Familial Defective Apo Research Articles

Autosomal recessive hypercholesterolemia: genetics and clinical aspects

In the last 10 years, new types of genetic hypercholesterolemia, different from familial hypercholesterolemia (FH) or familial defective apo B (FDB), have been reported; these forms have both autosomal dominant or recessive inheritance. Starting in 1995, we have described a new recessive type of hypercholesterolemia, named ARH. This disease, initially reported in one Sardinian family, is characterized by a severe increase of low-density lipoproteins (LDL) in plasma, xanthomas, xanthelasmas, and premature coronary heart disease. The family showed presence of consanguinity, bimodal distribution of cholesterol levels, and absence of vertical transmission. We have demonstrated that this hypercholesterolemia is due to a marked reduction of LDL catabolism that is caused by a selective reduction of LDLs uptake by the liver. The gene responsible for ARH maps to chromosome 1p35. It codes for an adaptor protein that interacts with the NPXY sequence in the cytoplasmic tail of LDL-R, and is necessary for the functioning of LDL-R in liver, but not in fibroblasts. Only two mutations were identified in Sardinia; a single base pair insertion in exon 4 (ARH1), and a nonsense mutation at codon 22 (ARH2). The high frequency of ARH in the island is probably due to a combination of genetic drift, consanguinity, and geographic isolation.

A novel LCAT mutation (Phe 382→Val) in a kindred with familial LCAT deficiency and defective apolipoprotein B-100

We studied a four-generation family (17 subjects) with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. A 30-year-old Caucasian male with corneal clouding and HDL cholesterol <0.1 mmol/l was a compound heterozygote for a novel mutation (Phe 382→Val), a previously reported mutation (Thr 321→Met) and a common variant (Thr 208→Ser) of the gene. Immunoreactive LCAT concentration (1.2 μg/ml), α-LCAT activity (13 nmol/ml per h) and cholesterol esterification rate (CER) (14 nmol/ml per h) in his plasma were, respectively, 14, 8 and 14% of the mean values in healthy subjects. The proband and 13 of his relatives also had familial defective apo B (FDB, Arg 3500→Gln). Six subjects had LCAT Phe 382→Val in combination with FDB. Plasma lipoprotein(a) (Lp(a)) was 24 nmol/l in the proband and 46–211 nmol/l in his father and siblings, consistent with expression of the 16 kringle 4 isoform. The proband had no signs of coronary heart disease (CHD), but his father, a paternal uncle and a female cousin had CHD before age 38 years.

Molecular basis of familial hypercholesterolemia in Brazil: Identification of seven novel LDLR gene mutations.

Low-density lipoprotein receptor (LDLR) gene mutations cause familial hypercholesterol-emia (FH), one of the most common single gene disorders. The spectrum of LDLR mutations in Brazil is not known. The aim of this study was the characterization of LDLR mutations in 35 unrelated Brazilian patients with heterozygous FH. The promoter region, the 18 exons and the flanking intron sequences of the LDLR gene were screened by PCR-SSCP analysis and by DNA sequencing. In addition, we have screened the apolipoprotein B gene (APOB) for known mutations (R3500Q and R3531C) that cause Familial defective apo B-100 (FDB) by PCR-RFLP procedure. We found two nonsense (E92X and C371X) and six missense LDLR mutations (R236W, G322S, G352D, A370T, C675W and C677Y), that were previously described in FH patients from other populations. We also found five novel missense [G(-20)R, T476P, V503G, D580H and S652R] and two novel frame shift LDLR mutations (FsR757 and FsS828). Four patients were found to carry two different mutations in the LDLR gene: G352D and A370T (one patient), S652R and C675W (one patient) and T476P and V503G (two patients). APOB mutations were not found. These findings demonstrate that there is a broad spectrum of mutations in the LDLR gene in FH individuals from Brazil.

