Lucia Magnolo

Characterization of Three Kindreds With Familial Combined Hypolipidemia Caused by Loss-of-Function Mutations of ANGPTL3

Background— Angiopoietin-like protein 3 (ANGPTL3) affects lipid metabolism by inhibiting the activity of lipoprotein and endothelial lipases. Angptl3 knockout mice have marked hypolipidemia, and heterozygous carriers of ANGPLT3, loss-of-function mutations were found among individuals in the lowest quartile of plasma triglycerides in population studies. Recently, 4 related individuals with primary hypolipidemia were found to be compound heterozygotes for ANGPTL3 loss-of-function mutations. Methods and Results— We resequenced ANGPTL3 in 4 members of 3 kindreds originally identified for very low levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (0.97±0.16 and 0.56±0.20 mmol/L, respectively) in whom no mutations of known candidate genes for monogenic hypobetalipoproteinemia and hypoalphalipoproteinemia had been detected. These subjects were found to be homozygous or compound heterozygous for ANGPTL3 loss-of-function mutations (p.G400VfsX5, p.I19LfsX22/p.N147X) associated with the absence of ANGPTL3 in plasma. They had reduced plasma levels of triglyceride-containing lipoproteins and of HDL particles that contained only apolipoprotein A-I and pre-&bgr;–high-density lipoprotein. In addition, their apolipoprotein B–depleted sera had a reduced capacity to promote cell cholesterol efflux through the various pathways (ABCA1-, SR-BI–, and ABCG1-mediated efflux); however, these subjects had no clinical evidence of accelerated atherosclerosis. Heterozygous carriers of the ANGPTL3 mutations had low plasma ANGPTL3 and moderately reduced low-density lipoprotein cholesterol (2.52±0.38 mmol/L) but normal plasma high-density lipoprotein cholesterol. Conclusions— Complete ANGPTL3 deficiency caused by loss-of-function mutations of ANGPTL3 is associated with a recessive hypolipidemia characterized by a reduction of apolipoprotein B and apolipoprotein A-I–containing lipoproteins, changes in subclasses of high-density lipoprotein, and reduced cholesterol efflux potential of serum. Partial ANGPTL3 deficiency is associated only with a moderate reduction of low-density lipoprotein.

Identification of patients with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia in Tunisia.

BACKGROUND Abetalipoproteinemia (ABL) and Homozygous Familial Hypobetalipoproteinemia (Ho-FHBL) are rare monogenic diseases characterised by very low plasma levels of cholesterol and triglyceride and the absence or a great reduction of apolipoprotein B (apoB)-containing lipoproteins. ABL results from mutations in the MTP gene; Ho-FHBL may be due to mutations in the APOB gene. METHODS We sequenced MTP and APOB genes in three Tunisian children, born from consanguineous marriage, with very low levels of plasma apoB-containing lipoproteins associated with severe intestinal fat malabsorption. RESULTS Two of them were found to be homozygous for two novel mutations in intron 5 (c.619-3T>G) and in exon 8 (c.923 G>A) of the MTP gene, respectively. The c.619-3T>G substitution caused the formation of an abnormal mRNA devoid of exon 6, predicted to encode a truncated MTP of 233 amino acids. The c.923 G>A is a nonsense mutation resulting in a truncated MTP protein (p.W308X). The third patient was homozygous for a novel nucleotide deletion (c.2172delT) in exon 15 of APOB gene resulting in the formation of a truncated apoB of 706 amino acids (apoB-15.56). CONCLUSIONS These mutations are expected to abolish the apoB lipidation and the assembly of apoB-containing lipoproteins in both liver and intestine.

