Identification and Molecular Analysis of Two ApoB Gene Mutations Causing Low Plasma Cholesterol Levels
Background Familial hypobetalipoproteinemia (FHB) is an autosomal codominant disorder characterized by abnormally low plasma levels of apoB and LDL cholesterol. Heterozygotes for FHB almost always have plasma LDL cholesterol levels <70 mg/dL and are asymptomatic. Because the low cholesterol levels may protect FHB heterozygotes from coronary heart disease, the mechanisms for FHB are of considerable interest.
Methods and Results The plasma lipoproteins of 29 subjects with LDL cholesterol levels <70 mg/dL were examined by SDS-PAGE. One subject who had virtually undetectable levels of LDL cholesterol had a truncated apoB, apoB-44.4, in his lipoproteins; a second subject with an LDL cholesterol level of 44 mg/dL had apoB-55 in his lipoproteins. The apoB-44.4 (2014 amino acids in length) resulted from a frameshift caused by an 11-bp insertion in exon 26 of the apoB gene; the apoB-55 (2494 amino acids) was caused by a nonsense mutation in exon 26 of the apoB gene. The apoB-55 mutation occurred at a CpG dinucleotide pair, a mutational hot spot, and was identical to a mutation described previously in a subject with hypobetalipoproteinemia. Our subject with apoB-55, however, had a different haplotype than the subject described previously, suggesting that the two apoB-55 mutations may have arisen independently. Of note, the apoB-55 proband’s father, who had very low cholesterol levels and who probably carried the apoB-55 mutation, had significant coronary and aortic atherosclerosis at autopsy.
Conclusions In a study of adults with low LDL cholesterol levels, we discovered two subjects with truncated apoB proteins and identified the responsible mutations. ApoB gene mutations causing truncated apoB are not particularly rare in subjects with low cholesterol levels. The role of these mutations in preventing atherosclerosis deserves further study.
The B apolipoproteins, apoB-48 and apoB-100, play important roles in lipoprotein metabolism. Whereas apoB-48 has an obligatory role in the formation of chylomicrons in the intestine,1 apoB-100 is synthesized by the liver and secreted in triglyceride-rich VLDL particles.2 VLDL undergoes extensive metabolism in the plasma and is eventually transformed into cholesteryl ester–enriched LDL. Genetic, epidemiological, and experimental studies have indicated that high plasma levels of LDL cholesterol are causally related to the development of coronary heart disease.2
For many years, it has been suggested that individuals with low levels of LDL cholesterol might be protected from the development of coronary artery disease. In recent years, it has been shown that familial hypobetalipoproteinemia (FHB), a condition characterized by low plasma levels of apoB and LDL cholesterol, can be caused by apoB gene mutations.3 4 5 6 7 8 Nearly all the mutations identified as causing FHB interfere with the formation of a full-length apoB-100 molecule. Heterozygotes for FHB typically are asymptomatic and have LDL cholesterol levels of 20 to 50 mg/dL; homozygotes typically have almost undetectable levels of LDL cholesterol. The clinical phenotype of FHB homozygotes is highly variable but in severe cases may include neurological complications resulting from the intestinal malabsorption of vitamin E.5 Although it is reasonable to postulate that the low cholesterol levels in FHB might protect affected subjects from coronary artery disease, little definitive data on this issue exist.
We have recently screened a group of 29 subjects with LDL cholesterol levels that were <70 mg/dL. We identified two subjects with apoB gene mutations that led to the formation of truncated apoB proteins. In this study, we have characterized these mutations on the biochemical and molecular levels.
Human Subjects and Lipoprotein Analysis
Subjects in the Framingham Offspring Study and an adult volunteer for a metabolic research study had fasting lipoprotein profiles measured as described previously.8 9 In 29 subjects with LDL cholesterol levels <70 mg/dL, VLDL, LDL, and HDL fractions were examined by SDS-PAGE as described previously.8 In two subjects, truncated apoBs, apoB-55, and apoB-44.4 (see “Results”) were identified. The ratio of the amount of apoB-100 to the amount of the truncated apoB in the VLDL was determined from SDS-polyacrylamide gels through gel scanning and peak integration with an LKB Ultrascan XL laser densitometer.
