Apolipoprotein CII (apo CII) gene expression defect in an individual with familial apo CII deficiency

We have examined the expression of the apolipoprotein CII (apo CII) gene in an individual with familial apo CII (apo CII) deficiency. Total RNA was prepared from this patient's liver tissue and analysed in Slot Blot and Northern Blot experiments using a cloned apo CII cDNA as a probe. In this patient, there is at least a four-fold decrease in the level of apo CII mRNA, when compared to liver tissue from a control individual. The residual apo CII mRNA detected in this patient is of normal length. These results suggest that the failure to detect apo CII protein in this patient's serum is not due to a failure to transcribe or process apo CII mRNA, but probably to a defect in the translation of the apo CII message. This defect results in partial degradation of the apo CII message leading to the much reduced levels which we have observed.     

The isolation of a genomic clone containing the apolipoprotein CII gene and the detection of linkage disequilibrium between two common DNA polymorphisms around the gene

Summary We report the isolation of a genomic clone containing the apolipoprotein CII (apo CII) gene and 5 and 3 flanking sequences. A detailed restriction map of the gene has been constructed and DNA fragments that are unique, or low copy number sequences in the genome have been identified. One of these detects a common restriction fragment length polymorphism (RFLP) with the enzyme BglI. Marked linkage disequilibrium is observed between this RFLP and that detected with the apo CII cDNA clone, even though the two variable restriction enzyme sites are approximately 12 kb apart. However, the usefulness of the apo CII gene as a marker for linkage studies is increased by the use of both RFLPs.     

Expression of normal and mutagenized apolipoprotein CII in procaryotic cells. Structure-function relationship

A full-length human apo CII cDNA clone has been constructed by completing the 5' end of an incomplete cDNA with a 44 bp long synthetic oligonucleotide. This apo CII cDNA insert was cloned into the pSP19 expression vector and transcribed and translated in vitro. Its N-terminal signal sequence (23 amino-acid residues) was accurately cleaved during cotranslational translocation through endoplasmic reticulum membranes to yield the mature apo CII. Mature apo CII was expressed on a preparative scale as fusion protein apo CII-beta-galactosidase with the full-length apo CII cDNA integrated into the pUR291 vector. Furthermore it was expressed in E. coli transformed with the pKK233-2 apo CII clone. The preform was accurately processed by the host cell. C-Terminal apo CII deletion mutants generated by partial Bal31 digestion of the pKK233-2 apo CII vector yielded well-defined truncated apo CII polypeptides on a preparative scale which allowed the determination of the polypeptide domain responsible for the activation of the serum lipoprotein lipase.     

Apolipoprotein (apo) E genotypes by polymerase chain reaction and allele-specific oligonucleotide probes: no detectable linkage disequilibrium between apo E and apo CII

Summary Allelic sequence variation in the apolipoprotein (apo) E gene has been analysed by means of synthetic oligonucleotide probes that detect single base pair substitutions in the codons for amino acid positions 112 and 158, substitutions that are responsible for the common isoforms. Use of the polymerase chain reaction procedure to amplify a sequence of 330 base pairs of the human apo E gene has permitted the development of a robust method for apo E genotyping. This technique has been used to determine the apo E genotype in 95 individuals in whom the genotype for an apo CII TaqI restriction fragment length polymorphism has also been determined. No strong linkage disequilibrium between the two gene loci was detected. This suggests that the metabolic effects of variation, in the apo E and apo CII genes, as detected by the polymorphisms used here, would operate in a statistically independent manner.     

Linkage relationships of the gene for apolipoprotein CII with loci on chromosome 19

Two common restriction fragment length polymorphisms detected with cloned gene probes for apolipoprotein CII (apo CII) have been used to study the inheritance of the gene in families segregating for loci on chromosome 19. Lod scores for APOC2 with the gene for complement component 3 (C3) exclude close linkage and give a maximum at a male recombination fraction of 0.25-0.30. Lod scores for APOC2 and FHC, the gene causing familial hypercholesterolaemia, are negative in males and suggest the genes may not be linked. However, it appears that APOC2 may be closely linked to the blood group loci Lutheran (Lu) and Secretor (Se), and probably less closely linked to Lewis (Le). These data are consistent with the gene order: FHC-----C3-----(Lu, Se, APOC2)     

Human apolipoproteins AI, AII, CII and CIII. cDNA sequences and mRNA abundance

The structure and function of the genes encoding the polypeptide components of plasma lipoproteins are of interest because of the central role they play in the regulation of lipid metabolism. We have now completed our previous studies on the human apoAI gene and furthermore isolated and sequenced cDNA clones for apoAII , CII and CIII. The nucleotide sequences show the signal peptides of apoAII , CII and CIII to be 18, 22 and 20 amino acids in length, respectively, and in addition that prepro apoAII bears a classical propeptide structure of 5 amino acids. The amino acid homology detected between apoCII and pro- apoAI is discussed, as is the gene arrangement of the 5' non-coding region of apoAI mRNA. The relative liver mRNA levels of the 4 apolipoproteins analysed in this study have been estimated and compared with their corresponding plasma products. The data reported here provide an essential basis for further studies of structural and functional alleles of apo AI, AII, CII and CIII genes.     

