Mutation in Apolipoprotein B Associated with Hypobetalipoproteinemia Despite Decreased Binding to the Low Density Lipoprotein Receptor

Mutations in apolipoprotein B (APOB) may reduce binding of low density lipoprotein (LDL) to the LDL receptor and cause hypercholesterolemia. We showed that heterozygotes for a new mutation in APOB have hypobetalipoproteinemia, despite a reduced binding of LDL to the LDL receptor. APOB R3480P heterozygotes were identified among 9,255 individuals from the general population and had reduced levels of apoB-containing lipoproteins. Most surprisingly, R3480P LDL bound with lower affinity to the LDL receptor than non-carrier LDL in vitro, and these results were confirmed by turnover studies of LDL in vivo. In very low density lipoprotein (VLDL) turnover studies, the amount of VLDL converted to LDL in R3480P heterozygotes was substantially reduced, suggesting that this was the explanation for the hypobetalipoproteinemia observed in these individuals. Our findings emphasized the importance of combining in vitro studies with both human in vivo and population-based studies, as in vitro studies often have focused on very limited aspects of complex mechanisms taken out of their natural context. Mutations in apolipoprotein B (APOB) may reduce binding of low density lipoprotein (LDL) to the LDL receptor and cause hypercholesterolemia. We showed that heterozygotes for a new mutation in APOB have hypobetalipoproteinemia, despite a reduced binding of LDL to the LDL receptor. APOB R3480P heterozygotes were identified among 9,255 individuals from the general population and had reduced levels of apoB-containing lipoproteins. Most surprisingly, R3480P LDL bound with lower affinity to the LDL receptor than non-carrier LDL in vitro, and these results were confirmed by turnover studies of LDL in vivo. In very low density lipoprotein (VLDL) turnover studies, the amount of VLDL converted to LDL in R3480P heterozygotes was substantially reduced, suggesting that this was the explanation for the hypobetalipoproteinemia observed in these individuals. Our findings emphasized the importance of combining in vitro studies with both human in vivo and population-based studies, as in vitro studies often have focused on very limited aspects of complex mechanisms taken out of their natural context. Low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; VLDL, very LDL; IDL, intermediate density lipoprotein; FCR, fractional catabolic rate; PR, production rate; HDL, high density lipoprotein. particles are cleared from plasma mainly by binding to high affinity LDL receptors and subsequent internalization and degradation in the liver. Affinity of LDL to the receptor is dependent on intact structural and functional domains in both the receptor and the ligand, apoB-100 (1Goldstein J.L. Hobbs H.H. Brown M.S. Scriver C.R. Beaudet A.L. Valle D. Sly W.S. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Diseases. McGraw-Hill Inc., New York2001: 2863-2913Google Scholar). ApoB-100, a 513-kDa glycoprotein composed of 4536 amino acid residues, is the predominant protein component of VLDL, IDL, and LDL (2Havel R.J. Kane J.P. Scriver C.R. Beaudet A.L. Valle D. Sly W.S. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Diseases. McGraw-Hill Inc., New York2001: 2717-2752Google Scholar). Studies of the three-dimensional structure of apoB-100 by immunoelectron microscopy have suggested that apoB-100 enwraps VLDL, IDL, and LDL particles like a belt, completing the encirclement by about amino acid residue 4050, and that the carboxyl-terminal end forms a bow that crosses backwards over the chain between residues 3000 and 3500 (3Chatterton J.E. Phillips M.L. Curtiss L.K. Milne R. Fruchart J.C. Schumaker V.N. J. Lipid Res. 1995; 36: 2027-2037Abstract Full Text PDF PubMed Google Scholar). Chatterton et al. (3Chatterton J.E. Phillips M.L. Curtiss L.K. Milne R. Fruchart J.C. Schumaker V.N. J. Lipid Res. 1995; 36: 2027-2037Abstract Full Text PDF PubMed Google Scholar) speculated that the carboxyl-terminal sequence of apoB-100 could act as a negative regulator of LDL receptor binding, and they proposed that in VLDL particles the bow would inhibit apoB-100 binding to the LDL receptor. In contrast, after lipolysis had transformed VLDL into IDL and finally LDL, the carboxyl-terminal bow moved sufficiently to allow interaction of apoB-100 with the LDL receptor. Furthermore, mutagenesis studies in mice by Borén et al. (4Borén J. Lee I. Zhu W. Arnold K. Taylor S. Innerarity T.L. J. Clin. Investig. 