Enzymatic Amplification of DNA and DNA Sequencing
To determine the mutation in the apoB gene responsible for apoB-44.4, an 867-bp segment of exon 26 of the apoB gene (apoB cDNA nucleotides 5794 through 6660) was enzymatically amplified using Taq DNA polymerase, oligonucleotides B44-1 (5′ CACTGaATTcCAGCAATGTCT 3′, apoB cDNA nucleotides 5794 through 5814) and B44-2 (5′ TATATGTCTGCAGTTGAGATAG 3′, complementary to apoB cDNA nucleotides 6660 through 6639), and 0.5 μg genomic DNA prepared from white blood cells. Two base mismatches (lower-case letters above) were included in oligonucleotide B44-1 to generate an EcoRI site. Oligonucleotide B44-2 contained a native Pst I site. To determine the mutation in the apoB gene responsible for apoB-55, a 1040-bp segment of exon 26 of the apoB gene (apoB cDNA nucleotides 7151 through 8190) was enzymatically amplified with oligonucleotides B55-1 (5′ CCATGAaTTcATCGAGAGGTATGAAGTAG 3′, apoB cDNA nucleotides 7151 through 7179) and B55-2 (5′ CGGGCCACTGCAGCTCACTGTTC 3′, complementary to apoB cDNA nucleotides 8190 through 8168). Two base mismatches (lowercase letters above) were included in B55-1 to create an EcoRI site; B55-2 contained a native Pst I site. For both mutations, the amplified DNA was purified from a polyacrylamide gel, digested with EcoRI and Pst I, and cloned into pGEM for DNA sequencing. DNA sequencing was performed with the dideoxy chain-termination technique on an ABI 373A DNA Sequencer (Perkin-Elmer, Applied Biosystems).10
FHB Associated With ApoB-44.4
The proband of the apoB-44.4 kindred was a healthy, well-developed, and asymptomatic 48-year-old man with total and HDL cholesterol levels of 34 and 25 mg/dL, respectively. The LDL cholesterol levels were undetectable. His nonfasting triglyceride level was 83 mg/dL. The extraordinarily low total and LDL cholesterol levels are typical for a compound heterozygote for FHB and are much lower than those observed in adults with the heterozygous form of FHB.5 SDS-PAGE of the VLDL, LDL, and HDL fractions revealed the presence of a truncated apoB, apoB-44.4 (Fig 1A⇓ and 1B⇓). Both the VLDL and LDL fractions of the proband also contained a full-length apoB-100. It is noteworthy, however, that the amount of apoB-100 in the VLDL fraction of the apoB-44.4 proband was very low. The ratio of apoB-100 to apoB-44.4 in the VLDL, as assessed by laser densitometry gel scanning, was very low, 0.31:1. In our prior studies of subjects with the heterozygous form of FHB.8 11 12 13 14 15 we documented that the amount of apoB-100 invariably exceeds the amount of the truncated apoB in the VLDL fraction, with a typical ratio of apoB-100 to truncated apoB in the VLDL of 5:1 or 10:1 and occasionally greater. The fact that the apoB-44.4 proband had an extremely low level of apoB-100 in his VLDL fraction represents additional evidence in favor of the argument that he is a compound heterozygote for FHB, with one mutant apoB allele yielding apoB-44.4, and the other yielding very low levels of a full-length apoB-100.5
A review of the medical records of the proband’s only known relatives (his deceased mother, two sons, and a half brother) revealed that all had total cholesterol levels that were less than the fifth percentile for age- and sex-matched control subjects, strongly suggesting that each of these individuals had the heterozygous form of FHB. A half brother of the proband had total and LDL cholesterol levels of 103 and 45 mg/dL, respectively, and had apoB-44.4 in his VLDL (Fig 1B⇑). These cholesterol levels are typical of those observed in adults with the heterozygous form of FHB5 and are significantly higher than those observed in the apoB-44.4 proband. In comparing the VLDL of the half brother to that of the apoB-44.4 proband (Fig 1B⇑), we see one noteworthy similarity and one noteworthy difference. The similarity was that both subjects had approximately the same amount of apoB-44.4 in their VLDL fractions (per milliliter of plasma). The difference was that the VLDL of the half brother contained a much greater amount of apoB-100 relative to apoB-44.4. As judged by gel scanning, the ratio of apoB-100 to apoB-44.4 in the VLDL of the half brother was 19.4:1. Thus, the ratio of apoB-100 to apoB-44 in the VLDL fractions of the proband and the proband’s half brother differed by >60-fold, without significantly different amounts of apoB-44.4 in their VLDL fractions. We strongly suspect that the reason for this difference is that the proband was a compound heterozygote for FHB and possessed, in addition to the mutant apoB-44.4 allele, a second mutant allele associated with very low levels of apoB-100.