Reduced C8 beta messenger RNA expression in families with hereditary C8 beta deficiency

Individuals with functional C8 beta deficiency are at increased risk for systemic neisserial infections. Studies by others have shown that the structural gene for this protein appears intact in deficient individuals. We studied affected individuals from 10 unrelated families to determine the basis for their defect. Using chain-specific antisera, C8 beta was undetectable on immunoblots of their sera. The polymerase chain reaction was used to probe cDNA synthesized from RNA isolated from human liver cells, HepG2 cells, peripheral blood monocytes, and fibroblasts to identify a readily available cell source expressing C8 beta message. Cells from each of these sources expressed C8 beta message. The identity of the amplified product was confirmed and this approach was used to probe cDNA synthesized from RNA harvested from monocytes or fibroblasts obtained from two unrelated families with C8 beta deficiency. C8 beta mRNA was readily detectable in C8 beta sufficient and heterozygous family members but required Southern blotting and hybridization to the 32P-labeled C8 beta probe for detection in the homozygous deficient probands. These results suggest that C8 beta-deficient individuals produce less C8 beta-specific mRNA than do normals and that the underlying basis for this deficiency is an abnormality in intracellular events that precede secretion.     

Primary structure of the bovine analogues to human apolipoproteins CII and CIII. Studies on isoforms and evidence for proteolytic processing

Two major isoforms of the bovine analogue to human apolipoprotein (apo) CII were purified from plasma. They were both as effective as human apo CII in activating lipoprotein lipase. Amino acid sequencing revealed that one form contained 79 amino acid residues, and corresponded to human pro apo CII. The other form lacked the first six residues at its N-terminus. This was apparently due to cleavage of the -Gln-Asp- linkage in the sequence H2N-Ala-His-Val-Pro-Gln-Gln-Asp-Glu-, analogous to cleavages described for human apo AI and apo CII. Previous studies with human apo CII have shown that the ability to activate lipoprotein lipase resides in the C-terminal third of the molecule. This was highly conserved in the bovine analogue: of the 30 last residues, 21 are identical. Five residues in this part of human apo CII have been reported to be essential for activation of lipoprotein lipase. Only one of these, Tyr63, is present in the bovine sequence. The bovine structure contains a threonine at position 61, instead of serine in the human, and the four last residues are -Ser-Gly-Lys-Asp instead of the allegedly necessary -Lys-Gly-Glu-Glu. Three differently sialylated isoforms of the bovine analogue to human apolipoprotein CIII were also isolated and partially sequenced. All three lacked the first three N-terminal residues as compared to sequences from other species (man, dog and rat). Sequence differences were more pronounced at the ends than in the central parts of the apo CIII molecules.     

Isolation and characterisation of a cDNA clone for human apolipoprotein CI and assignment of the gene to chromosome 19

Summary We have synthesised a mixed oligonucleotide 17 bases long and used it to isolate cDNA clones for apolipoprotein CI (apo CI) from an adult liver cDNA library. The partial sequence of one of these clones confirms its identity. We have used this probe and Southern blotting techniques to identify the human apo CI gene in DNA from a series of rodent x human somatic cell hybrids. Our Results provide evidence for the assignment of this gene to human chromosome 19.     

Apolipoprotein C-II deficiency: identification of a structural variant ApoC-II Padova

Apolipoprotein(apo) C-II DNA, RNA and protein from a patient with a familial deficiency of apoC-II were evaluated and compared to normal individuals. No major defect of the apoC-II gene could be detected by Southern blot hybridization. Northern and slot blot analyses of total liver RNA documented normal levels of a normal sized apoC-II mRNA. Immunohistochemical studies of the liver of the apoC-II deficient patient revealed a normal to slightly elevated intracellular content of the C-II apolipoprotein. Plasma apoC-II was 3 to 5% of normal apoC-II levels and exhibited abnormal electrophoretic mobility on two dimensional gel electrophoresis and immunoblotting. We postulate that at the molecular level, the deficiency of apoC-II in the plasma of this patient results from a structural defect in the coding portion of the apoC-II gene leading to either defective secretion of cellular apoC-II or increased catabolism of a structurally defective apoC-II in plasma.     