1998; 101: 1084-1093Crossref PubMed Scopus (183) Google Scholar) suggested that the region where the carboxyl-terminal bow of apoB-100 crosses the main chain of apoB-100 is located around residue 3500 in LDL. In humans, three point mutations have been reported around this site in the APOB gene. All three mutations result in the loss of arginines and in varying degrees of decreased receptor affinity in vitro, reflected in the varying effects on lipid and lipoprotein levels in vivo; R3500Q and R3500W are associated with moderate to severe hypercholesterolemia, whereas R3531C has no effect or a marginal effect on lipid levels in the general population in vivo (5Soria L.F. Ludwig E.H. Clarke H.R.G. Vega G.L. Grundy S.M. McCarthy B.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 587-591Crossref PubMed Scopus (470) Google Scholar, 6Pullinger C.R. Hennessy L.K. Chatterton J.E. Liu W. Love J.A. Mendel C.M. Frost P.H. Malloy M.J. Schumaker V.N. Kane J.P. J. Clin. Investig. 1995; 95: 1225-1234Crossref PubMed Scopus (169) Google Scholar, 7Borén J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 8Tybjærg-Hansen A. Steffensen R. Meinertz H. Schnohr P. Nordestgaard B.G. N. Engl. J. Med. 1998; 338: 1577-1584Crossref PubMed Scopus (167) Google Scholar). Mutations resulting in truncations of apoB-100 are known to abolish LDL receptor binding when located amino-terminal to the putative binding site at amino acids 3359–3369 (site B), indicating that this site is crucial for receptor interaction. In contrast, apoB truncations enhance LDL receptor binding when located carboxyl-terminal to the binding site, indicating that the negative regulatory effect of the bow of apoB-100 can be abolished by removal of the carboxyl-terminal tail (9Parhofer K.G. Barrett P.H.R. Bier D.M. Schonfeld G. J. Clin. Investig. 1992; 89: 1931-1937Crossref PubMed Scopus (61) Google Scholar, 10Gabelli C. Bilato C. Martini S. Tennyson G.E. Zech L.A. Corsini A. Albanese M. Brewer Jr., H.B. Crepaldi G. Baggio G. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1189-1196Crossref PubMed Scopus (23) Google Scholar, 11Linton M.F. Farese Jr., R.V. Young S.G. J. Lipid Res. 1993; 34: 521-541Abstract Full Text PDF PubMed Google Scholar). A mutation in APOB, R3480P, located in the region where the carboxyl-terminal bow crosses over the main apoB-100 chain, and briefly mentioned in a previous paper (12Nissen H. Hansen P.S. Færgeman O. Hørder M. Clin. Chem. 1995; 41: 419-423Crossref PubMed Scopus (31) Google Scholar), led us to the following hypothesis. Because this mutation causes a substitution of the basic side chain of arginine with the non-polar of proline in an α-helical region, the R3480P mutation might have an impact on both the secondary and tertiary structure of apoB-100, and thus on the affinity of apoB-100 for the LDL receptor via a change in the negative regulatory effect of the carboxyl-terminal bow. This then could affect LDL levels, catabolism, and production in R3480P heterozygotes. To study the effect of the R3480P mutation in apoB-100 on the levels of lipids, lipoproteins, and apolipoproteins and to characterize the effect of the mutation on LDL metabolism, we screened 13,427 Danish individuals (9,255 individuals from the general population (The Copenhagen City Heart Study) and 4,172 patients with ischemic cardiovascular disease or hyperlipidemia) for the presence of this mutation. In R3480P heterozygotes, we determined lipids, lipoproteins, and apolipoproteins, and we compared these with the corresponding values in non-carriers in the general population of the same age, sex, and APOE genotype. The relative LDL receptor affinity of R3480P LDL and non-carrier LDL was determined in vitro in a competitive receptor-binding assay in normal human fibroblasts. In in vivo turnover studies of iodinated LDL, we compared fractional catabolic rate (FCR) and production rate (PR) of LDL from R3480P, R3480W, R3500Q, and R3531C heterozygotes with simultaneously injected iodinated LDL from non-carriers. Finally, in in vivo turnover studies of iodinated VLDL, we