Familial apolipoprotein A-I and C-III deficiency, variant II

The biochemical, clinical, and genetic features were examined in the proband (homozygote) and heterozygotes (n = 17) affected with familial apolipoprotein A-I and C-III deficiency, variant II (previously described as apolipoprotein A-I absence). The proband was a 45-year-old white female with mild corneal opacification and significant three-vessel coronary artery disease (CAD), who died shortly after bypass surgery. Autopsy findings included significant atherosclerosis in the coronary and pulmonary arteries and the abdominal aorta as well as extracellular stromal lipid deposition in the cornea. No reticuloendothelial lipid deposits in the liver, bone marrow, or spleen were noted (unlike Tangier disease). Laboratory features included marked high density lipoprotein (HDL) deficiency and undetectable plasma apolipoproteins (apo) A-I and C-III. The percentage of plasma cholesterol in the unesterified form was normal at 30%. The activity and mass of lecithin:cholesterol acyltransferase (LCAT) were 42% and 36% of normal, respectively, and the cholesterol esterification rate was 43% of normal. Deficiencies of plasma vitamin E and essential fatty acid (linoleic, C18:2) were also noted. Evaluation of plasma lipoproteins and apolipoproteins in 37 kindred members revealed 17 heterozygotes with HDL cholesterol values below the 10th percentile of normal. Of these, all had apoA-I levels more than one standard deviation below the normal mean, and 37.5% had a similar decrease in apoC-III values. Mean (+/- SD) plasma HDL cholesterol, apoA-I, and apoC-III values (mg/dl) in heterozygotes were 54.0%, 62.4%, and 79.2% of normal, respectively. No evidence of CAD was observed in 10 heterozygotes 40 years of age or less; however, CAD was detected in 3 of 7 heterozygotes over 40 years of age, one of whom died at age 56 years of complications of myocardial infarction and stroke. The inheritance pattern in this kindred was autosomal codominant. ApoA-I isolated from a heterozygote had an isoelectric focusing pattern and amino acid composition similar to normal. Utilizing DNA isolated from two obligate heterozygotes, no abnormalities in the apoA-I or apoC-III genes were detected by Southern blot analysis utilizing specific probes following restriction enzyme digestion. The data indicate that familial apolipoprotein A-I and C-III deficiency, variant II, is similar to variant I (described by Norum et al. 1982. N. Engl. J. Med. 306: 1513-1519), but differs at the clinical level (lack of xanthomas), the biochemical level (lack of detectable apoA-I, lower apoA-II level), and at the gene level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a new mouse model for human apolipoprotein A-I/C-III/A-IV deficiency

Human data raised the possibility that coronary heart disease is associated with mutations in the apolipoprotein gene cluster APOA1/C3/A4 that result in multideficiency of cluster-encoded apolipoproteins and hypoalphalipoproteinemia. To test this hypothesis, we generated a mouse model for human apolipoprotein A-I (apoA-I)/C-III/A-IV deficiency. Homozygous mutants (Apoa1/c3/a4(-/-)) lacking the three cluster-encoded apolipoproteins were viable and fertile. In addition, feeding behavior and growth were apparently normal. Total cholesterol (TC), high density lipoprotein cholesterol (HDLc), and triglyceride levels in the plasma of fasted mutants fed a regular chow were 32% (P < 0.001), 17% (P < 0.001), and 70% (P < 0.01), respectively, those of wild-type mice. When fed a high-fat Western-type (HFW) diet, Apoa1/c3/a4(-/-) mice showed a further decrease in HDLc concentration and a moderate increase in TC, essentially in non-HDL fraction. The capacity of Apoa1/c3/a4(-/-) plasma to promote cholesterol efflux in vitro was decreased to 75% (P < 0.001), and LCAT activity was decreased by 38% (P < 0.01). Despite the very low total plasma cholesterol, the imbalance in lipoprotein distribution caused small but detectable aortic lesions in one-third of Apoa1/c3/a4(-/-) mice fed a HFW diet. In contrast, none of the wild-type mice had lesions. These results demonstrate that Apoa1/c3/a4(-/-) mice display clinical features similar to human apoA-I/C-III/A-IV deficiency (i.e., marked hypoalphalipoproteinemia) and provide further support for the apoa1/c3/a4 gene cluster as a minor susceptibility locus for atherosclerosis in mice.     

Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Association between coronary heart disease and the apolipoprotein A-I/C-III/A-IV complex in a Japanese population

Several studies have reported that a variant allele (S₂) of the apolipoprotein (apo) A-I/C-III/A-IV complex is associated with hyperlipoproteinemia in some populations and that the frequency of this allele is two- to fivefold higher in patients with premature coronary heart disease (CHD) than in healthy controls. In the present study in a Japanese population, we were unable to confirm the association of the S₂ allele with either coronary heart disease or elevated serum apo C-III levels, as has been previously reported in Caucasians. No genotype difference was observed among the severity of coronary heart disease, as determined by the number of involved vessels (one, two and three vessel disease), compared to controls. In addition, the frequency of the S₂ allele among Japanese, in both CHD (0.328) and controls (0.369), was quite different from that in many other populations.     

Familial apolipoprotein E deficiency and type III hyperlipoproteinemia due to a premature stop codon in the apolipoprotein E gene.

A kindred with apolipoprotein E deficiency and a truncated lower molecular weight apoE mutant, designated apoE-3Washington, has been identified. Gel electrophoresis demonstrated complete absence of the normal apoE isoproteins and the presence of a small quantity of a lower molecular weight apoE. Plasma apoE levels in the proband were approximately 4% of normal. This marked deficiency of apoE resulted in delayed uptake of chylomicron and very low density lipoprotein (VLDL) remnants by the liver, elevated plasma cholesterol levels, mild hypertriglyceridemia, and the development of type III hyperlipoproteinemia. Sequence analysis of the patient's apoE gene revealed a single nucleotide substitution of an A for a G, which converted amino acid 210 of the mature protein, tryptophan (TGG), to a premature chain termination codon (TAG), thus leading to the synthesis of a truncated E apolipoprotein of 209 amino acids with a molecular mass of 23.88 kDa. Northern blot analysis of differentiated monocyte-derived macrophages demonstrated a mutant mRNA indistinguishable in size from normal apoE mRNA. The nucleotide substitution also resulted in the formation of a new restriction site for Mae I. Using this enzyme we were able to establish that the proband is a homozygote and that her two offsprings are heterozygous for the epsilon-3Washington allele. These data demonstrate that the striking deficiency of apoE-3Washington results in a moderate form of type III hyperlipoproteinemia. The clinical presentation also suggests a dispensable role of apoE in the nervous system and in immunoregulation.     

Human apolipoprotein A-I and C-III genes reside in the p11----q13 region of chromosome 11

Apolipoprotein (apo) A-I is a major protein of high density lipoproteins (HDL). The gene for apoA-I has been localized to the p11 leads to q13 region of chromosome 11 by filter hybridization analysis of mouse-human hybrid cell cDNAs containing chromosome 11 translocations utilizing a cloned human apoA-I cDNA probe. The known linkage of apoA-I and apoC-III also permitted the simultaneous assignment of the apoC-III gene to the same region on chromosome 11. Comparison with previously established gene linkages on the mouse and human genome suggests that apoA-I + apoC-III may be linked to the esterase A4 and uroporphyrinogen synthase genes which are present on the long arm of human chromosome 11. The localization of the apoA-I + apoC-III genes in the p11----q13 region of chromosome 11 represents a definitive chromosomal assignment of a human apolipoprotein gene, and will now enable more detailed analysis of the geneomic organization and linkages of the apolipoprotein genes.     

Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice reduces atherogenesis in response to a high fat-high cholesterol diet

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)-gamma1, and Bcl-2. We have found that treatment of A431 cells with tumor necrosis factor-alpha in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase-3. Among various caspases, caspase-1, caspase-3 and caspase-7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD-cmk and DEVD-fmk in vitro. We also investigated the effect of caspase-dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC-gamma1. Cleavage of EGFR by caspase-3 significantly impaired the tyrosine phosphorylation of PLC-gamma1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti-apoptotic signaling, to abrogate the activation of EGFR-dependent downstream survival signaling molecules.     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Localization of the human catalase and apolipoprotein A-I genes to chromosome 11

Studies of the catalase and apolipoprotein A-I genes are pertinent to the understanding of human disease. Not only are these genes involved in acatalasemia and atherosclerosis, respectively, but they are also important gene markers for chromosome 11, deletions of which are involved in the development of Wilms tumor. We have used in situ hybridization to localize these genes to specific bands on chromosome 11. Hybridization with a catalase cDNA yielded a significant number of cells (38%) exhibiting label at band 11p13. A high percentage of metaphase cells (50%) hybridized with a human genomic fragment containing the gene for apolipoprotein A-I displayed labeling at 11q13.     

The nucleotide and derived amino acid sequence of human apolipoprotein A-IV mRNA and the close linkage of its gene to the genes of apolipoproteins A-I and C-III

Both cDNA and genomic clones encoding human apolipoprotein (apo-) A-IV have been isolated and characterized. Southern blot analyses of apo-A-IV gene-containing cosmids revealed that the apo-A-IV gene is linked to the apo-A-I and apo-C-III genes within a 20-kilobase span of chromosome 11 DNA. The apo-A-IV gene is located about 14 kilobases downstream from the apo-A-I gene in the same orientation, with the apo-C-III gene located between them in the opposite orientation. The nucleotide sequence of the corresponding human apo-A-IV mRNA was determined, and the derived amino acid sequence showed that mature plasma apo-A-IV contained 376 residues. Throughout most of its length, human apo-A-IV was found to contain multiple tandem 22-residue repeated segments having amphipathic, alpha-helical potential. Amino acid substitutions within these homologous segments were generally conservative in nature. A comparison of the sequences of human and rat apo-A-IV revealed a 79% identity of amino acid positions in the amino-terminal 60 residues and a 58% identity in the remainder of the sequences, with the human protein containing 5 extra residues near the carboxyl terminus. An examination of the distribution of apo-A-IV mRNA in different tissues of the rat, marmoset, and man showed that apo-A-IV mRNA was abundant in both the liver and small intestine of the rat, but abundant in both the liver and small intestine of the marmoset and man. It was expressed in only trace amounts in all other tissues that were examined. These findings on the structure and expression of apo-A-IV and the close linkage of its gene to those of apo-A-I and apo-C-III suggest a regulatory relationship between the three genes.     

Genetic variation in mouse apolipoprotein A-IV due to insertion and deletion in a region of tandem repeats.

We have detected three unique apolipoprotein A-IV (apoA-IV) charge isoforms in strains of commensal mice. The cDNA sequences for one representative of each isoform (Mus domestesticus strains C57BL/6J and 129/J and Mus castaneus) revealed a polymorphism within a series of four imperfect repeats encoding the sequence Glu-Gln-Ala/Val-Gln. Insertions or deletions of 12 nucleotides within this repetitive region have given rise to three genotypes characterized by three (129), four (C57BL/6), or five (M. castaneus) copies of the repeat unit. To ascertain the extent of this variation among other species of the Mus genus, we sequenced this region of apoA-IV cDNAs from eight additional M. domesticus inbred strains and from five wild-derived Mus species. All eight additional M. domesticus strains examined had four repeat units, as found in C57BL/6. Among wild-derived mice, however, one species (Mus spretus) had three repeats, two species (Mus cookii and Mus cervicolor) had four repeats, and two species (Mus hortulanus and Mus minutoides) had five repeats. A lack of correlation between the number of repeat units and the phylogeny of Mus species indicates that independent mutations may have occurred throughout the evolution of specific mouse lineages. We suggest that the repetitive nature of the polymorphic sequence may predispose this region to slippage errors during DNA replication, resulting in frequent deletion/insertion mutations.     

