PCSK9-D374Y mediated LDL-R degradation can be functionally inhibited by EGF-A and truncated EGF-A peptides: An in vitro study.

BACKGROUND AND AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) through the LDLR epidermal growth factor-like repeat A (EGF-A) domain and induces receptor internalization and degradation. PCSK9 has emerged as a novel therapeutic target for hypercholesterolemia. Clinical studies with PCSK9 inhibiting antibodies have demonstrated strong LDL-c lowering effects, but other therapeutic approaches using small molecule inhibitors for targeting PCSK9 functions may offer supplementary therapeutic options. The aim of our study was to evaluate the effect of synthetic EGF-A analogs on mutated (D374Y) PCSK9-D374Y mediated LDLR degradation in vitro. METHODS: Huh7 human hepatoma cells were transiently transfected to overexpress the gain-of-function D374Y PCSK9 mutation, which has been associated with severe hypercholesterolemia in humans. RESULTS: Transient transfection of cells with PCSK9-D374Y expression vector very effectively enhanced degradation of mature LDLR in Huh7. Treatment with both EGF-A and EGF-A truncated peptides inhibited this effect and showed increased LDLR protein in Huh7 cells transfected with PCSK9-D374Y in a clear concentration dependent manner. Huh7 transfected cells treated with increasing concentration of EGF-A analogs also showed an increase internalization of labeled Dil-LDL. CONCLUSIONS: The result of our study shows that EGF-A analogs are able to effectively hamper the enhanced degradation of LDLR in liver cells expressing PCSK9-D374Y.

Genetic epidemiology of autosomal recessive hypercholesterolemia in Sicily: Identification by next-generation sequencing of a new kindred.

BACKGROUND: Autosomal recessive hypercholesterolemia (ARH) is a rare inherited lipid disorder. In Sardinia, differently from other world regions, the mutated allele frequency is high. It is caused by mutations in the low-density lipoprotein receptor adaptor protein 1 gene. Fourteen different mutations have been reported so far; in Sardinia, 2 alleles (ARH1 and ARH2) explain most of the cases. Four ARH patients, all carriers of the ARH1 mutation, have been identified in mainland Italy and 2 in Sicily. OBJECTIVE: The objectives of the study were to improve the molecular diagnosis of familial hypercholesterolemia (FH) and to estimate the frequency of the ARH1 allele in 2 free-living Sicilian populations. METHODS: We sequenced by targeted next-generation sequencing 20 genes related to low-density lipoprotein metabolism in 50 hypercholesterolemic subjects. Subjects from 2 free-living populations from Northern (Ventimiglia Heart Study, 848 individuals) and Southern Sicily (Zabut Zabùt Aging Project, 1717 individuals) were genotyped for ARH1 allele. RESULTS: We identified 1 homozygous carrier of the ARH1 mutation among the 50 hypercholesterolemic outpatients. Population-based genotyping of ARH1 in 2565 subjects allowed the identification of 1 heterozygous carrier. The overall estimated allele frequency of ARH1 in Sicily was 0.0002 (0.02%). CONCLUSIONS: The identification of a new case of ARH in Sicily among 50 clinically diagnosed FH highlights the importance of next-generation sequencing analysis as tool to improve the FH diagnosis. Our results also indicate that ARH1 carrier status is present in ∼1:2500 of Sicilian inhabitants, confirming that ARH is extremely rare outside Sardinia.

Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

BACKGROUND: Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. OBJECTIVE: The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. METHODS: We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. RESULTS: The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. CONCLUSIONS: We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG.

Myristic acid is associated to low plasma HDL cholesterol levels in a Mediterranean population and increases HDL catabolism by enhancing HDL particles trapping to cell surface proteoglycans in a liver hepatoma cell model.

BACKGROUND: HDL-C plasma levels are modulated by dietary fatty acid (FA), but studies investigating dietary supplementation in FA gave contrasting results. Saturated FA increased HDL-C levels only in some studies. Mono-unsaturated FA exerted a slight effect while poly-unsaturated FA mostly increased plasma HDL-C. AIMS: This study presents two aims: i) to investigate the relationship between HDL-C levels and plasma FA composition in a Sicilian population following a "Mediterranean diet", ii) to investigate if FA that resulted correlated with plasma HDL-C levels in the population study and/or very abundant in the plasma were able to affect HDL catabolism in an "in vitro" model of cultured hepatoma cells (HepG2). RESULTS: plasma HDL-C levels in the population correlated negatively with myristic acid (C14:0, ß = -0.24, p < 0.01), oleic acid (C18:1n9, ß = -0.22, p < 0.01) and cis-11-Eicosenoic (C20:1n9, ß = -0.19, p = 0.01) and positively with palmitoleic acid (C16:1, ß = +0.19, p = 0.03). HepG2 cells were conditioned with FA before evaluating HDL binding kinetics, and only C14:0 increased HDL binding by a non-saturable pathway. After removal of heparan sulphate proteoglycans (HSPG) by heparinases HDL binding dropped by 29% only in C14:0 conditioned cells (p < 0.05). C14:0 showed also the highest internalization of HDL-derived cholesteryl esters (CE, +32% p = 0.01 vs. non-conditioned cells). CONCLUSIONS: C14:0 was correlated with decreased plasma HDL-C levels in a Mediterranean population. C14:0 might reduce HDL-C levels by increasing HDL trapping to cell surface HSPG and CE stripping from bound HDL. Other mechanisms are to be investigated to explain the effects of other FA on HDL metabolism.