estimated the amount of VLDL from R34380P heterozygotes converted to LDL compared with the conversion of non-carrier VLDL. This design with simultaneous injection of differently labeled R3480P LDL (or VLDL) and non-carrier LDL (or VLDL) excludes all variation because of differences between recipients, because the two types of lipoprotein are metabolized by the exact same pathways in the same recipient. Thus, even small differences in metabolism because of differences in LDL (or VLDL) particles can be detected accurately. Subjects—The Copenhagen City Heart Study (1991–1994) comprised an almost equal number of women (55%) and men stratified into 10-year age groups from 20 to 80 years and above, drawn randomly from the Copenhagen Central Population with the of a of the Danish general population M. A. G. Schnohr P. J. J. Med. 1989; 41: Scholar, P. G. P. H. M. Heart J. 2001; 9,255 individuals were for the A population comprised 4,172 patients with a of ischemic disease ischemic disease or In 13,427 individuals 9,255 were for the and of these were and of Danish The studies were by and Danish as Copenhagen and and and Copenhagen and was to the of studies were by the Danish of R3480P mutation was by the substitution of for at in in of the APOB on C. M. D. J. PubMed Scopus Google Scholar, E.H. C. J. B. McCarthy B.J. PubMed Scopus Google Scholar). from of the 13,427 individuals was to and with an of by with The in the an site for The R3500Q and R3531C mutations were identified as A. Steffensen R. Meinertz H. Schnohr P. Nordestgaard B.G. N. Engl. J. Med. 1998; 338: 1577-1584Crossref PubMed Scopus (167) Google a with was identified of this apoB-100 region in individuals to be used as non-carriers in turnover The presence of all mutations was confirmed by APOE was determined as R. Nordestgaard B.G. B. Schnohr P. A. J. Lipid Res. 41: Full Text Full Text PDF PubMed Google Scholar). and and were used to plasma levels of and apoB A. Steffensen R. Meinertz H. Schnohr P. Nordestgaard B.G. N. Engl. J. Med. 1998; 338: 1577-1584Crossref PubMed Scopus (167) Google Scholar). The of LDL and apoB were determined as J. Lipid Res. Full Text PDF PubMed Google Scholar, B.G. A. B. Arterioscler. Thromb. 1992; PubMed Google Scholar, J.P. in Scholar). plasma of three mutation number number and and for age in 10-year age sex, and APOE lipoprotein were in a and Inc., at density with and were in VLDL IDL LDL and In of receptor binding were as by Arnold et al. K. Innerarity T.L. A Scholar). In human were in and To LDL were to with human LDL with of R3480P or non-carrier LDL, was to the in and human of at the was in were for differences in by the protein in and the number of of The number of of protein was for The amount of LDL to of normal LDL after a was determined K. Innerarity T.L. A Scholar). In LDL and VLDL LDL turnover studies were in vivo to and of LDL from R3480P, R3480W, R3500Q, and R3531C heterozygotes LDL from non-carriers simultaneously injected in the same this of LDL production rate that production R3480P LDL, findings in low LDL receptor were with turnover studies in high LDL receptor VLDL turnover studies were in vivo to the of VLDL, and the amount of VLDL converted to LDL from R3480P heterozygotes VLDL from non-carriers was simultaneously injected in the same recipient. and of LDL to and the for turnover studies were as K. Nordestgaard B.G. B. K. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar, G. Grundy S.M. J. Lipid Res. Full Text PDF PubMed Google Scholar). VLDL turnover studies were in the same that in apolipoprotein B was in lipoprotein with B.G. A. B. Arterioscler. Thromb. 1992; PubMed Google Scholar), of plasma protein with acid as in the LDL study K. Nordestgaard B.G. B. K. