Homozygous familial hypobetalipoproteinemia. Increased LDL catabolism in hypobetalipoproteinemia due to a truncated apolipoprotein B species, apo B-87Padova.

Mutations on the apolipoprotein (apo) B gene that interfere with the full-length translation of the apoB molecule are associated with familial hypobetalipoproteinemia (FHBL), a disease characterized by the reduction of plasma apoB and LDL cholesterol. In this report, we describe an FHBL kindred carrying a unique truncated apoB form, apoB-87Padova. Sequence analysis of amplified genomic DNA identified a single G deletion at nucleotide 12032, which shifts the translation reading frame and causes a termination at amino acid 3978. Two homozygous subjects and seven heterozygous relatives were studied. Although homozygous individuals had only trace amounts of LDL, they were virtually free from the symptoms typical of homozygous FHBL subjects. We investigated the in vivo turnover of radiolabeled normal apoB-100 LDL and apoB-87 LDL in one homozygous patient and two normal control subjects. ApoB-87 LDL showed a similar metabolism in all three subjects, with a fractional catabolic rate more than double that of normal LDL. The rate of entry of apoB-87 in the LDL compartment was also markedly decreased compared with normal apoB-100. The increased in vivo catabolism of apoB-87 LDL was paralleled in vitro by a 2.5-fold increased ability of these particles to inhibit the uptake and degradation of normal apoB-100 LDL by normal human cultured fibroblasts. These results indicate that apoB-87 LDL has an enhanced ability to interact with the LDL receptor, the increased apoB catabolism contributes to the hypobetalipoproteinemia and may explain the mild expression of the disease in the two homozygous individuals.

Ontogenetic regulation of apolipoprotein B mRNA editing during human and rat development in vivo.

The solubilization and delivery of lipids in plasma rely on both forms of apolipoprotein B (apo B): apo B-100 and apo B-48. Apo B-48 is the translational product of apo B-100 mRNA that undergoes peritranscriptional conversion of C----U, replacing codon CAA (glutamine 2,153) with the inframe stop codon (UAA). We examined mRNA editing activity in the human and the rat by reverse transcription-polymerase chain reaction primer-extension analysis of intestine and liver total RNA. In rat intestine the percentage of apo B transcripts that undergo editing increases dramatically the day before birth (from approximately 1% to 80%), whereas the rat liver acquires an adult level of editing activity during the third postnatal week (rising from approximately 8% to 30%), when weaning is completed, bile acid composition matures, and plasma thyroid hormone levels peak. In contrast to the rat, the human intestine acquires adult levels of apo B mRNA editing relatively early in fetal development, rising from 10% at 10 weeks to approximately 80% by the end of the second trimester. Our results establish that apo B mRNA editing is 1) developmentally regulated in a tissue- and species-specific manner; 2) fully developed prenatally in both human and rat intestine, suggesting a crucial role of apo B-48 in mammalian fetal adaptation to extrauterine life; and 3) acquired early in human fetal intestine, implying a potential role for apo B-48 in prenatal lipid metabolism.

Both apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine.

Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of lipoprotein particles in plasma by interacting with the LDL receptor. Utilizing human intestinal biopsy organ cultures, we have studied the synthesis of intestinal apoB in man. Intestinal organ cultures from normal adults (n = 6) were incubated in the presence of protease inhibitors in media supplemented with [35S]methionine. Media from these cultures were evaluated by sequential NaDodSO4 polyacrylamide gel electrophoresis, radioautography, and Western blot analyses, and intestinal biopsies were studied using immunohistochemistry. The relative abundance of apoB-100 and apoB-48 mRNA was assessed using reverse transcriptase-polymerase chain reaction followed by primer extension. Although apoB-48 was the principal isoprotein that was newly synthesized by intestinal organ cultures, apoB-100 was also synthesized and secreted by human intestinal organ cultures with 16 +/- 3% of the intestinal apoB mRNA coding for apoB-100. These results establish that apoB-100 is produced by the human intestine. The synthesis of the atherogenic apoB-100 by the intestine has pathophysiologic implications for the development of diet-induced atherosclerosis.

A base mutation of the C-erbA beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. Molecular heterogeneity in two other kindreds.