Identification of the mutation responsible for a case of plasmatic apolipoprotein CII deficiency (Apo CII-Bari)

We studied a case of familial Apolipoprotein CII deficiency. By Southern hybridization, amplification and sequence analysis, the genetic defect was identified. It consists in a point mutation C- greater than G in the third exon of the gene causing a premature stop codon. Truncated at the aa. 36 of the mature form, the protein loses its functional domains, becomes inefficient and cannot be detected in the plasma, because of its high instability. The mutation destroys an RsaI site, present in the normal gene sequence. This point mutation is useful in the diagnosis of this Apolipoprotein CII deficiency.     

There are two gene sequences for human apolipoprotein CI (apo CI) on chromosome 19, one of which is 4 kb from the gene for apo E

A cDNA probe corresponding to the mRNA sequence for apolipoprotein E (apo E) was used to screen two independently-constructed human genomic libraries. Two recombinants (lambda E-2, and lambda E2-1), isolated using the apo E cDNA probe, also contain part or all of the apo CI gene. Hybridisation studies using both apo E and apo CI cDNA probes show that these two genes are in the same orientation and separated by 4 kb.     

The isolation of genomic recombinants for the human apolipoprotein B gene and the mapping of three common DNA polymorphisms of the gene —a useful marker for human chromosome 2

Summary We have used four independently isolated cDNA probes for human apolipoprotein B (apo B), to isolate overlapping genomic recombinants for the 3 portion of the apo B gene. The cDNA clones and a unique fragment from the genomic recombinant have been used to identify the human apo B gene in DNA from a series of roden x human somatic cell hybrids. Our results provide evidence for the assignment of this gene to the short arm of human chromosome 2 (p23-pter). We have used the cDNA probes to identify three common DNA polymorphisms. The first, detected with the restriction enzyme XbaI and our probe pAB4, has a rare allele frequency of 0.48. The other two polymorphisms are detected with the probe pAB3. The enzyme MspI detects at least three alleles, with frequencies of 0.67, 0.16 and 0.15, while that detected with the enzyme EcoRI has a rare allele frequency of 0.12. The relative position of these polymorphisms has been mapped using the genomic recombinants.Investigation of a small number of haplotypes indicares that there is linkage equilibrium between the polymorphisms, which have a total polymorphism information content (PIC) value of more than 0.8. These polymorphisms will provide useful markers for genetic studies on chromosome 2 and for the analysis of the involvement of variants of the apo B gene in the development of hyperlipidaemia.     

Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: Presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form

Pseudohypoaldosteronism is a syndrome characterized by salt wasting and a failure to thrive due to the resistance towards the action of aldosterone. Aldosterone levels and plasma renin activity are extremely elevated and aldosterone binding sites in peripheral mononuclear leukocytes have regularly shown to be reduced or absent. Sporadic as well as familial cases have been identified and an autosomal dominant as well as an autosomal recessive mode of inheritance has been described. A defect in the aldosterone receptor has been postulated, however, molecular genetic analysis in selected patients has not revealed a mutation in the sequence of the coding region of the cDNA of the mineralocorticoid receptor gene. In the present study we have used a fluorescence-labeled antibody to detect possible receptor expression in monocytes from patients with various clinical forms of pseudohypoaldosteronism. Patients with the sporadic as well as with the autosomal dominant form were clearly immunopositive despite being negative in terms of aldosterone receptor binding. In contrast in two patients with the autosomal recessive form there was no detectable receptor protein, consistent with the results obtained in the aldosterone binding studies. These results suggest that the pathogenesis of pseudohypoaldosteronism is heterogeneous not only regarding the mode of inheritance but also in terms of receptor binding. Thus, in a subgroup of patients the inability of the receptor to bind ligand may be due to a defect involving other, probably cellular factors rather than a deficiency or a defect in the mineralocorticoid receptor system itself.     

Genetic determination of high-density lipoprotein-cholesterol and apolipoprotein A-1 plasma levels in a family study of cardiac catheterization patients.

Plasma levels of two lipoprotein risk factors, high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-1 (apo A-1), have been shown to be negatively associated with the risk of developing coronary artery disease, and several reports have examined familial factors in HDL-C and apo A-1 levels. A number of studies suggest that shared genes influence familial resemblance of these lipoprotein levels far more than do shared environments. Possible mechanisms for the inheritance of these two risk factors (HDL-C and apo A-1 plasma levels) are explored using data from 390 individuals in 69 families ascertained through probands undergoing diagnostic cardiac catheterization. Segregation analysis was used to test a series of specific models of inheritance. Evidence for single-locus control of apo A-1 levels, with Mendelian transmission of a dominant allele leading to elevated apo A-1 levels, was seen in these families, although there was additional correlation among sibs present. This locus accounted for 48.6% and 37.2% of the total variation in apo A-1 levels in males and females, respectively. Similar evidence of segregation at a single locus controlling HDL-C levels was not seen in these families.     

