Hypobetalipoproteinemia Caused by Apob-76, a Newtruncation

CASE REPORT

Clinical Characterization of a Case with Familial Hypobetalipoproteinemia Caused by Apo B-76, a NewTruncation of Apolipoprotein B, Combined with Apo E2/E2 Phenotype Kazuo Takahashi, Minoru Hikita, Kouichi Taira, Junji Kobayashi, Hideaki Bujo* and Yasushi Saito

Abstract B molecule and that result in the synthesis of truncated forms of apo B (1). Natural mutations giving rise to truncated apo B Wereport a 43-year-old Japanese manwith hypobeta- varying in size from apo B-2 to apo B-89 have been described lipoproteinemia likely due to apolipoprotein (apo) B-76, a (1). In several reports, both increased rates of catabolism and new truncation of apo B, and with homozygousity for the low production rates for the truncated apo B molecules have apo E2 isoform. He had no history suggestive of fat malab- been implicated as the cause of low LDLcholesterol levels (3- sorption and no sign of neurological disorder. His fasting 6). baseline serum low-density lipoprotein (LDL) cholesterol ApoE is a constituent of plasma chylomicron remnants, and apo B levels were approximately half of normal. His very low density lipoprotein (VLDL), intermediate density li- plasma apo E level was elevated and its phenotype showed poprotein (IDL), and high density lipoprotein (HDL) and plays the E2/E2 homozygote. SDS-polyacrylamide gel electro- an important role in lipoprotein metabolism (7-10). The' three phoresis of delipidated LDLfraction revealed a newtrun- major isoforms of apo E (E2, E3, and E4), as identified by cated apo B, designated as apo B-76 according to the centile isoelectric focusing, are coded for by three alleles (e2, e3, and system of nomenclature. The postprandial lipid metabo- e4) (ll, 12). The E2 isoform shows poor binding of remnants lism of the patient showedan almost normal response after to hepatic lipoprotein receptors and delayed clearance from fat loading. plasma (13-15). Most of the patients with type III hyperlip- (Internal Medicine 40: 1015-1019, 2001) idemia are homozygous for the E2 isoform ( 16). However, only 1-4% of E2/E2 homozygotes develops type III hyperlipidemia; Key words: low density lipoprotein, polyacrylamide gel elec- additional environmental factors may be involved in the devel- trophoresis, postprandial lipid metabolism opment of the disorder. Here, we report a 43-year-old Japanese manwith hypobetalipoproteinemia likely due to apo B-76, a new truncation of apo B, and with homozygousity for the apo E2 isoform. Introduction Case Report Familial hypobetalipoproteinemia, an autosomal codominant disorder characterized by a low plasma cholesterol concentra- Case report tion, can be caused by mutations in the apo B gene (1). Indi- The patient, a 43-year-old Japanese man, was referred to viduals with the homozygousform of hypobetalipoproteinemia our hospital for obesity treatment. Developments during in- have extremely low levels of LDLcholesterol (<10 mg/dl) and fancy and growth after birth were within normal limits. He had often display a severe clinical phenotype with intestinal fat no history of persistent diarrhea or loose stool suggestive of fat malabsorption and neurological disease associated with the malabsorption. Hebecameobese after the age of 30 years. He deficiency of fat-soluble vitamins (1 , 2). Heterozygotes have was diagnosed to have hypertension at the age of 37 and has moderately low LDLcholesterol levels (<40 mg/dl) and are been on medicationsince then. Onexamination, the patient's usually asymptomatic. Most of the mutations causing height was 169 cm and weight was 125 kg (body mass index hypobetalipoproteinemia have been nonsense or frameshift 43.8 kg/m2). Systolic blood pressure and diastolic blood pres- mutations that interfere with the translation of a full-length apo sure were 160 and 82 mmHg,respectively. There were no signs

From the Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, Chiba and *the Department of Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba Received for publication August 15, 2000; Accepted for publication June 1 1 , 2001 Reprint requests should be addressed to Dr. Kazuo Takahashi, the Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-0856