Novel mutations of CETP gene in Italian subjects with hyeralphalipoproteinemia

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that catalyses the transfer of cholesteryl esters from HDL to the other plasma lipoproteins. Genetic deficiency of CETP is one of the known causes of elevation of plasma HDL-C (primary hyperalphalipoproteinemia, HALP). We sequenced CETP gene in a group of 24 Italian subjects with primary HALP (HDL-C>80 mg/dl) suspected to have CETP deficiency. Two unrelated subjects both coming from the same geographical district, were found to be heterozygous for a nucleotide substitution in exon 6 (c.544C>T) and another subject was found to be heterozygous for a C>T transition in exon 9 (c.802C>T). Both mutations introduce a premature stop codon and are predicted to cause the production of truncated proteins (Q165X and R268X, respectively) devoid of function. The fourth proband was found to carry a T>C substitution in intron 15 (c.1407+2T>C) predicted to abolish the function of the donor splice site. To define the effect of this mutation on CETP pre-mRNA splicing we analysed CETP mRNA in COS-1 cells expressing a CETP minigene harbouring the mutation. The analysis of minigene transcript in COS-1 cells showed that IVS15+2T>C mutation caused the formation of an abnormal mRNA in which exon 14 joins directly to exon 16, predicted to encode a truncated peptide of 435 amino acids. In mutation carriers plasma CETP activity was found to be reduced by 38-60%. These are the first mutations in the CETP gene found in Italian subjects with HALP.

Variable phenotypic expression of homozygous familial hypobetalipoproteinaemia due to novel APOB gene mutations

Homozygous familial hypobetalipoproteinaemia (Ho‐FHBL) is a rare co‐dominant disorder characterized by extremely low levels of low‐density lipoprotein cholesterol (LDL‐C) and apolipoprotein B (apoB). Most patients with Ho‐FHBL have mutations in APOB gene resulting in truncated apoBs. Some patients are asymptomatic, while others have fatty liver, intestinal fat malabsorption and neurological dysfunctions. We investigated three adult subjects with severe hypobetalipoproteinaemia and a family history of FHBL. Proband FHBL‐47 had liver cirrhosis with hepatocarcinoma and a renal carcinoma but no clinical manifestations related to FHBL. He was a compound heterozygote for a 7‐bp deletion in exon 21 and a base insertion in exon 26 resulting in truncated apoBs (apoB‐22.46/apoB‐66.51). Proband FHBL‐53, with severe hepatic steatosis and fibrosis, had a nonsense mutation in exon 19 resulting in a truncated apoB (apoB‐20.61) and a rare nucleotide substitution in intron 14 (c.2068‐4T>A). The latter was also present in her daughter, found to have low plasma LDL‐C and apoB. Proband FHBL‐82 had chronic diarrhoea and steatorrhoea. She was found to be homozygous for a nonsense mutation in exon 24 resulting in a truncated apoB (apoB‐26.65). In adult subjects, the presence of chronic liver disease and chronic diarrhoea, when associated with severe hypobetalipoproteinaemia, may lead to the diagnosis of Ho‐FHBL.

Abnormal apolipoprotein B pre-mRNA splicing in patients with familial hypobetalipoproteinaemia

Background: Familial hypobetalipoproteinaemia (FHBL) is a codominant disorder characterised by fatty liver and reduced plasma levels of low-density lipoprotein (LDL) and its protein constituent apolipoprotein B (apoB). FHBL is linked to the APOB gene in some but not all known cases. In a group of 59 patients with FHBL genotyped for APOB gene mutations, we found three novel splice-site mutations: c.904+4A→G in intron 8, c.3843−2A→G in intron 24 and c.4217−1G→T in intron 25. Objective: To assess the effects of these mutations on apoB pre-mRNA splicing. Methods: ApoB mRNA was analysed in the liver of one proband and in cells expressing APOB minigenes harbouring the mutations found in the other probands. Results: In the liver of the c.3843−2A→G carrier, an apoB mRNA devoid of exon 25 was identified, predicted to encode a truncated peptide of 1260 amino acids. The analysis of minigene transcripts in COS-1 cells showed that the c.904+4A→G mutation caused the formation of an mRNA devoid of exon 8, predicted to encode a short apoB of 247 amino acids. The minigene harbouring the c.4217−1G→T mutation in intron 25 generated an mRNA in which exon 25 joined to a partially deleted exon 26, resulting from the activation of an acceptor site in exon 26; this mRNA is predicted to encode a truncated protein of 1380 amino acids. All these truncated apoBs were not secreted as constituents of plasma lipoproteins. Conclusion: These findings demonstrate the pathogenic effect of rare splice-site mutations of the APOB gene found in FHBL.

Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia.

Monogenic hypobetalipoproteinemias include three disorders: abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) with recessive transmission and familial hypobetalipoproteinemia (FHBL) with dominant transmission. We investigated three unrelated Tunisian children born from consanguineous marriages, presenting hypobetalipoproteinemia associated with chronic diarrhea and retarded growth. Proband HBL-108 had a moderate hypobetalipoproteinemia, apparently transmitted as dominant trait, suggesting the diagnosis of FHBL. However, she had no mutations in FHBL candidate genes (APOB, PCSK9 and ANGPTL3). The analysis of MTTP gene was also negative, whereas SAR1B gene resequencing showed that the patient was homozygous for a novel mutation (c.184G>A), resulting in an amino acid substitution (p.Glu62Lys), located in a conserved region of Sar1b protein. In the HBL-103 and HBL-148 probands, the severity of hypobetalipoproteinemia and its recessive transmission suggested the diagnosis of ABL. The MTTP gene resequencing showed that probands HBL-103 and HBL-148 were homozygous for a nucleotide substitution in the donor splice site of intron 9 (c.1236+2T>G) and intron 16 (c.2342+1G>A) respectively. Both mutations were predicted in silico to abolish the function of the splice site. In vitro functional assay with splicing mutation reporter MTTP minigenes showed that the intron 9 mutation caused the skipping of exon 9, while the intron 16 mutation caused a partial retention of this intron in the mature mRNA. The predicted translation products of these mRNAs are non-functional truncated proteins. The diagnosis of ABL and CMRD should be considered in children born from consanguineous parents, presenting chronic diarrhea associated with hypobetalipoproteinemia.

Homozygous familial hypobetalipoproteinemia: A Turkish case carrying a missense mutation in apolipoprotein B.

The autosomal co-dominant disorder familial hypobetalipoproteinemia (FHBL) may be due to mutations in the APOB gene encoding apolipoprotein B (apoB), the main constituent peptide of chylomicrons, very low and low density lipoproteins. We describe an 11month-old child with failure to thrive, intestinal lipid malabsorption, hepatic steatosis and severe hypobetalipoproteinemia, suggesting the diagnosis of homozygous FHBL, abetalipoproteinemia (ABL) or chylomicron retention disease (CMRD). The analysis of candidate genes showed that patient was homozygous for a variant (c.1594 C>T) in the APOB gene causing arginine to tryptophan conversion at position 505 of mature apoB (Arg505Trp). No mutations were found in a panel of other potential candidate genes for hypobetalipoproteinemia. In vitro studies showed a reduced secretion of mutant apoB-48 with respect to the wild-type apoB-48 in transfected McA-RH7777 cells. The Arg505Trp substitution is located in the βα1 domain of apoB involved in the lipidation of apoB mediated by microsomal triglyceride transfer protein (MTP), the first step in VLDL and chylomicron formation. The patients condition improved in response to a low fat diet supplemented with fat-soluble vitamins. Homozygosity for a rare missense mutation in the βα1 domain of apoB may be the cause of both severe hypobetalipoproteinemia and intestinal lipid malabsorption.

Functional analysis of two novel splice site mutations of APOB gene in familial hypobetalipoproteinemia.

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low density lipoprotein cholesterol (LDL-C) and its protein constituent apolipoprotein B (apoB), which may be due to mutations in APOB gene, mostly located in the coding region of this gene. We report two novel APOB gene mutations involving the acceptor splice site of intron 11 (c.1471-1G>A) and of intron 23 (c.3697-1G>C), respectively, which were identified in two patients with heterozygous FHBL associated with severe fatty liver disease. The effects of these mutations on APOB pre-mRNA splicing were assessed in COS-1 cells expressing the mutant APOB minigenes. The c.1471-1G>A APOB minigene generated two abnormal mRNAs. In one mRNA the entire intron 11 was retained; in the other mRNA exon 11 joined to exon 12, in which the first nucleotide was deleted due to the activation of a novel acceptor splice site. The predicted products of these mRNAs are truncated proteins of 546 and 474 amino acids, designated apoB-12.03 and apoB-10.45, respectively. The c.3697-1G>C APOB minigene generated a single abnormal mRNA in which exon 23 joined to exon 25, with the complete skipping of exon 24. This abnormal mRNA is predicted to encode a truncated protein of 1220 amino acids, designated apoB-26.89. These splice site mutations cause the formation of short truncated apoBs, which are not secreted into the plasma as lipoprotein constituents. This secretion defect is the major cause of severe fatty liver observed in carriers of these mutations.