Isolation of the human HDL apoprotein A1 gene.

Apo A1 is the major apoprotein of the human plasma high density lipoprotein (HDL). We have isolated apo A1 cDNA and genomic clones and used them to study the gene organisation as defined by its restriction enzyme map. These studies showed that apo A1 is coded by a unique gene. Cross hybridisation was not observed with functionally related apoprotein genes. Increased levels of HDL have been correlated with certain protection against coronary heart disease. If there is a genetic component that contributes to the variable levels of HDL found in the population, it may be possible to correlate these differences with distinct gene organisation patterns.     

The apolipoprotein(a) gene resides on human chromosome 6q26–27, in close proximity to the homologous gene for plasminogen

Summary Apolipoprotein(a) [apo(a)], the glycoprotein associated with the lipoprotein(a) [Lp(a)] subfraction of plasma lipoproteins, has been shown to exhibit heritable molecular weight isoforms ranging from 400–700 kDa. Increased serum concentrations of Lp(a) correlate positively with the risk of atherosclerosis. Variations in Lp(a) plasma levels among individuals are inherited as a codominant quantitative trait. As part of an effect to define the basis of these variations and further clarify the expression of the protein, we have determined the chromosomal location of the human apo(a) gene. Blot hybridization analysis of DNA from a panel of mouse-human somatic cell hybrids with an apo(a) cDNA probe revealed a complex pattern of bands, all of which segregated with chromosome 6. In situ hybridization yielded a single peak of grain density located on chromosome 6q26–27. Apo(a) cDNA sequences exhibit striking homology to those of the plasma protease plasminogen, and, therefore, we have reexamined the chromosome assignment of the plasminogen gene. We conclude that both the apo(a) and plasminogen genes reside on human chromosome 6q22–27, consistent with a gene duplication mechanism for their evolutionary origin. The results are of significance for the genetic control of apo(a) expression and genetic influences predisposing to atherosclerosis.     

Genetic independence of two forms of carbonic anhydrase from bovine erythrocytes]

The two major forms of bovine erythrocyte carbonic anhydrase have been designated as CI and CII because their high activity of the C type. Separation of both forms and isolation of CI from the ethanol chloroform extract of the hemolysate were obtained by either column chromatography on DEAE-cellulose DE 23 or on DEAE-sephadex A-50. But pure preparations of the CII form were only obtained from DEAE-sephadex A-50 which separated CII from a minor component CIv1. Comparative studies of the CI and CII forms and of the minor component CIv1 strongly suggest that CIv1 is a conformational variant of CI and CII are genetic variants differing at least in their primary structure by one Arg yields Gln substitution 56 residues from the N-acetylated terminus. Based on the large variability of the proportion of the two isozymes in heterozygous individuals, the modality of the inheritance of these enzymes is discussed.     

Two amino acid substitutions in apolipoprotein B are in complete allelic association with the antigen group (x/y) polymorphism: Evidence for little recombination in the 3'' end of the human gene

We report the identification of an A-to-G base change, in exon 29 of the apolipoprotein B (apo B) gene, that results in the substitution of serine for asparagine at residue 4311 of mature apo B100. In a recent publication, Huang et al. have reported a C-to-T base change in exon 26 that causes the substitution of leucine for proline at residue 2712 of apo B. We have found complete linkage disequilibrium between the alleles at both these sites and an immunochemical polymorphism of LDL designated antigen group (x/y) (Ag(x/y)) in a sample of 118 Finnish individuals. This implies that either one of these substitutions--or both of them combined--could be the molecular basis of the Ag(x/y) antigenic determinants, with the allele encoding serine4311 plus leucine2712 representing the Ag(x) epitope, and that encoding asparagine4311 plus proline2712 the Ag(y) epitope. In a sample of 90 healthy Swedish individuals the Leu2712/Ser4311 allele is associated both with reduced serum levels of LDL-cholesterol and apo B and with raised levels of HDL. However, these differences are of smaller effect than those associated with the XbaI RFLP of the apo B gene in this sample. We have also genotyped 523 individuals from European, Asian, Chinese, and Afro-Caribbean populations and have found complete association between the sites encoding residues 2712 and 4311 in all of these samples, although there are large allele frequency differences between these populations. In addition, there is strong linkage disequilibrium with allelic association between the alleles of these sites and those of the XbaI RFLP in all the populations examined. Taken together, these data suggest that, since the divergence of the major ethnic groups, there has been little or no recombination in the 3' end of the human apo B gene.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.