One of the two offspring of the apoB-44.4 proband, a 12-year-old boy, agreed to give a sample of blood for this study. This subject did not have apoB-44.4 in his lipoproteins (Fig 1B⇑), but his cholesterol levels were quite low. The total and LDL cholesterol levels were 76 and 8 mg/dL, respectively. These levels are less than the fifth percentile for age- and sex-matched control subjects and are very similar to the cholesterol levels in other FHB heterozygotes of a similar age.12 The 12-year-old son did not inherit the apoB-44.4 allele from his father but almost certainly inherited a mutant allele associated with markedly reduced amounts of apoB-100.
To determine the molecular defect accounting for the production of apoB-44.4, we cloned an enzymatically amplified segment of exon 26 into pGEM. Sequencing revealed that 5 of 10 pGEM clones contained a duplication of 11 bp: cDNA nucleotides 6231 through 6241 inserted between cDNA nucleotides 6243 and 6244. This 11-bp duplication produced three novel amino acids followed by a premature stop codon (Fig 1C⇑). The apoB-44.4 mutation creates two new restriction sites, BsaBI and Mam I, both at apoB cDNA nucleotide 6232. The apoB-44.4 is predicted to contain 2014 amino acids.
FHB Associated With ApoB-55
In screening 28 Framingham offspring, we identified a 57-year-old man who had apoB-55 in his plasma lipoproteins (Fig 2A⇓). The ratio of apoB-100 to apoB-55 in the VLDL, as determined by gel scanning, was 34.9. The proband was asymptomatic and had total, HDL, and LDL cholesterol levels of 108, 62, and 44 mg/dL, respectively. The triglyceride concentration was 21 mg/dL. Funduscopic examination of the proband revealed a normal retina with no signs of retinitis pigmentosa. To identify the responsible mutation, the relevant segment of exon 26 was amplified and subcloned into pGEM for sequencing. Of 9 pGEM clones, 5 contained a C-to-T transition at apoB cDNA nucleotide 7692 that resulted in a nonsense mutation at codon 2495. The single nucleotide substitution creates a new Bsa I restriction site (at apoB cDNA nucleotide 7687). Restriction fragment digestion of amplified DNA from the apoB-55 proband revealed that neither of his two apoB alleles contained the polymorphic Xba I site in exon 26 of the apoB gene (at apoB cDNA nucleotide 7674).
A son of the proband with apoB-55 had total, HDL, and LDL cholesterol levels of 194, 33, and 139 mg/dL, respectively. The mother of the proband had total cholesterol measurements of 194, 223, 230, and 250 mg/dL. Analysis of their plasma VLDL and LDL fractions revealed that neither carried the apoB-55 mutation. Medical records revealed that two of six first-degree relatives (a brother and daughter who were not available for blood sampling) had total cholesterol levels less than the fifth percentile for age- and sex-matched control subjects, suggesting that they probably carried the apoB-55 mutation. The proband’s father had Parkinson’s disease; as a result of this disorder, he fell and died from head injuries in 1965 at 71 years of age. Framingham Heart Study records revealed that he had total cholesterol levels of 126 mg/dL in 1950, 124 mg/dL in 1952, 110 mg/dL in 1954, 129 mg/dL in 1956, 124 mg/dL in 1958, 128 mg/dL in 1960, 125 mg/dL in 1962, and 143 mg/dL in 1964. These very low cholesterol levels strongly suggest that he carried the apoB-55 mutation. Interestingly, although the proband’s father did not have symptoms of coronary heart disease, an autopsy revealed multiple large atheroma in the left main, left anterior descending, circumflex, and right coronary arteries. In addition, the aorta and iliac arteries had calcified and ulcerated atheroma. The only atherosclerotic risk factor of the proband’s father was borderline hypertension, with diastolic blood pressure readings of 88 to 96 mm Hg over a 16-year period. He did not smoke, have diabetes mellitus, or have a family history of premature atherosclerotic heart disease.