Novel CREB3L3 Nonsense Mutation in a Family With Dominant Hypertriglyceridemia.

OBJECTIVE: Cyclic AMP responsive element-binding protein 3-like 3 (CREB3L3) is a novel candidate gene for dominant hypertriglyceridemia. To date, only 4 kindred with dominant hypertriglyceridemia have been found to be carriers of 2 nonsense mutations in CREB3L3 gene (245fs and W46X). We investigated a family in which hypertriglyceridemia displayed an autosomal dominant pattern of inheritance. APPROACH AND RESULTS: The proband was a 49-year-old woman with high plasma triglycerides (≤1300 mg/dL; 14.68 mmol/L). Her father had a history of moderate hypertriglyceridemia, and her 51-year-old brother had triglycerides levels as high as 1600 mg/dL (18.06 mmol/L). To identify the causal mutation in this family, we analyzed the candidate genes of recessive and dominant forms of primary hypertriglyceridemia by direct sequencing. The sequencing of CREB3L3 gene led to the discovery of a novel minute frame shift mutation in exon 3 of CREB3L3 gene, predicted to result in the formation of a truncated protein devoid of function (c.359delG-p.K120fsX20). Heterozygosity for the c.359delG mutation resulted in a severe phenotype occurring later in life in the proband and her brother and a good response to diet and a hypotriglyceridemic treatment. The same mutation was detected in a 13-year-old daughter who to date is normotriglyceridemic. CONCLUSIONS: We have identified a novel pathogenic mutation in CREB3L3 gene in a family with dominant hypertriglyceridemia with a variable pattern of penetrance.

Homozygous familial hypobetalipoproteinemia: two novel mutations in the splicing sites of apolipoprotein B gene and review of the literature.

OBJECTIVE: Familial hypobetalipoproteinemia (FHBL) is autosomal codominant disorder of lipoprotein metabolism characterized by low plasma levels of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB) below the 5(th) percentile of the distribution in the population. Patients with the clinical diagnosis of homozygous FHBL (Ho-FHBL) are extremely rare and few patients have been characterized at the molecular level. Here we report the medical history and the molecular characterization of one paediatric patient with clinical features of Ho-FHBL. METHODS: A one month old infant with failure to thrive, severe hypocholesterolemia and acanthocytosis was clinically and genetically characterized. Molecular characterization of the proband and her parents was performed by direct sequencing of the APOB gene and functional role of the identified mutations was assessed by the minigene methodology. RESULTS: The proband was found carrying two novel splicing mutations of the APOB gene (c.3696+1G > C and c.3697-1G > A). CHOK1H8 cells expressing minigenes harbouring the mutations showed that these two mutations were associated with the retention of intron 23 and skipping of exon 24, resulting in two truncated apoB fragments of approximate size of 26-28 % of ApoB-100 and the total absence of apoB. CONCLUSION: We describe the first case of Ho-FHBL due to two splicing mutations affecting both the donor and the acceptor splice sites of the same intron of the APOB gene occurring in the same patient. The clinical management of the proband is discussed and a review of the clinical and genetic features of the published Ho-FHBL cases is reported.

A novel APOB mutation identified by exome sequencing cosegregates with steatosis, liver cancer, and hypocholesterolemia.

OBJECTIVE: In familial hypobetalipoproteinemia, fatty liver is a characteristic feature, and there are several reports of associated cirrhosis and hepatocarcinoma. We investigated a large kindred in which low-density lipoprotein cholesterol, fatty liver, and hepatocarcinoma displayed an autosomal dominant pattern of inheritance. APPROACH AND RESULTS: The proband was a 25-year-old female with low plasma cholesterol and hepatic steatosis. Low plasma levels of total cholesterol and fatty liver were observed in 10 more family members; 1 member was affected by liver cirrhosis, and 4 more subjects died of either hepatocarcinoma or carcinoma on cirrhosis. To identify the causal mutation in this family, we performed exome sequencing in 2 participants with hypocholesterolemia and fatty liver. Approximately 22 400 single nucleotide variants were identified in each sample. After variant filtering, 300 novel shared variants remained. A nonsense variant, p.K2240X, attributable to an A>T mutation in exon 26 of APOB (c.6718A>T) was identified, and this variant was confirmed by Sanger sequencing. The gentotypic analysis of 16 family members in total showed that this mutation segregated with the low cholesterol trait. In addition, genotyping of the PNPLA3 p.I148M did not show significant frequency differences between carriers and noncarriers of the c.6718A>T APOB gene mutation. CONCLUSIONS: We used exome sequencing to discover a novel nonsense mutation in exon 26 of APOB (p.K2240X) responsible for low cholesterol and fatty liver in a large kindred. This mutation may also be responsible for cirrhosis and liver cancer in this family.