Familial defective apo B 100 in a South European population

Familial defective apo B 100 in a South European population

Unusual inheritance of severe primary hypercholesterolemia

Primary hypercholesterolemia, due to an increase of plasma low density lipoproteins (LDL), is a broad term which includes both monogenic disorders (caused by mutations of a single gene with a large effect) and polygenic conditions (involving several genes, each with a relatively small effect). There are two well defined monogenic conditions which cause an increased level of plasma LDL: familial hypercholesterolemia (FH) and familial defective apo B100 (FDB). FH is a classical example of an autosomal co-dominant disorder with a clear gene-dose effect. The FH phenotype (increased plasma LDL, presence of tendon xanthomas and premature CHD) is more severe in homozygotes than in heterozygotes. FH is due to lack of functional receptor for LDL on the cell surface whereby LDL cannot be taken up and catabolised by the cells, especially hepatocytes. More than 300 mutations of LDL-receptor gene have been found in FH subjects. The phenotypic expression of FH in homozygotes and, to some extent, in heterozygotes is related to the residual LDL-receptor activity in tissues. LDL-receptor mutations that completely abolish the receptor function (receptor negative) usually result in a more severe clinical expression of FH [1]. FDB is due to point mutations in apo B gene (such as the substitution of arginine at position 3500 with glutamine, R3500Q) which cause the presence of LDL that bind poorly to the LDL-receptor [2]. Usually the elevation of plasma LDL in FBD-heterozygotes is less severe than that observed in FH heterozygotes [3]. A few FDB homozygotes have been reported; their clinical phenotype appears to be less severe than that observed in FH-homozygotes [4,5].

Identification and haplotype analysis of apolipoprotein B-100 Arg3500→Trp mutation in hyperlipidemic Chinese

DNA screening for apolipoprotein (apo) B-100 mutations was performed in hyperlipidemic Chinese. The apo B-100 gene segment surrounding previously identified familial defective apo B-100 (FDB) mutations was amplified by PCR and subjected to single-strand conformation polymorphism (SSCP) analysis. One subject's aberrant SSCP band was cloned and sequenced to study the molecular lesions. A recurrent ArgCGG-to-TrpTGG mutation (R3500W) in the codon 3500 of the apo B-100 gene was identified. The C-to-T transition creates a NlaIII site and permits rapid restriction analysis of the mutation. A total of 373 hyperlipidemic patients and 309 controls were screened for R3500W. Nine unrelated subjects were shown to be heterozygous for the mutation, and no R3500W carriers were found in the control group (P = 0.004). Six polymorphic markers, including five restriction fragment length polymorphisms and one hypervariable repeat region, were used for haplotype analysis on the mutant allele. In two families, the R3500W mutation could be unambiguously assigned to a unique haplotype XbaI-/MaeI+/MspI+/EcoRI+/ Eco57I+/34 3'HVR repeats; in the other seven unrelated heterozygotes, this finding was consistent when an unequivocal haplotype was deduced. The results suggest that all R3500W alleles are identical by descent in our population. The fact that the same mutant allele was identified in other Asians with FDB indicates a common Asian origin for the R3500W mutations.

Evaluation of a clinically applicable mutation screening technique for genetic diagnosis of familial hypercholesterolemia and familial defective apolipoprotein B.

We have recently developed a simple mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) technique for detection of mutations in the coding and regulatory regions of the low density lipoprotein receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene leading to familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. To evaluate the assay, 14 Danish families suspected of FH were studied. In ten families, the DGGE assay detected seven different point mutations, including mutations undescribed prior to establishing the assay. In addition, in one of these ten families and in one of the remaining four families, Southern blotting detected the FH-DK3 exon 5 deletion. Based on segregation analysis and clinical data, the FH diagnosis was dubious in the remaining three families without DGGE or Southern blotting detectable mutations. In conclusion, a simple DGGE based mutation screening assay may detect underlying mutations in most FH/FDB families, thus allowing its routine use in genetic counselling of FH-families.

VLDL and IDL apolipoprotein B-100 kinetics in familial hypercholesterolemia due to impaired LDL receptor function or to defective apolipoprotein B-100