In this article, we describe two new kindreds with FHB, each associated with a truncated form of apoB. The apoB-44.4 mutation was identified in a 48-year-old man with undetectable levels of LDL cholesterol and a total cholesterol level of 34 mg/dL. These remarkably low cholesterol levels are typical of those found in compound heterozygotes for FHB and are significantly lower than levels typically observed in FHB heterozygotes.5 We therefore strongly suspect that the apoB-44.4 proband was a compound heterozygote for FHB, with one allele yielding apoB-44.4 and a second allele associated with markedly decreased levels of apoB-100. Another reason for suspecting that the apoB-44.4 proband was a compound heterozygote for FHB was supplied by examination of the VLDL fractions in the family. In the VLDL of FHB heterozygotes, we previously documented that the amount of apoB-100 invariably exceeds the amount of the truncated apoB.5 8 11 13 14 15 Consistent with these prior findings, the ratio of apoB-100 to apoB-44.4 in the VLDL of a half brother of the apoB-44.4 proband (a FHB heterozygote) was 19.4:1. In contrast, a high ratio of apoB-100 to apoB-44.4 was not observed in the VLDL of the apoB-44.4 proband. Despite having a similar absolute amount of VLDL–apoB-44.4 (compared with his half brother), the ratio of apoB-100 to apoB-44.4 in the proband’s VLDL fraction was 0.31:1. We strongly suspect that the low levels of apoB-100 in the VLDL fraction of the proband were due to a second mutant allele associated with very low levels of a full-length apoB-100. This idea was supported by analysis of the proband’s 12-year-old son. That subject almost certainly inherited the mutant allele associated with low levels of apoB-100; he clearly had FHB (LDL cholesterol level of 8 mg/dL), but he did not inherit the apoB-44.4 mutation. Because of the small size of the apoB-44.4 family, we could not perform the haplotyping studies required to prove that the allele yielding low levels of apoB-100 was truly a mutant apoB allele, as opposed to a mutant allele of some other dominantly inherited, yet-to-be-identified cholesterol-lowering gene.16 However, one could argue that the latter possibility is unlikely. If the proband had some other non-apoB cholesterol-lowering gene, one might expect that he would have low levels of both apoB-44.4 and apoB-100. This was not the case; comparison of the VLDL fractions of the proband and the half brother revealed that the proband had a selective decrease in the amount of apoB-100. Moreover, there have been several prior reports of well-characterized FHB compound heterozygotes who had one mutant apoB allele yielding a truncated apoB and a second mutant apoB allele yielding low levels of apoB-100.3 6 12 13 Like the apoB-44.4 proband in this study, the VLDL of these compound heterozygotes also contained low levels of apoB-100 relative to the level of the truncated apoB.
The mutation responsible for the production of apoB-44.4 was an 11-bp insertion within exon 26 of the apoB gene. Insertional mutations are much less common than deletions. Except for a single mutation involving an insertion of one adenine into a stretch of seven consecutive adenines,17 no prior insertional mutations have been described in the apoB gene. In contrast, more than 10 short deletions have been identified in the apoB gene.5 Insertional mutations are likewise rare in other genetic diseases. Of 82 different mutations causing cystic fibrosis, only 6 were insertions.18 Similarly, of 150 different mutations in the LDL receptor gene causing familial hypercholesterolemia, only 9 were insertions.19
The second case of hypobetalipoproteinemia was a 57-year-old man with an LDL cholesterol level of 44 mg/dL, a level quite typical for the heterozygous form of FHB. He had apoB-55 in his lipoproteins, the result of a nonsense mutation in exon 26 of the apoB gene. The same mutation was previously identified in an 80-year-old man with heterozygous hypobetalipoproteinemia and atypical retinitis pigmentosa.7 In that case, the retinitis pigmentosa appeared to be etiologically unrelated to the FHB, and the absence of retinitis pigmentosa in our subject supports this conclusion. Of note, the two independently identified subjects with apoB-55 did not share an identical apoB haplotype. In our subject with apoB-55, both apoB alleles lacked the polymorphic Xba I site in exon 26 of the apoB gene; in the other patient with the apoB-55 mutation, both alleles contained the Xba I polymorphism. It is quite possible that the two apoB-55 mutations occurred independently, as the mutation occurred at a CpG dinucleotide, a mutational hot spot.20 21 In any case, this report documents the first example of the same mutation causing hypobetalipoproteinemia in more than one kindred.
