic
19.
Clin Chim Acta ; 189(2): 153-7, 1990 Aug 15.
Article in English | MEDLINE | ID: mdl-2397597

ABSTRACT

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.


Subject(s)
Apolipoproteins E/genetics , Polymorphism, Genetic , Alleles , Base Sequence , Blotting, Southern , DNA/genetics , DNA Probes , DNA-Directed DNA Polymerase , Humans , Molecular Sequence Data , Polymerase Chain Reaction
20.
Eur J Clin Invest ; 19(5): 438-41, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2511020

ABSTRACT

Ten men with Klinefelter's syndrome were studied to assess the effect of testosterone replacement on plasma lipids and apolipoproteins. Measurements taken before the insertion of a testosterone ester implant were compared with those obtained 1 week and 4 weeks later. Mean plasma testosterone, androstenedione, total cholesterol and calculated LDL-cholesterol increased significantly after 1 and 4 weeks. No significant changes were seen in total plasma concentrations of HDL-cholesterol, HDL-cholesterol subfractions 2 and 3 or in apoplipoproteins A-I, A-II or B. A significant correlation was seen between total cholesterol and plasma oestradiol concentrations (Rs = 0.61; P less than 0.001). A significant negative correlation was seen between the concentrations of total testosterone and total triglyceride (Rs = -0.56; P less than 0.005) but not with the other lipid parameters. Testosterone replacement is associated with slight but potentially adverse changes in plasma cholesterol levels.


Subject(s)
Apolipoproteins/blood , Lipids/blood , Testosterone/pharmacology , Adult , Cholesterol/blood , Coronary Disease/etiology , Estrogens/blood , Humans , Klinefelter Syndrome/blood , Klinefelter Syndrome/complications , Klinefelter Syndrome/drug therapy , Male , Risk Factors , Testosterone/blood
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