Mutations in the apolipoprotein (apo) B, E (LDL) receptor gene and in the apolipoprotein B-100 gene are the cause of familial hypercholesterolemia (FH) and of familial defective apo B-100 (FDB), respectively. Whether these abnormalities lead to altered production or uptake of very low density lipoprotein (VLDL) or intermediate density lipoprotein (IDL) has not been established previously. Therefore VLDL and IDL apo B-100 kinetics were measured in seven subjects with FH, in six subjects with FDB, and in five normocholesterolemic controls using primed-constant infusions of [1-13C]leucine. Absolute production rates (APR) of VLDL apoB were higher in FH than in controls (27.1 ± 1.9 vs. 17.9 ± 2.1 mg/kg/day P < 0.03). VLDL APR in FDB were between those of FH and controls (24.3 ± 4.8 mg/kg/day), and demonstrated a relatively large inter-individual variability. The increase in VLDL APR in FH resulted in higher fasting serum triglyceride concentrations than in controls (P < 0.05), whereas in FDB triglycerides were between those observed in FH and in controls. A significant correlation was observed between VLDL apoB APR and serum triglycerides in FH and in FDB; the correlation coefficient for all subjects was r = 0.84 (P < 0.0001), indicating that the major determinant of serum triglyceride concentrations was VLDL apoB APR. IDL apoB APR was lower in FH and in FDB compared to controls (P < 0.03 P < 0.02, respectively): and its fractional catabolic rate (FCR) was slightly lower in FH and in FDB, resulting in similar plasma IDL apoB concentrations in all three groups of subjects. IDL apoB APR in FH were negatively correlated with LDL cholesterol concentrations (r = –0.89; P < 0.001); LDL cholesterol concentrations correlated positively with the part of VLDL that did not appear in IDL (r = 0.82 P < 0.02), bypassing therefore the delipidation cascade. In conclusion the data demonstrate increased VLDL apoB production rates in FH. VLDL and IDL kinetics differ when LDL concentrations are elevated either due to a LDL receptor defect or due to defective apolipoprotein B-100.—Zulewski H., R. Ninnis, A. R. Miserez, M. W. Baumstark, and U. Keller. VLDL and IDL apolipoprotein B-100 kinetics in familial hypercholesterolemia due to impaired LDL receptor function or to defective apolipoprotein B-100.

Familial defective apolipoprotein B-100 in hypercholesterolemic Chinese Canadians: identification of a unique haplotype of the apolipoprotein B-100 allele

Familial defective apo B-100 (FDB) is an autosomal dominant condition resulting in hypercholesterolemia. It is generally observed in 1–6% of hypercholesterolemic subjects in Caucasian populations studied. There are, thus far, no reports characterizing the frequency and phenotype of FDB in a Chinese population. We report on the frequency of the FDB (Arg (3500)→Gln) mutation and the associated haplotype among 160 hypercholesterolemic (TC≥6.2 mmol/l) Chinese Canadians including 36 subjects with a clinical diagnosis of familial hypercholesterolemia (FH). Screening for the FDB mutation was done using a mutagenic polymerase chain reaction and haplotype analysis was undertaken using eight diallelic markers and the 3′HVR marker. One Chinese Canadian clinical FH heterozygote was positive for the FDB Arg (3500)→Gln mutation while none of the remaining non-FH hypercholesterolemic subjects ( n=124) were carriers of this mutation. Haplotype analysis in the patient positive for this mutation revealed a unique haplotype which differed from both the common haplotype of this mutation observed in Caucasians and from the only other haplotype reported in a Chinese individual. The associated haplotype included a 9-base pair deletion in the signal peptide region and the presence of three restriction sites absent in previously reported haplotypes. These data suggest that the apo B-100 Arg (3500)→Gln mutation does not appear to be a significant factor contributing to moderate hypercholesterolemia in a Chinese population residing in Canada. However, this mutation was rarely observed among Chinese individuals with a clinical diagnosis of FH. The presence among Chinese individuals of two different haplotypes associated with this mutation, which are different from what has been described among Caucasians is compatible with multiple recurrent origins for this mutation in the Chinese population.

Denaturing gradient-gel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed Asian cohort: two cases of arginine3500 → tryptophan mutation associated with a unique haplotype

The Arg-to-Trp substitution at codon 3500 in the apolipoprotein (apo) B-100 gene is established as a cause of familial defective apo B-100 (FDB), a functional mutation, resulting in reduced LDL receptor binding and manifest hypercholesterolemia. In a search for similar mutations in 163 Malaysians, we screened the putative receptor-binding region (codons 3456-3553) of the apo B-100 gene by PCR amplification and denaturing gradient-gel electrophoresis. Four single-base mutations were detected and confirmed by DNA sequencing. Two females, a Chinese and a Malay, had the same CGG3500-->TGG mutation, resulting in an Arg3500-to-Trp substitution. This is the second published report of such an independent mutation involving the same codon as the established Arg3500-to-Gln mutation. The two other mutations detected, CTT3517-->CTG and GCC3527-->GCT, resulted in degenerate codons with no amino acid substitutions. All four mutations were associated with a unique apo B haplotype, different from those found in Caucasian FDB patients but concurring with that previously reported for two other Asians with FDB.