Gene and gene-product variation in the apolipoprotein A-I/C-III/A-IV cluster in the Dogrib Indians of the Northwest Territories.

The Dogrib, an Amerindian tribe residing in the Northwest Territories of Canada, were typed for DNA and protein polymorphism at the apolipoprotein A-I/C-III/A-IV gene cluster. Variation was seen at three previously described RFLPs detected with the enzymes SstI, PstI, and XmnI, though frequencies of these polymorphisms differ significantly from those reported in other populations. They exhibit no variation at two previously reported PvuII sites. No variation was seen in the APO A-I or APO A-IV gene products, with the Dogrib showing the most common isoelectric-focusing/immunoblot patterns of other world populations. Haplotype frequencies computed from inferred haplotypes and by maximum likelihood estimation did not differ significantly. The extent of nonrandom association of these sites is highly significant (P less than .00001), though pairwise analysis shows significance between the SstI and XmnI sites only. Levels of fasting triglyceride and fasting total cholesterol were determined for each individual. Analysis of covariance shows that fasting triglyceride levels in women vary significantly with the XmnI genotype. These results suggest that genetic variation at the APO A-I/C-III/A-IV gene cluster may be a useful tool for the study of quantitative lipoprotein variation in the Dogrib.     

Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I

In this report, we have identified two apolipoproteins (apo), apoD and apoA-IV, that, together with the previously identified apoA-I and apoE, accumulate in the regenerating peripheral nerve. These four apolipoproteins were identified in regenerating rat sciatic nerves by their molecular weights, their isoelectric points, and their recognition by specific antibodies. Antibodies were also used to document the changing concentrations of these apolipoproteins in homogenates of regenerating sciatic nerves collected 1 day to 6 weeks after a denervating crush injury. By 3 weeks after injury, at their peak accumulation, apoA-IV and apoA-I had increased 14- and 26-fold, respectively, relative to their concentrations in the normal nerve. Apolipoproteins D and E, in contrast, increased over 500- and 250-fold, respectively, by 3 weeks. These same apolipoproteins also accumulated in the regenerating sciatic nerves of two other species, the rabbit and the marmoset monkey. Immunocytochemistry showed that apoD was produced by astrocytes and oligodendrocytes in the normal central nervous system, and by neurolemmal or fibroblastic cells in the normal peripheral nervous system. Metabolic labeling of both apoD and apoE by [35S]methionine during an in vitro incubation of regenerating rat sciatic nerve segments confirmed that these apolipoproteins are synthesized by the nerve. Neither apoA-IV nor apoA-I was metabolically labeled, however, suggesting that they enter the nerve from the plasma. The results from this study provide evidence that several different apolipoproteins from various sources may play a role in lipid transport within neural tissues.     

A method to screen apolipoprotein polymorphisms in whole plasma: description of apolipoprotein A-IV variants in dyslipidemias and a reassessment of apolipoprotein A-I in Tangier disease