Internal Medicine Vol. 40, No. 10 (October 2001) 1015 Takahashi et al of clinically overt neurological disorder or atherosclerosis. Results Blood analysis of the patient showed a red blood cell count of Lipids and lipoproteins 4.6xlO6 /ml without acanthocytes, and hemoglobin of 13.5 g/ ide,The patient'sand HDLcholesterol,plasma levels wereof 98,total 85,cholesterol,and 32 mg/dl,triglycer-respec- dl. Blood chemical values, including total protein, aspartate tively,apo AI, whenAll, heB, visitedCII, CIII,our hospitaland E concentrationsfor the first weretime. 99,The28,plasma35, aminotransferase, alanine aminotransferase, lactic dehydroge- 3.9, 6.3, and 8.0 mg/dl, respectively (normal range: 137±26, Polyacrylamide32±6, 79±20, 3.4+1.3,gel electrophoretic7.5±3.0, 4.1±1.2analysismg/dl,ofrespectively).the patient's nase, alkaline phosphatase, bilirubin, urea nitrogen, creatinine, plasma showed a trace amount of LDLand a broad peak be- creatinine kinase, and electrolytes, were all normal. The 75 g teroltween LDLand andapo VLDLB in (Fig.the LDL1). fractionThe concentrationsshowed approximatelyof choles- oral glucose tolerance test showed a normal pattern. There was VLDL,one-halfIDL,theandnormalLDL fractionslevel and (Tablethe apo1).B was distributed in no evidence of endocrinological disorder. Computerized tomog- Identification of a truncated apo B from the patient's plasma raphy at the umbilicus level showed 248 cm2 of visceral fat containedTo determinea truncatedwhether theapoplasmaB, the lipoproteinsVLDLand LDLoffractionsthe patientwere area as excessive intra-abdominal visceral fat accumulation. examinedscribed above.by SDS-polyacrylamideThe stained polyacrylamidegel electrophoresisgel revealed aastrun-de- Lipid and lipoprotein analysis Various lipoprotein fractions were isolated using the stan- dard ultracentrifugation technique according to the method of Havel et al (17). Plasma and lipoprotein cholesterol, triglyceride, and phospholipid levels were measured by enzymatic methods. Plasmaapolipoprotein concentrations weredeterminedby single radial immunodifusion methods. The plasma lipoprotein profile was analyzed by polyacrylamide gel electrophoresis (18). The separated lipoprotein fractions were stained with Sudan Black and detected with a densitometer. Plasma apo E isoform was determined by isoelectric focusing (IEF) on a polyacrylamide gel (19). Determination of the truncated apolipoprotein B in VLDLand LDL fractions To determine whether the plasma lipoproteins of the patient contained a truncated apo B, the VLDLand LDLfractions were examined by SDS polyacrylamide gel electrophoresis, as described elsewhere (20). The prepared lipoprotein fractions were delipidated in 3: 1 ether/ethanol containing 20 mMbutylated hydroxytoluene and then dissolved in SDSsample buffer. Samples were heated at 100°C for 5 minutes and separated on 3% polyacrylamide gels containing 0. 1% SDS. After electrophoresis, apolipoproteins were visualized by siver staining. To determine the molecular weight of the truncated apo Bfrom the patient, we used the following published values for the HDLLDL VLDL molecular weights of the normal apo B: B-100, 550,000 and B-48, 264,000 (20). Figure 1. Patient's lipoprotein sub fraction by polyacrylamide Vitamin A and fat loading test gel electrophoresis. The plasma lipoprotein profile was analyzed As described previously, the oral vitamin A and fat loading by polyacrylamide gel electrophoresis. The separated lipoprotein fractions were stained with Sudan Black and detected with test wascarried out on the patient, as well as an apo E2/E2 a densitometer. homozygote, and a control (n=5) (21). Briefly, after a 12 hours overnight fast, the subjects ingested 40 g/m2 fresh cream, as the fat source, containing 50,000 units/m2 of vitamin A. After the fat loading, peripheral blood samples were obtained at 2 Table 1. Lipid and Lipoprotein Profiles of the Subject hours intervals up to 6 hours. Plasma triglyceride level was (mg/dl) Plasma VLDL IDL LDL HDL2 HDL3 measured by enzymatic methods. The lipid was extracted from each sample and the concentration of retinyl palmitate (RP) Cholesterol 115.0 19.0 8.6 37.1 22.2 26.4 was measured by high performance liquid chromatography (2 1 ). Triglycerides 87. 1 6 1.3 4.9 8. 1 6.0 5.7 The significance of the metabolic response after the fat load- Phospholipids 146.0 23.3 7.5 24.9 31.8 36.2 ing was analyzed by assessing the curve of triglyceride and RP Apo B 33.5 5.9 5.0 17.8 n.d. n.d. values. n.d.: not determined.