Truncated forms of apoB can cause low levels of LDL cholesterol and may be common causes of FHB. Intuitively, one would expect that these mutations would prevent the development of coronary artery disease; therefore, it is interesting that the father of the apoB-55 proband, who almost certainly carried the mutation, had significant atherosclerotic disease at autopsy. In the future, noninvasive studies of the carotid arteries could provide important information on the incidence of atherosclerosis in subjects with FHB.
This work was supported by NHLBI grants HL-02626 (F.K.W.) and HL-41633 (S.G.Y.). Dr Welty was a Research and Teaching Scholar of the American College of Physicians. We gratefully acknowledge the assistance of Dr Richard Karas in this research. We express deep appreciation to the apoB-44.4 and apoB-55 families for their participation in this research.
- Received March 23, 1995.
- Revision received July 10, 1995.
- Accepted August 18, 1995.
- Copyright © 1995 by American Heart Association
Chen S-H, Habib G, Yang C-Y, Gu Z-W, Lee BR, Weng S-A, Silberman SR, Cai S-J, Deslypere JP, Rosseneu M, Gotto AM Jr, Li W-H, Chan L. Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon. Science. 1987;238:363-366.
Young SG. Recent progress in understanding apolipoprotein B. Circulation. 1990;82:1574-1594.
Collins DR, Knott TJ, Pease RJ, Powell LM, Wallis SC, Robertson S, Pullinger CR, Milne RW, Marcel YL, Humphries SE, Talmud PJ, Lloyd JK, Miller NE, Muller D, Scott J. Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia. Nucleic Acids Res. 1988;16:8361-8375.
Huang L-S, Ripps ME, Korman SH, Deckelbaum RJ, Breslow JL. Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination. J Biol Chem. 1989;264:11394-11400.
Pullinger CR, Hillas E, Hardman DA, Chen GC, Naya-Vigne J-M, Iwasa JA, Hamilton RL, Lalouel J-M, Williams RR, Kane JP. Two apolipoprotein B gene defects in a kindred with hypobetalipoproteinemia, one of which results in a truncated variant, apoB-61, in VLDL and LDL. J Lipid Res. 1992;33:699-710.
Welty FK, Hubl ST, Pierotti VR, Young SG. A truncated species of apolipoprotein B (B67) in a kindred with familial hypobetalipoproteinemia. J Clin Invest. 1991;87:1748-1754.
Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74:5463-5467.
Farese RV Jr, Garg A, Pierotti VR, Vega GL, Young SG. A truncated species of apolipoprotein B, B-83, associated with hypobetalipoproteinemia. J Lipid Res. 1992;33:569-577.
Young SG, Bertics SJ, Curtiss LK, Dubois BW, Witztum JL. Genetic analysis of a kindred with familial hypobetalipoproteinemia: evidence for two separate gene defects: one associated with an abnormal apolipoprotein B species, apolipoprotein B-37; and a second associated with low plasma concentrations of apolipoprotein B-100. J Clin Invest. 1987;79:1842-1851.
Young SG, Bertics SJ, Curtiss LK, Witztum JL. Characterization of an abnormal species of apolipoprotein B, apolipoprotein B-37, associated with familial hypobetalipoproteinemia. J Clin Invest. 1987;79:1831-1841.
Young SG, Pullinger CR, Zysow BR, Hofmann-Radvani H, Linton MF, Farese RV Jr, Terdiman JF, Snyder SM, Grundy SM, Vega GL, Malloy MJ, Kane JP. Four new mutations in the apolipoprotein B gene causing hypobetalipoproteinemia, including two different frameshift mutations that yield truncated apolipoprotein B proteins of identical length. J Lipid Res. 1993;34:501-507.
Hobbs HH, Leitersdorf E, Leffert CC, Cryer DR, Brown MS, Goldstein JL. Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors. J Clin Invest. 1989;84:656-664.
Groenewegen WA, Krul ES, Averna MR, Pulai J, Schonfeld G. Dysbetalipoproteinemia in a kindred with hypobetalipoproteinemia due to mutations in the genes for apoB (apoB-70.5) and apoE (apoE2). Arterioscler Thromb. 1994;14:1695-1704.
Pearson P, Francomano C, Foster P, Bocchini C, Li P, McKusick V. The status of online mendelian inheritance in man (OMIM) medio 1994. Nucleic Acids Res. 1994;22:3470-3473.