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). catabolic rate (FCR) of LDL, as the of the of LDL was to the of Med. Biol. PubMed Scopus Google Scholar), where the of Scholar). rate was by the of LDL by the of LDL and was as of LDL was the of plasma LDL and plasma estimated by at after injection of labeled LDL. Because plasma LDL levels were lower in R3480P heterozygotes than in we used the LDL levels of R3480P heterozygotes and of non-carriers to the estimated of LDL. The amount of VLDL converted to LDL was by the of VLDL with the of VLDL of VLDL was estimated from human in vivo VLDL turnover studies a in the of P. Barrett us the that the VLDL catabolic rate of VLDL as the of the of VLDL to LDL was determined with the a The of two VLDL and and two LDL and was injected into and and were drawn from and were estimated for the the removal of VLDL from the removal of VLDL from the removal of LDL from the of VLDL from to the from to the fractional catabolic rate of VLDL or the of VLDL converted to LDL in and the between and for the two lipoprotein were taken as the of plasma estimated by at after injection of the labeled VLDL and the plasma at of LDL from VLDL was by the of VLDL with the fractional catabolic rate of VLDL the R3480P heterozygotes identified in the general population were all for the APOE all were to individuals with the the were with and for Scholar, for Inc., Scholar). To the effect of R3480P on in heterozygotes identified in the general we converted values for for heterozygotes to their 10-year age and by sex, and we compared with the values of in the general population as a as A. Steffensen R. Meinertz H. Schnohr P. Nordestgaard B.G. N. Engl. J. Med. 1998; 338: 1577-1584Crossref PubMed Scopus (167) Google Scholar, P. G. in Google Scholar). The or in a in R3480P heterozygotes was by the for non-carriers in the general population for age 10-year age sex, and APOE from the of the R3480P the or for a were compared with the normal with by a All values are and a than or equal to is to of the APOB R3480P of 9,255 in the general population and of 4,172 patients with ischemic cardiovascular disease or and were for the R3480P mutation in apoB of APOB R3480P in the with non-carriers in the general population of the same age, sex, and APOE R3480P and to be associated with lower than levels of plasma LDL IDL VLDL and and with than and of the R3480P heterozygotes had a of or of ischemic a with and had a at age of for R3480P and compared with non-carriers in the general population of the same age, sex, and apolipoprotein of at of of years of age, or in a new Because all were simultaneously in all of the Copenhagen City Heart was to to a in the 10-year age by and APOE thus for known effects of age, sex, and APOE on lipid and lipoprotein for R3480P was associated with lower LDL apoB and compared with non-carriers in the general population of the same age, sex, and APOE R3480P was associated with and compared with non-carriers in the general A had values for these to for R3480P heterozygotes VLDL and age as as and of to between R3480P heterozygotes and non-carriers in the general population of ischemic ischemic and between R3480P heterozygotes and non-carriers in the general population The R3480P mutation was associated with in levels of LDL VLDL and of of and and with in levels of and the of of and when compared with individuals in the general population of the same age 10-year age sex, and APOE change in lipids, lipoproteins, and apolipoproteins in for R3480P non-carriers in the general change to to B to to to to to to in a new by R3480P was associated with decreased in VLDL and IDL when compared with non-carriers of the same age 10-year age sex, and APOE VLDL and IDL VLDL and IDL and whereas was a an with lower LDL levels R3480P was associated with differences in levels between the two groups R3480P was associated with in in plasma compared with non-carriers as as with in in VLDL, IDL, LDL, and to Finally, R3480P was associated with in plasma apoB and compared with non-carriers and and LDL and of of the LDL were in R3480P heterozygotes and non-carriers of apoB the LDL, IDL, and VLDL by on a showed no differences between R3480P heterozygotes and non-carrier indicating that the R3480P mutation was associated with forms of LDL of R3480P LDL to with non-carrier LDL for binding to normal human was compared with non-carrier LDL A of non-carrier LDL from human of R3480P LDL protein compared with of non-carrier LDL LDL of R3480P LDL and non-carrier LDL and determined simultaneously in or in that R3480P LDL is metabolized than non-carrier LDL in both with low LDL receptor and in with high receptor In with fractional catabolic rate of R3480P LDL was on decreased by compared with non-carrier LDL in Furthermore, because plasma LDL levels were reduced in R3480P heterozygotes compared with non-carriers in the