Screening of the 3′ two-thirds of the coding area of the apo B gene in Finnish hypercholesterolemic patients: Report of six new genetic variants

Hypercholesterolemia clustering in families not explained by either low density lipoprotein (LDL)-receptor mutations producing familial hypercholesterolemia (FH), or the apolipoprotein B (apo B) Arg3500→Gln mutation with familial defective apo B (FDB), is common in the Finnish population. In search of previously unknown apo B mutations, we screened exons 26 to 29 of the apo B gene in 68 Finnish severely hypercholesterolemic (≥8 mmol/l) non-FH, non-FDB patients, using a single-strand conformation polymorphism analysis based screening method. Four rare and two polymorphic previously unreported DNA variations were detected. The rare variants were a three-nucleotide deletion, with the deletion of Asp2186, an A11961→G change leading to a Thr3918→Ala change, a T12922→G change causing a Val4238→Ala substitution, and a neutral T12935→C change leading to a new RsaI cutting site. The polymorphic G12937→C and G13569→A changes leading to Arg4243→Thr and Ala4454→Thr substitutions, respectively, had minor allele frequencies of 0.03 and 0.02. None of these variants seemed to explain the hyperlipidemia in these patients. A major Finnish mutation causing severe hypercholesterolemia is unlikely to exist in the 3′ two-thirds of the coding area of the apo B gene.

Statin therapy in a kindred with both apolipoprotein B and low density lipoprotein receptor gene defects

We studied an extended family of similar genetic and environmental background to determine whether there is a difference in response to statin therapy in those subjects with heterozygous familial hypercholesterolaemia (FH Afrikaner-1 (FH1) or FH Afrikaner-2 (FH2)) compared to those with familial defective apo B-100 (FDB), or both FH plus FDB. Fasting lipid profiles and Lp(a) levels were done on 18 members of the family and then repeated following 6 weeks of therapy with simvastatin 20 mg daily. Statin therapy reduced LDL-cholesterol (LDL-C) by 31% in those with FH ( n=7); 29.8% in FDB ( n=5) and 25.4% in those with both FDB and FH ( n=5). There was no response to statin therapy in the single subject with both FH1, FH2, as well as FDB. Lp(a) levels did not change significantly either within or between any of the groups following statin therapy (FH from 6.5 (1.2–72.3) to 5.3 (1.2–52.3), FDB from 6.1 (4.70–71) to 8.2 (5.7–79) and FDB plus FH from 4.5 (2.6–17.4) to 3.1 (1.9–24) mg/dl). Statins are equally effective in lowering LDL-C in related subjects with heterozygous FH, FDB or both FDB plus FH. The ability of statins to lower LDL-C in FDB is probably due to increased hepatic uptake of lipoprotein precursors of LDL that can bind via apo E receptors. Lp(a) concentration is not reduced by drugs that stimulate LDL receptor activity implying that LDL receptors do not contribute greatly to normal clearance of Lp(a) in hypercholesterolaemic subjects with defects in receptor-mediated endocytosis of LDL.

Clinically applicable mutation screening in familial hypercholesterolemia.

Mutations in the LDL receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene result in the clinically indistinguishable phenotypes designated familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. Introduction of genetic diagnosis in phenotypic FH families may remove the diagnostic inaccuracies known from traditional clinical/biochemical FH diagnosis and allow more differentiated prognostic evaluations and genetic counseling of FH/FDB families. Previous genetic screening methods for FH have, however, been too cumbersome for routine use, however. To overcome these problems, we designed a mutation screening assay based on the highly sensitive denaturing gradient gel electrophoresis (DGGE) technique. The setup allows within 24 hr to pinpoint if and where a potential mutation is located in the LDLR promoter, the 18 LDLR gene exons and corresponding intronic splice site sequences, or in the codon 3500 region of apo B-100. The pinpointed region is subsequently sequenced. As an evaluation of the sensitivity, we demonstrated the ability of the assay to detect 27 different mutations or polymorphisms covering all the examined regions, except LDLR exon 16. In conclusion, a simple, but sensitive, clinically applicable mutation screening assay based on the DGGE principle may reveal the underlying mutation in most FH/FDB families and offer a tool for a more differentiated prognostic and therapeutic evaluation in FH/FDB.

Does the presence of the 3500 mutant apolipoprotein B-100 in low density lipoprotein particles affect their atherogenicity?