1016 Internal Medicine Vol. 40, No. 10 (October 2001) Hypobetalipoproteinemia with Apo B-76 cated apo B (Fig. 2). The molecular weight of the abnormal Determination of apo E phenotype apo B as determined by regression analysis was found to be To demonstrate the mechanism of a high plasma level of 4 1 8,000. According to the centile system of nomenclature (20), apo E and the formation of IDLfraction, weanalyzed the apo the abnormal apo B was designated as apo B-76. E phenotype by IEF. Apo E phenotype of the patient showed homozygousity for the E2 isoform (data not shown). Kinetic property of the patient in response to a high fat meai To analyze the post-prandial lipid metabolism in the patient, wemeasuredthe concentration of triglyceride andRPafter vitamin A and fat loading as described in the Methods section. Wealso performed the test on a subject with homozygousity for the apo E2 isoform whodid not have hypertriglyceridemia and a control (n=5). Figure 3A shows the time course of plasma triglyceride concentrations after the fat loading test. The plasma triglyceride levels of the patient increased slightly at 4 and 6 hours after fat loading, but the changes in the triglyceride levels did not differ significantly from the control. Onthe con- trary, the triglyceride levels of the subject with apo E2/E2 homozygote increased remarkably until 6 hours after fat loading. Moreover, plasma RP levels of the patient as well as the triglyceride levels increased slightly at 4 hours after fat loading, but the changes of the RP levels did not differ from the control (Fig. 3B). The RP levels of the subject with apo E2/E2 homozygote increased remarkably until 6 hours after fat loading. Discussion In this report, we present a case of hypobetalipoproteinemia combined with homozygousity for the apo E2 isoform. Famil- ial hypobetalipoproteinemia can be caused by mutations in the apo B gene that interfere with the translation of a full-length apo B molecule (1). Frequently, a truncated apo B molecule can be detected in the plasma of affected subjects. The patient had a truncated apo B in VLDLand LDLfractions. The molecular weight of the truncated apo B was estimated to be 76% of the full-length apo B-100. Various truncated forms of apo B Q Apo B-100 have been found to segregate with the hypobetalipoproteinemia phenotype in more than 30 kindred. Then, on the basis of this protein sizing, direct sequencing of exon 26 was carried out on

Figure 2. Analysis of patient's apo B by SDS-polyaerylamide Apo B-48 gel electrophoresis. A. 3 %polyacrylamide gel stained with silver. VLDLand LDLfractions were isolated by ultracentrifugation and delipidated. Then, the samples were separated by SDS- polyacrylamide gel electrophoresis and visualized by silver staining. Lanes 1: VLDLfraction obtained from the patient, 2: VLDL fraction from control, 3: LDLfraction from the patient, 4: LDL fraction from control. Large arrow: apo B-100, small arrow: apo B-48, arrowhead: apo B-76, truncated apo B from the patient. B. Plot of the mobility of apo B-100 and B-48 on 3% polyacry- 1 2 3 4 lamide gel. The plot is delivered from polyacrylamide gel stained with silver shown in Fig. 2A. The migration length of the patient's Distance from the top (cm) truncated apo B is indicated by the closed circle. The molecular weight of the truncated apo B (418,000) was estimated from the B regression curve.

1017 Internal Medicine Vol. 40, No. 10 (October 2001) Takahashi et al

(mg/dl) teins among the different lipoprotein classes. Apo B-76 was c 300 | r- 1 r 1 o present primarily in the LDLfraction but also in VLDLand 1 ji -* IDL fractions. It is known that the lipid-binding domains are