The Janus-faced manifestations of homozygous familial hypobetalipoproteinemia due to apolipoprotein B truncations

Familial hypobetalipoproteinemia is a codominant disorder characterized by low plasma levels of low-density lipoprotein cholesterol and apolipoprotein B (apoB), which in ∼50% of the cases is due to mutations in APOB gene. In most cases, these mutations cause the formation of truncated apoBs of various sizes, which have a reduced capacity to bind lipids and form lipoprotein particles. Here, we describe 2 children with severe hypobetalipoproteinemia found to be homozygous for novel APOB gene mutations. The first case (HBL-201) was an asymptomatic 13-year-old boy incidentally found to have slightly elevated serum transaminases associated with hepatic steatosis. He was homozygous for a truncated apoB (2211 amino acids, apoB-48.74) whose size is similar to that of wild-type apoB-48 (2152 amino acids) produced by the intestine. ApoB-48.74 is expected to be incorporated into chylomicrons in the intestine but might have a reduced capacity to form secretion-competent very low-density lipoprotein in the liver. The second patient (HBL-96) was a 6-month-old girl suspected to have abetalipoproteinemia, for the presence of chronic diarrhea, failure to thrive, extremely severe hypobetalipoproteinemia, and low plasma levels of vitamin E and vitamin A. She was homozygous for a nonsense mutation (Gln513*) resulting in a short truncated apoB (apoB-11.30), which is not secreted into the plasma. In this patient, the impaired chylomicron formation is responsible for the severe clinical manifestations and growth retardation. In homozygous familial hypobetalipoproteinemia, the capacity of truncated apoBs to form chylomicrons is the major factor, which affects the severity of the clinical manifestations.

Novel missense variants in LCAT and APOB genes in an Italian kindred with familial lecithin:cholesterol acyltransferase deficiency and hypobetalipoproteinemia

Background Lecithin:cholesterol acyltransferase (LCAT) is responsible for cholesterol esterification in plasma. Mutations of LCAT gene cause familial LCAT deficiency, a metabolic disorder characterized by hypoalphalipoproteinemia. Apolipoprotein B (apoB) is the main protein component of very-low-density lipoproteins and low-density lipoprotein (LDL). Mutations of APOB gene cause familial hypobetalipoproteinemia, a codominant disorder characterized by low plasma levels of LDL cholesterol and apoB. Objective This was a genetic and biochemical analysis of an Italian kindred with hypobetalipoproteinemia whose proband presented with hypoalphalipoproteinemia and severe chronic kidney disease. Methods Plasma lipids and apolipoproteins, cholesterol esterification, and high-density lipoprotein (HDL) subclass distribution were analyzed. LCAT and APOB genes were sequenced. Results The proband had severe impairment of plasma cholesterol esterification and high preβ-HDL content. He was heterozygote for the novel LCAT P406L variant, as were two other family members. The proband’s wife and children presented with familial hypobetalipoproteinemia and were heterozygotes for the novel apoB H1401R variant. Cholesterol esterification rate of apoB H1401R carriers was reduced, likely attributable to the low amount of circulating LDL. After renal transplantation, proband’s lipid profile, HDL subclass distribution, and plasma cholesterol esterification were almost at normal levels, suggesting a mild contribution of the LCAT P406L variant to his pretransplantation severe hypoalphalipoproteinemia and impairment of plasma cholesterol esterification. Conclusion LCAT P406L variant had a mild effect on lipid profile, HDL subclass distribution, and plasma cholesterol esterification. ApoB H1401R variant was identified as possible cause of familial hypobetalipoproteinemia and resulted in a reduction of cholesterol esterification rate.