A sensitive and rapid immunological detection method was used to screen for apolipoprotein A-IV variants. Antibodies to human lymph chylomicron or plasma apolipoprotein A-IV, and plasma apolipoprotein A-I were raised in rabbits. Antibodies to apolipoprotein A-I or apolipoprotein A-IV were shown to be monospecific to their respective antigens by reactivity against human chylomicron apolipoproteins by immunoblot analysis. Plasma samples were obtained from dyslipidemic subjects from the Lipid Research Clinic of Columbia University. The plasma samples were isoelectrically focused (pH 4-6) on slab gels. Plasma proteins were then transferred to nitrocellulose paper for immunoblotting. Apolipoprotein A-IV polymorphism was determined by specific immunological detection of apolipoprotein A-IV. Identical apolipoprotein A-IV isoprotein patterns were observed when either antibodies to lymph or plasma apolipoprotein A-IV were used for immunoblotting. All the dyslipidemic plasma samples screened contained the two major and one or two minor isoproteins of normal plasma. In two instances, new apolipoprotein A-IV variants having an additional isoform were detected. One subject was hypertriglyceridemic (triacylglycerols = 342 mg/dl, cholesterol = 251 mg/dl) and had an additional major acidic apolipoprotein A-IV isoform. Another subject with mild hypocholesterolemia (triacylglycerols = 209 mg/dl, cholesterol = 120 mg/dl) was found to have additional major and minor basic apolipoprotein A-IV isoforms. The specificity of this technique allows detection of polymorphism of apolipoproteins of similar isoelectric points by use of a single dimension isoelectric focusing gel. This technique also demonstrated the presence of altered apolipoprotein A-I isoforms in the plasma of a patient with Tangier disease. These isoforms were previously identified as isoforms 2 and 4 of normal plasma by use of two-dimensional gel electrophoresis. However, by use of this new technique and careful evaluation of previously published two-dimensional gels, we now identify these apolipoprotein A-I isoforms as being more acidic than those of normal plasma.     

Haplotype analysis of the apolipoprotein gene cluster on human chromosome 11

Members of the apolipoprotein gene cluster (APOA1/C3/A4/A5) on human chromosome 11q23 play an important role in lipid metabolism. Polymorphisms in both APOA5 and APOC3 are strongly associated with plasma triglyceride concentrations. The close genomic locations of these two genes as well as their functional similarity have hindered efforts to define whether each gene independently influences human triglyceride concentrations. In this study, we examined the linkage disequilibrium and haplotype structure of 49 SNPs in a 150-kb region spanning the gene cluster. We identified a total of five common APOA5 haplotypes with a frequency of greater than 8% in samples of northern European origin. The APOA5 haplotype block did not extend past the 7 SNPs in the gene and was separated from the other apolipoprotein gene in the cluster by a region of significantly increased recombination. Furthermore, one previously identified triglyceride risk haplotype of APOA5 (APOA5*3) showed no association with three APOC3 SNPs previously associated with triglyceride concentrations, in contrast to the other risk haplotype (APOA5*2), which was associated with all three minor APOC3 SNP alleles. These results highlight the complex genetic relationship between APOA5 and APOC3 and support the notion that APOA5 represents an independent risk gene affecting plasma triglyceride concentrations in humans.     

Effect of apolipoprotein activators on the specificity of lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency.

Although it is known that plasma lecithin:cholesterol acyltransferase (LCAT) is activated by several apolipoproteins (apo) including A-I, C-I, D, A-IV, and E, it is not clear what the physiological importance of having different apolipoprotein activators is. One possible explanation is that the activation by different apolipoproteins may result in the utilization of different species of phosphatidylcholine (PC), leading to the formation of different species of cholesteryl esters (CE). In order to determine this possibility, we analyzed the molecular species composition of PC and CE in two patients with familial deficiency of apoA-I and apoC-III. The LCAT activity, assayed by three different procedures, was found to be 36-63% of the control value. The lower LCAT activity, however, was due to deficiency of the enzyme rather than the absence of apoA-I. The patients' plasma was relatively enriched with sn-2 18:2 PC species reflecting the partial deficiency of LCAT activity. The fatty acid composition of plasma CE was not significantly different from that of controls. HPLC analysis of labeled CE formed after incubation of plasma with [C14]cholesterol showed no significant difference in the species of CE synthesized by the LCAT reaction. The transfer of pre-existing as well as newly formed CE from HDL to the apoB-containing lipoproteins was accelerated compared to control plasma. These results show that the absence of apoA-I does not significantly affect either the activity or the specificity of LCAT, and that the other apolipoprotein activators can substitute adequately for it.