^ distributed rather uniformly along the sequence of the apo B o 200 - j^*^ protein, longer apo B proteins are therefore capable of binding more lipid-enriched lipoprotein particles (22, 23). For most mutations, it is unknownwhich biochemical-metabolic pro- cesses are affected. Wealso could not investigate the explana- tion for the low LDLcholesterol level of the patient. However, in the case with apo B-75, the kinetic analysis showedthat the production of lipoprotein particles containing apo B-75 was * 0I » 1 « impaired and the clearance of apo B-75 containing LDLpar- ticles was higher than apo B-100 containing particles due to 0 2 4 6 (h) the enhanced LDL-receptor binding of apo B-75 (6). Then, both Time after fat loading low production rates and increased rates of catabolism for the G"g/dl) truncated apo B molecules have been implicated as the cause c , , , , , 2 i i i of a low LDLconcentration (6). Aninteresting aspect of this study is that the postprandial lipid metabolism of the patient showed an almost normal response after the fat loading despite his clinical characteristics such as intra-abdominal visceral fat accumulation and homozygousity for apo E2 isoform. Wepreviously reported that postprandial lipid metabolism is impaired in subjects with intra-abdominal visceral fat accumulation (2 1 ). Moreover, Rail et al revealed that apo E2 of hypo-, normo-, and hypercholesterolemic subjects showsthe samesevere functional abnormali- ties in the binding of remnants to hepatic lipoprotein receptors and in the clearance from plasma (24). These data suggested | 0 2 4 6 (h) that the existence of the truncated apo Bmight affect the post- Time after fat loading prandial lipid metabolism. Krul et al demonstrated the in vivo B kinetics of lipoproteins in the subject with truncated apo B-75 and showed that VLDL,IDL, and LDLparticles containing not only apo B-75 but also apo B-100 were present at a lower Figure 3. Analysis of lipid metabolism after vitamin Aand fat loading. The subjects ingested 40 g/m2 fresh cream and 50,000 concentration than in normolipidemic control subjects (6). In units/m2 of vitamin A. Peripheral blood samples were obtained addition, their kinetic analysis suggested that VLDLand IDL at 2 hours intervals up to 6 hours after fat loading. A. Time- particles containing apo B- 100 might be preferentially cleared dependent changes of plasma triglyceride concentration after from plasma rather than being converted to LDL(6). Further fat loading. Closed circle: patient, open circle: control open tri- experiments are necessary to clarify this hypothesis. angle: normolipidemic subject with apo E2/E2 homozygote. B. Time-dependent changes of plasma RP concentration after fat References loading. Plasma RP was measured by high performance liquid chromatography. Closed circle: patient, open circle: control open 1) Linton MF, Farese RVJr, Young SG. Familial hypobetalipoproteinemia. triangle: normolipidemic subject with apo E2/E2 homozygote. J Lipid Res 34: 521-541, 1993. 2) Kane JP, Havel RJ. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins. in: The Metabolic Basis of Inher- ited Disease. 6th ed. Scriver CR, Beaudet AL, Sly WS, and Valle D. Eds. McGraw-Hill, New York, 1989: 1 139-1 164. DNAfrom the patient. Unfortunately, DNAsequencing revealed 3) Hardman DA, Pullinger CR, Hamilton RL, Kane JP, Malloy MJ. Molecu- that the patient had no mutation between nucleotide positions lar and metabolic basis for the metabolic disorder normotriglyceridemic 10107 (residues 3300) and 1 1459 (residues 3750) of apo B abetalipoproteinemia. J Clin Invest 88: 1722-1729, 1991. 4) Parhofer KG, Daugherty A, Kinoshita M, Schonfeld G. Enhanced clear- CDNA.At least we denied the mutations resulting in the syn- ance from plasma of low-density lipoproteins containing a truncated thesis of apo B-75 (deletion ofC at 10366) and apo B-82 (C to apolipoprotein, apo B-89. J Lipid Res 31: 2001-2007, 1990. A transversion at 1 141 1), which were positioned closest to apo 5) Parhofer KG, Barrett PH, Bier DM, Schonfeld G. Lipoproteins contain- B-76 (data not shown). Therefore, apo B-76 reported here is ing the truncated apolipoprotein, apo B-89, are cleared from human plasma the first, to our knowledge, to be identified in a Japanese sub- more rapidly than apo B-100-containing lipoproteins in vivo. J Clin In- ject. vest 89: 1931-1937, 1992. 6) Krul ES, Parhofer KG, Barrett PH, Wagner RD, Schonfeld G. Apo B-75, Weexamined the distribution of the truncated apo B pro- a truncation of apolipoprotein B associated with familial hypobeta-

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