Familial apolipoprotein E deficiency and type Ill 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 a p 0 E 3 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ , 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 111 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 e-QWarhlngton allele. I These data demonstrate that the striking deficiency of apoE-3Washington results in a moderate form of type 111 hyperlipoproteinemia. The clinical presentation also suggests a dispensable role of apoE in the nervous system and in immunoregu1ation.-Lohse, P., H. B. Brewer 111, M. S. Meng, S. I. Skarlatos, J. C. LaRosa, and H. B. Brewer, Jr. Familial apolipoprotein E deficiency and type 111 hyperlipoproteinemia due to a premature stop codon in the apolipoprotein E gene. J. Lipid Res. 1992. 33: 1583-1590. Supplementary key words nonsense mutation lipoprotein metabolism atherosclerosis dysbetalipoproteinemia three common alleles that code for the isoproteins apoE-2, apoE-3, and apoE-4 (for review see ref. 6). T h e e 3 allele has the highest frequency in the population and apoE-3 is therefore considered to be the parent isoprotein. Homozygosity for ap0E-2(Arg,,~+Cys) predisposes to the development of type I11 hyperlipoproteinemia presumably due to a reduced binding affinity of apoE-2 for the LDL receptor of less than 2% that of apoE-3 (7). This leads to a delayed metabolic clearance of apoE-containing particles in vivo. ApoE-4 (Cysl 12+Arg) demonstrates normal binding to the LDL receptor in vitro (7), but is catabolized more rapidly when compared to apoE-3 as shown by in vivo kinetic studies (for review see refs. 8 and 9). Type 111 hyperlipoproteinemia is characterized by elevated plasma cholesterol and triglyceride levels and the accumulation of cholesterol and apoE-enriched lipoprotein remnants of both intestinal and hepatic origin (broad-/3 or floating-fl disease). Clinical features include tuboeruptive as well as palmar xanthomas and the development of premature coronary and peripheral atherosclerosis (for review see refs. 8, 10-12). A very rare form of type 111 hyperlipoproteinemia is due to apolipoprotein E deficiency. T h e only kindred described thus far had no detectable plasma apoE (13) and markedly decreased levels (14, 15) of two abnormal apoE mRNAs (14). Cloning of the apoE gene from the apoEdeficient patient and subsequent DNA sequence analysis revealed an A to G substitution in the acceptor splice site Apolipoprotein E is a 299 amino acid glycoprotein of 34.2 kDa (1). It is a constituent of chylomicrons, VLDL, and HDL, and mediates the cellular uptake of these particles through a n interaction with LDL (apoB,E) and putative apoE receptors (2-5). ApoE is a genetically polymorphic plasma protein with Abbreviations: apo, apolipoprotein; bp, base pairs; HDL, high density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; VLDL, very low density lipoproteins. 'To whom reprint requests should be addressed at: Bldg. 10, Room 7N117, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Journal of Lipid Research Volume 33, 1992 1583 by gest, on O cber 9, 2017 w w w .j.org D ow nladed fom of the third intron of the apoE gene (16). This mutation caused aberrant splicing of the third intron, thus producing two apoE mRNAs containing either the entire or the 3'-portion of the third intron. Translation stop codons within these intronic sequences led to the production of apoE peptides of approximately 10,000 Da, which were not immunoprecipitable with a polyclonal apoE antibody (14, 16). The nucleotide substitution also created a new restriction site for the enzyme Sac I1 (16). Using a polymerase chain reaction product that contained the 3'-end of intron 3 and the 5'-end of exon 4 and the restriction fragment length polymorphism for Sac 11, we were able to demonstrate that the proband is homozygous for this mutation (P. Lohse, unpublished data). In the present report we describe a new kindred with apoE deficiency due to a G to A substitution in the apoE gene, which replaces amino acid 210, tryptophan (TGG), with a premature stop codon (TAG). This leads to the synthesis of a truncated apoE with a predicted molecular weight of 23,880 and is proposed to be the basis for the type I11 hyperlipoproteinemia observed in the homozygous patient. MATERIALS AND METHODS