general population and LDL production rate was reduced by in R3480P heterozygotes when compared with non-carriers for mutations in of R3500Q LDL and R3531C LDL and non-carrier LDL and R3480P LDL and LDL and and non-carrier LDL determined in human non-carriers after simultaneous catabolic rate and production rate of R3480P LDL and non-carrier LDL determined simultaneously in human non-carriers and in in is the of in is the of catabolic in is the of in is the of catabolic rate rate in is the of in is the of catabolic rate catabolic in in in is the of in is the of catabolic rate rate LDL in is the of in a new with known in vivo turnover studies were in R3500Q LDL, R3531C LDL, non-carrier LDL, R3480P LDL, and LDL non-carrier LDL simultaneously injected in the same The in compared with non-carriers were as for R3500Q and for R3531C LDL and no for non-carrier LDL and for R3480P LDL and and for LDL and LDL LDL fractional catabolic rate and LDL production rate between heterozygotes for R3500Q, R3480P, and all identified in The Copenhagen City Heart LDL were in R3500Q heterozygotes, in R3531C heterozygotes, and decreased in R3480P heterozygotes compared with non-carriers in the general LDL fractional catabolic rate was decreased for all mutations compared with the was for R3500Q heterozygotes, intermediate for R3480P and heterozygotes, and for R3531C heterozygotes. contrast, LDL production rate was in R3500Q and R3531C heterozygotes compared with was decreased in R3480P and heterozygotes. VLDL was for R3480P and non-carrier VLDL injected simultaneously into three non-carriers Because VLDL levels were lower in R3480P heterozygotes than in conversion of VLDL to LDL on these values was and for R3480P heterozygotes and corresponding to a in heterozygotes catabolic rate and conversion rate to LDL of R3480P VLDL non-carrier VLDL determined simultaneously in human fractional catabolic rate converted to LDL fractional catabolic rate converted to LDL fractional catabolic rate converted to LDL in fractional catabolic rate converted to LDL in a new In this we the of a new mutation in APOB, R3480P, located in a region of apoB-100 for of binding of VLDL, IDL, and LDL to the LDL receptor (4Borén J. Lee I. Zhu W. Arnold K. Taylor S. Innerarity T.L. J. Clin. Investig. 1998; 101: 1084-1093Crossref PubMed Scopus (183) Google Scholar, 7Borén J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, H. Hansen P.S. Færgeman O. Hørder M. Clin. Chem. 1995; 41: 419-423Crossref PubMed Scopus (31) Google Scholar). Our that APOB R3480P at a of in in a general population of and is associated with hypobetalipoproteinemia, despite an to with the LDL receptor. The explanation for this to be a in the amount of VLDL converted to LDL in these individuals in vivo. Because we identified R3480P heterozygotes among 9,255 randomly individuals from the general population and among 4,172 patients with ischemic cardiovascular disease or results that R3480P is associated with these In of identified in the general population had on lower levels of LDL apolipoprotein and VLDL and levels to be when compared with individuals in the general population for age, sex, and APOE genotype. Thus, R3480P is the mutation in APOB associated with a moderate hypobetalipoproteinemia results were by of plasma from three R3480P heterozygotes and non-carriers from the general population for age, sex, and APOE and were years after the these findings suggested a decreased production an of apoB-100 in R3480P heterozygotes. Most surprisingly, R3480P LDL showed a in LDL receptor affinity in in vitro competitive receptor binding studies, suggesting a The in receptor affinity was confirmed in in vivo turnover studies of LDL, where fractional catabolic rate of R3480P LDL was on reduced by compared with non-carrier LDL in both and a reduced affinity of LDL for the LDL receptor in vivo as in the turnover studies, levels of all apoB-100 in plasma were lower than when compared with suggesting a in production rate of LDL. This was we as the LDL production rate in R3480P heterozygotes was reduced by compared with non-carriers. In of VLDL turnover studies showed that the amount of VLDL converted to LDL was reduced by in R3480P heterozygotes compared with non-carriers. Furthermore, an identified had hypobetalipoproteinemia as as