Apolipoprotein B-100 (apo B-100) is the protein component of low density lipoprotein (LDL) responsible for its binding and clearance by LDL receptors (LDL-R). In familial defective apo B-100 (FDB), a mutation in apo B-100 at residue 3500 markedly reduces its affinity for LDL-R, often causing accumulation of defective LDL particles, and an increased proneness to coronary artery disease (CAD). In FDB heterozygotes, about 70% of the LDL particles are mutant, which may alter their atherogenicity relative to LDL containing normal apo B. Therefore, we compared CAD in heterozygous FDB with CAD in heterozygous familial hypercholesterolemia (FH), since raised LDL is usually present from birth in both conditions, and in FH the LDL particles that accumulate have normal apo B, as the inherited defect involves the LDL-R. The clinical presentation of coronary atherosclerosis and its angiographic appearance were examined in FDB and FH patients matched for conventional cardiac risk factors (hypertension, smoking, sex) and serum lipid levels. There was no significant difference between the FDB and FH patients ( n = 11 pairs) in the type of cardiac symptoms or their ages of onset (50 ± 9 vs. 45 ± 11 years). Coronary angiographic appearance was also similar in both groups ( n = 9 pairs). These observations suggest that LDL particles with the 3500 mutation in apo B have the same atherogenicity as LDL particles with normal apo B.

The affinity of low-density lipoproteins and of very-low-density lipoprotein remnants for the low-density lipoprotein receptor in homozygous familial defective apolipoprotein B-100

We have identified two familial defective apo B-100 (FDB) homozygotes by DNA sequencing and have measured affinity of low-density lipoproteins (LDL) and very-low-density lipoprotein (VLDL) remnants for the LDL receptor in vitro. The patients were a 66-year-old man with coronary heart disease (plasma cholesterol level, 9.5 mmol/l before treatment) and his 69-year-old sister, without signs of cardiovascular disease (plasma cholesterol, 12.0 mmol/l before treatment). In both patients, treatment with statins caused a marked fall in plasma cholesterol level. Binding affinity of LDL from the two patients was 10%–20% of normal at 4 °C and 37 °C. Binding affinity of VLDL remnants was normal. We conclude that (1) residual affinity of LDL in homozygous FDB is high enough to permit significant catabolism via the LDL-receptor pathway, and (2) normal affinity of VLDL remnants permits normal hepatic clearance of precursors of LDL and increased clearance of LDL precursors when receptor activity is stimulated by statins. Residual affinity of LDL and normal affinity of remnants could explain why expression of the FDB mutation is generally milder than that of LDL receptor mutations causing familial hypercholesterolaemia.

Compound heterozygotes for familial defective apo B-100 (FDB) and familial hypercholesterolemia (FH)

Compound heterozygotes for familial defective apo B-100 (FDB) and familial hypercholesterolemia (FH)

Familial defective apo B-100 (FDB) in France: frequency and clinical presentation in families

Familial defective apo B-100 (FDB) in France: frequency and clinical presentation in families

Founder effect for familial defective apo B-100 (FDB) mutation in France

Founder effect for familial defective apo B-100 (FDB) mutation in France

Lipoprotein(a) levels in subjects with familial defective apo B-100 (FDB)

Lipoprotein(a) levels in subjects with familial defective apo B-100 (FDB)

Characteristics of 46 heterozygous carriers and 57 unaffected relatives in five Danish families with familial defective apolipoprotein B-100.

Plasma concentrations of cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein (apo) B, and lipoprotein(a) (Lp[a]) in 46 persons heterozygous for the apo B-3500 mutation causing familial defective apo B-100 (FDB) were compared with those in 57 non-FDB relatives. FDB patients had 50% to 70% higher mean concentrations of cholesterol, LDL cholesterol, and apo B than non-FDB relatives (P < 10(-4) for all three variables). Triglycerides were higher (P = .016) and HDL cholesterol was lower (P = .021) in FDB patients. The concentration ranges of these variables were broad in each family, and there was no between-family difference in means for cholesterol and LDL cholesterol. There was no phenotype-specific difference in Lp(a) concentrations between FDB patients and non-FDB relatives. Apo E4 is normally associated with higher concentrations of LDL and apo E2 with lower concentrations. This relation was partly reversed in FDB patients: apo E4 was associated with lower apo B concentrations and apo E2 with higher apo B concentrations. Tendon xanthomata were found in members of two of the five families. Six of 12 FDB patients > 50 years old had atherosclerotic disease. In contrast, all 18 non-FDB relatives > 50 years old were apparently healthy. A total of 8 FDB patients with atherosclerotic disease had 36% higher cholesterol concentrations, 28% higher apo B concentrations, 50% higher triglyceride concentrations, and 120% higher Lp(a) concentrations than FDB patients without clinical atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)