Generalized thyroid hormone resistance (GTHR) is a disorder of thyroid hormone action that we have previously shown to be tightly linked to one of the two thyroid hormone receptor genes, c-erbA beta, in a single kindred, A. We now show that in two other kindreds, B and D, with differing phenotypes, there is also linkage between c-erbA beta and GTHR. The combined maximum logarithm of the odds score for all three kindreds at a recombination fraction of 0 was 5.77. In vivo studies had shown a triiodothyronine (T3)-binding affinity abnormality in nuclear receptors of kindred A, and we therefore investigated the defect in c-erbA beta in this kindred by sequencing a major portion of the T3-binding domain in the 3'-region of fibroblast c-erbA beta cDNA and leukocyte c-erbA beta genomic DNA. A base substitution, cytosine to adenine, was found at cDNA position 1643 which altered the proline codon at position 448 to a histidine. By allelic-specific hybridization, this base substitution was found in only one allele of seven affected members, and not found in 10 unaffected members of kindred A, as expected for a dominant disease. Also, this altered base was not found in kindreds B or D, or in 92 random c-erbA beta alleles. These results and the fact that the mutation is predicted to alter the secondary structure of the crucial T3-binding domain of the c-erbA beta receptor suggest this mutation is an excellent candidate for the genetic cause of GTHR in kindred A. Different mutations in the c-erbA beta gene are likely responsible for the variant phenotypes of thyroid hormone resistance in kindreds B and D.

Characterization of single base substitutions in edited apolipoprotein B transcripts.

Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.

Expression of apolipoprotein B mRNAs encoding higher- and lower-molecular weight isoproteins in rat liver and intestine.

Two B apolipoproteins (apo) are present in human plasma, designated apoB-100 and apoB-48, and represent translational products from mature apoB mRNAs that differ by a single base. Either the glutamine codon encoded by the single-copy apoB gene at nucleotide 6666 is transcribed and translated to produce apoB-100 or an RNA-editing mechanism substitutes a uracil for cytosine, altering this glutamine codon (CAA) to a stop codon (UAA), prematurely terminating translation to produce apoB-48. In the present report, editing of rat apoB transcripts was evaluated by amplification of RNA with the polymerase chain reaction by use of primers based on the apoB cDNA cloned from a rat liver cDNA library. The combined results of this study show that (i) a single copy of the apoB gene exists in the rat; (ii) the rat apoB gene encodes only the glutamine codon for the synthesis of apoB of higher molecular weight (apoBH); (iii) rat apoB transcripts undergo RNA editing; (iv) apoBH and apoB of lower molecular weight (apoBL) in the rat represent structural equivalents of apoB-100 and apoB-48 in humans, respectively; (v) RNA editing occurs in both the liver and intestine of the rat; (vi) rat hepatic apoB RNA is more extensively edited than is human hepatic apoB RNA, which is consistent with the marked increase in apoBL secretion by the rat liver when compared with human; and (vii) the definitive identification of apoBH mRNA as well as apoBL mRNA in the rat intestine provides a mechanism for the biosynthesis of both apoBH and apoBL by the rat intestine.

Structural and functional analysis of the rat malic enzyme gene promoter.

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences +1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.

Alternative splicing generates messages encoding rat c-erbA proteins that do not bind thyroid hormone.

The nucleotide and predicted amino acid sequences of two variant cDNAs [rat brain thyroid hormone receptor (rTR alpha) vI and vII], isolated from a rat brain cDNA library by using the Pst I fragment of v-erbA, showed virtual identity with the rat brain thyroid hormone receptor (rTR alpha) [Thompson, C. C., Weinberger, C., Lebo, R. & Evans, R. M. (1987) Science 237, 1610-1614] in the putative DNA binding domain and in the first 180 amino acids of the hormone binding domain but no similarity except for 5 amino acids at the extreme 3' end. Isolation and sequencing of the 3' end of the gene coding for rTR alpha, vI and vII mRNAs revealed that the 3' heterogeneity is due to alternative splicing of the primary transcripts of the same gene. RNA transfer blot analyses with probes unique to rTR alpha, rTR alpha vI, and rTR alpha vII showed that only the variant mRNAs are abundantly expressed in rat brain, contrary to the previously reported high-level expression of rTR alpha. Since in vitro translation products of rTR alpha vI and rTR alpha vII did not bind thyroid hormones specifically, our findings explain the discrepancy between the reported abundance of the receptor mRNA and the low receptor levels determined by ligand binding studies in rat brain. These variant mRNAs are also expressed in kidney, heart, spleen, and liver, albeit at lower levels. The presence of an intact DNA binding domain in rTR alpha vI and rTR alpha vII suggests that the variants might have modulating functions in thyroid hormone action.