Low-density Lipoprotein Receptor Mutations Research Articles

3259Next-generation sequencing to confirm clinical FH in The Netherlands

Abstract Background Pathogenic variants in the genes encoding the low-density lipoprotein receptor (LDLR), apolipoprotein B100 (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9)) are known to cause familial hypercholesterolemia (FH), a disease characterized by high LDL-C levels. We set out to investigate which proportion of patients with extremely elevated LDL-C levels, who were referred for molecular analysis, carry a mutation in any of the three FH genes or other, putative FH genes. Methods Targeted next-generation sequencing of 28 genes involved in lipid metabolism was performed in 1475 clinical FH patients with LDL-C levels >5mmol/L (corresponding with possible, probable and definite FH score according to the Dutch Lipid Clinic Network criteria) and triglyceride levels <4.5 mmol/L. Genetic variants and copy number variants were curated consecutively using specialist analysis software. (Likely) pathogenic variants in LDLR, APOB, or PCSK9 were annotated as FH-causing variants. Results 14.3% of the 1475 patients with clinical FH were heterozygous carriers of a mutation in LDLR, APOB, or PCSK9. This number ranged from 8% in the lowest LDL-C group (5–5.99 mmol/L) to 56% in patients with the highest LDL-C levels (>8 mmol/L). Patients with LDLR, APOB, or PCSK9 variants had median LDL-C levels of 6.8 [5.7–7.9], 6.2 [5.1–7.0], and 6.4 [5.7–6.8] mmol/L [interquartile range], respectively. Mutation-negative FH patients had median LDL-C levels of 5.8 [5.2–6.5] mmol/L. Of the FH mutation-negative patients 7.4% had a (likely) pathogenic variant in the gene encoding lipoprotein lipase (LPL) and 1.3% in the apolipoprotein E (APOE) gene. The proportion of FH mutation-negative patients carrying an APOE variant increased from 1.2% in the lowest LDL-C group to 7.5% in patients with LDL-C >8 mmol/L. Mutation-positive FH patients were significantly younger (41.61±17.53 years vs 52.62±13.28 years P<0.001) and had a significant lower BMI (25.86±4.87 vs 26.55±4.07kg/m2, P=0.02) compared to mutation negative patients. Figure 1 Conclusions A genetic defect in LDLR, APOB or PCSK9 is identified in only 14.3% of the patients with a clinical FH phenotype (defined as LDL-C >5 mmol/L) referred for molecular testing in the Netherlands. Mutations in LPL and APOE were found in a minor proportion of the FH mutation-negative patients. Our finding that a mutation is only found 56% of patients who present with LDL-C levels above 8 mmol/L indicates that either misclassification of FH and/or other genetic defects may be present in this group. Acknowledgement/Funding Grant [016.156.445] from The Netherlands Organisation for Scientific Research (NWO)

P725Molecular genetics of familial hypercholesterolemia in Israel revisited

Abstract Objective Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the genes for LDL receptor (LDLR), apolipoprotein B (APOB) and Proprotein convertase subtilisin/kexin type9 (PCSK9). The purpose of the current investigation was to define the current spectrum of mutations causing FH in Israel. Methods New families were collected through the MEDPED (Make Early Diagnosis Prevent Early Death) FH program. Molecular analysis of the LDLR, PCSK9 and APOB genes were done using High Resolution Melt and direct sequencing in 67 index cases. A 6-SNP LDL-C gene score calculation for polygenic hypercholesterolaemia was done using TaqMan genotyping. Results Mean serum cholesterol was 7.48±1.89mmol/L and the mean serum LDL-C was 5.99±1.89mmol/L. Mutations in the LDLR and APOB gene were found in 24 cases (35.8%), with 16 in LDLR, none in PCSK9 and one, p.(R3527Q) in the APOB gene, which is the first APOB mutation carrier identified in the Israeli population. Of the LDLR mutations, two were novel; p.(E140A) and a promoter variant, c.-191C>A. The c.2479G>A p.(V827I) in exon 17 of the LDLR gene was found in 8 patients (33.3% of the mutations) with modestly elevated LDL-C but also in a compound heterozygous patient with a clinical homozygous FH phenotype, consistent with this being a “mild” FH-causing variant. A significantly higher 6-SNP LDL-C score was found in mutation-negative cases compared with a normal Caucasian cohort (p=0.03), confirming that polygenic inheritance of common LDL-C raising SNPs can produce an FH phenocopy. Conclusions The results indicate a different spectrum of genetic causes of FH from that found previously, in concordance with the heterogeneous and changing origins of the Israeli population, and confirm that a polygenic cause is also contributing to the FH phenotype in Israel.