both reduced production and of LDL, findings for R3480P heterozygotes. Finally, has been by to have reduced affinity for the LDL receptor in vitro J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). would that is a between the conversion rate of VLDL to LDL in VLDL turnover studies and the production rate of LDL in LDL turnover studies This is because of the levels of LDL and VLDL in the used for turnover studies and because of in vivo of VLDL. The used in turnover studies is very when the fractional catabolic rate of R3480P LDL (or VLDL) with non-carrier LDL (or VLDL) between the because the the is when the fractional catabolic are with lipoprotein levels in the to production rate of LDL and VLDL converted to LDL. the levels of LDL production and of VLDL converted to LDL in R3480P heterozygotes non-carriers have the fractional catabolic rate as as the lipoprotein levels in the general population are both very the that the observed hypobetalipoproteinemia in R3480P heterozygotes is by a in the conversion of VLDL to LDL. microscopy studies have that of apoB-100 enwraps the LDL like a and that the carboxyl-terminal a bow that crosses over this belt, the carboxyl-terminal of apoB-100 to amino acid 3500 (3Chatterton J.E. Phillips M.L. Curtiss L.K. Milne R. Fruchart J.C. Schumaker V.N. J. Lipid Res. 1995; 36: 2027-2037Abstract Full Text PDF PubMed Google Scholar). The to inhibit the interaction of apoB-100 VLDL with the LDL after conversion of VLDL to LDL, arginine 3500 with the normal interaction between LDL and receptor (4Borén J. Lee I. Zhu W. Arnold K. Taylor S. Innerarity T.L. J. Clin. Investig. 1998; 101: 1084-1093Crossref PubMed Scopus (183) Google Scholar). The loss of arginine at this site as in apolipoprotein this resulting in receptor-binding LDL. Thus, mutations to the receptor-binding region, residues (site B), may the affinity of apoB-100 to the LDL receptor. a has been of the carboxyl-terminal bow with the of apoB-100, enwraps the LDL J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). This that arginine 3500 with and that this interaction is for of the carboxyl-terminal tail of The that with arginines in to arginine 3500 in apoB-100 conversion of VLDL to LDL. mutations in apoB-100 have been to varying degrees of receptor binding and all are located to the receptor-binding R3500Q (5Soria L.F. Ludwig E.H. Clarke H.R.G. Vega G.L. Grundy S.M. McCarthy B.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 587-591Crossref PubMed Scopus (470) Google Scholar), R3500W D. K. M.J. J. Arterioscler. Thromb. Vasc. Biol. 1995; PubMed Scopus Google Scholar), R3531C C.R. Hennessy L.K. Chatterton J.E. Liu W. Love J.A. Mendel C.M. Frost P.H. Malloy M.J. Schumaker V.N. Kane J.P. J. Clin. Investig. 1995; 95: 1225-1234Crossref PubMed Scopus (169) Google Scholar), J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and R3480P, the mutation in this All these mutations result in the loss of an arginine and with of the carboxyl-terminal tail of the Copenhagen City Heart we have that with R3500Q identified in the general population had to levels of LDL and identified in the same population R3531C had LDL and apoB levels that from of non-carriers A. Steffensen R. Meinertz H. Schnohr P. Nordestgaard B.G. N. Engl. J. Med. 1998; 338: 1577-1584Crossref PubMed Scopus (167) Google Scholar). In with R3500Q was associated with a in of ischemic whereas was in R3531C heterozygotes. In the study we that R3480P is associated with a in apoB-100 lipoproteins, a reduced affinity of LDL for the LDL receptor both in vivo and in vitro, and with a reduced production of LDL from VLDL In with R3480P heterozygotes are at an of ischemic cardiovascular In we have identified the mutation in apoB associated with hypobetalipoproteinemia, and we have that the in APOB mutation is the result of production rate of VLDL, conversion of VLDL to LDL, and removal of LDL via the LDL receptor. Thus, the effects of a reduced affinity for the LDL receptor can be by mechanisms and result in a hypobetalipoproteinemia Our findings the importance of combining in vitro studies with both human in vivo and population-based studies, as in vitro studies often on very limited of complex mechanisms taken out of their natural context. We and for and P. Barrett for the used in the VLDL turnover