Prevalence of ANGPTL3 and APOB gene mutations in subjects with combined hypolipidemia.

OBJECTIVE: Mutations of the ANGPTL3 gene have been associated with a novel form of primary hypobetalipoproteinemia, the combined hypolipidemia (cHLP), characterized by low total cholesterol and low HDL-cholesterol levels. The aim of this work is to define the role of ANGPTL3 gene as determinant of the combined hypolipidemia phenotype in 2 large cohorts of 913 among American and Italian subjects with primary hypobetalipoproteinemia (total cholesterol<5th percentile). METHODS AND RESULTS: The combined hypolipidemia cut-offs were chosen according to total cholesterol and HDL-cholesterol levels reported in the ANGPTL3 kindred described to date: total cholesterol levels, <2nd percentile and HDL-cholesterol, levels<2nd decile. Seventy-eight subjects with combined hypolipidemia were analyzed for ANGPTL3 and APOB genes. We identified nonsense and/or missense mutations in ANGPTL3 gene in 8 subjects; no mutations of the APOB gene were found. Mutated ANGPTL3 homozygous/compound heterozygous subjects showed a more severe biochemical phenotype compared to heterozygous or ANGPTL3 negative subjects, although ANGPTL3 heterozygotes did not differ from ANGPTL3 negative subjects. CONCLUSION: These results demonstrated that in a cohort of subjects with severe primary hypobetalipoproteinemia the prevalence of ANGPTL3 gene mutations responsible for a combined hypolipidemia phenotype is about 10%, whereas mutations of APOB gene are absent.

Variable phenotypic expression of chylomicron retention disease in a kindred carrying a mutation of the Sara2 gene.

Chylomicron retention disease is a recessive inherited disorder characterized by fat malabsorption and steatorrhea and is associated with failure to thrive in infancy. We describe a kindred carrying a mutation of Sara2 gene causing a chylomicron retention phenotype. The proband was a 5-month-old baby, born of consanguineous, apparently healthy parents from Morocco, with failure to thrive. There was a large quantity of fats in feces and malabsorption of fat-soluble vitamins. Intestinal biopsies showed a diffused enterocyte vacuolization with large cytosolic lipid droplets. Chylomicron retention disease or Anderson disease was hypothesized, and the Sara2 gene was analyzed by direct sequencing. Analysis of the Sara2 gene in the proband identified a 2-nucleotide homozygous deletion in exon 3 leading to a premature stop codon (c.75-76 del TG-L28fsX34). The father was heterozygous for the same mutation, whereas the proband's mother was homozygous, suggesting a variable phenotypic expression of the molecular defect. More studies are needed to understand the reasons of the phenotypic variability of the same molecular defect in the same family.

Familial hypobetalipoproteinemia due to apolipoprotein B R463W mutation causes intestinal fat accumulation and low postprandial lipemia.

OBJECTIVE: Familial hypobetalipoproteinemia (FHBL) is characterized by inherited low plasma levels of apolipoprotein B (apoB)-containing lipoproteins. In this paper we investigated whether the already described APOB R463W missense mutation, a FHBL mutation able to impair the activity of microsomal triglyceride transfer protein (MTP), may cause intestinal fat accumulation and reduced postprandial lipemia. METHODS: Four out of five probands harboring APOB R463W mutation were compared with six healthy controls and six patients with celiac disease (CD). An oral fat load supplemented with retinyl palmitate (RP) was administered and a gastro-duodenal endoscopy with biopsy was performed. RESULTS: Plasma triglyceride area under curves was significantly reduced in FHBL probands compared to controls and CD patients; the proportion of absorbed RP was similar to that of CD patients. Only the intestinal biopsies of FHBL patients showed lipids accumulating within the duodenal mucosa. CONCLUSIONS: FHBL due to R463W apoB mutation is a cause of intestinal fat accumulation and postprandial lipid absorption impairment.

A novel loss of function mutation of PCSK9 gene in white subjects with low-plasma low-density lipoprotein cholesterol.

OBJECTIVES: The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African-Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. METHODS AND RESULTS: We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest (<5th) percentile, 44 in the highest (>95th) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delG) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. CONCLUSIONS: We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals.

Six novel mutations of the LDL receptor gene in FH kindred of Sicilian and Paraguayan descent.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.