Impaired adult hippocampal neurogenesis in a mouse model of familial hypercholesterolemia: A role for the LDL receptor and cholesterol metabolism in adult neural precursor cells

ObjectiveIn familial hypercholesterolemia (FH), mutations in the low-density lipoprotein (LDL) receptor (LDLr) gene result in increased plasma LDL cholesterol. Clinical and preclinical studies have revealed an association between FH and hippocampus-related memory and mood impairment. We here asked whether hippocampal pathology in FH might be a consequence of compromised adult hippocampal neurogenesis. MethodsWe evaluated hippocampus-dependent behavior and neurogenesis in adult C57BL/6JRj and LDLr−/− mice. We investigated the effects of elevated cholesterol and the function of LDLr in neural precursor cells (NPC) isolated from adult C57BL/6JRj mice in vitro. ResultsBehavioral tests revealed that adult LDLr−/− mice showed reduced performance in a dentate gyrus (DG)-dependent metric change task. This phenotype was accompanied by a reduction in cell proliferation and adult neurogenesis in the DG of LDLr−/− mice, suggesting a potential direct impact of LDLr mutation on NPC. Exposure of NPC to LDL as well as LDLr gene knockdown reduced proliferation and disrupted transcriptional activity of genes involved in endogenous cholesterol synthesis and metabolism. The LDL treatment also induced an increase in intracellular lipid storage. Functional analysis of differentially expressed genes revealed parallel modulation of distinct regulatory networks upon LDL treatment and LDLr knockdown. ConclusionsTogether, these results suggest that high LDL levels and a loss of LDLr function, which are characteristic to individuals with FH, might contribute to a disease-related impairment in adult hippocampal neurogenesis and, consequently, cognitive functions.

Evaluation of the role of STAP1 in Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is characterised by elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and a substantial risk for cardiovascular disease. The autosomal-dominant FH is mostly caused by mutations in LDLR (low density lipoprotein receptor), APOB (apolipoprotein B), and PCSK9 (proprotein convertase subtilisin/kexin). Recently, STAP1 has been suggested as a fourth causative gene. We analyzed STAP1 in 75 hypercholesterolemic patients from Berlin, Germany, who are negative for mutations in canonical FH genes. In 10 patients with negative family history, we additionally screened for disease causing variants in LDLRAP1 (low density lipoprotein receptor adaptor protein 1), associated with autosomal-recessive hypercholesterolemia. We identified one STAP1 variant predicted to be disease causing. To evaluate association of serum lipid levels and STAP1 carrier status, we analyzed 20 individuals from a population based cohort, the Cooperative Health Research in South Tyrol (CHRIS) study, carrying rare STAP1 variants. Out of the same cohort we randomly selected 100 non-carriers as control. In the Berlin FH cohort STAP1 variants were rare. In the CHRIS cohort, we obtained no statistically significant differences between carriers and non-carriers of STAP1 variants with respect to lipid traits. Until such an association has been verified in more individuals with genetic variants in STAP1, we cannot estimate whether STAP1 generally is a causative gene for FH.

Structure-Function Relationships of LDL Receptor Missense Mutations Using Homology Modeling.

Mutations in the low-density lipoprotein receptor (LDLR), which cause familial hypercholesterolemia (FH), present a variable clinical FH phenotype. To date, over 1600 FH-causing mutations have been found worldwide. The aim of this study was to investigate the structure-function relationships of LDLR mutations by using homology modeling. Structural analysis of 36 missense mutations of known receptor activity (33 severe, 1 mild, and 2 non-pathogenic phenotypes) using sequence comparison and homology modeling was performed. Severe phenotypes had less than 2% to 32% of residual LDLR activity. Mild phenotypes had 76-92% of residual LDLR activity. Finally, non-pathogenic phenotypes had normal residual LDLR activity. Sequence comparisons showed that most of the severe phenotypes were located within the fully conserved residues of LDLR, while most of the mild and non-pathogenic phenotypes were located within the poorly conserved residues. Homology modeling demonstrated several phenomena for severe phenotypes: disruption of disulfide bond formation, disturbance of the calcium binding sites, and perturbation of LDLR hydrophobic conserved packing. In contrast, mild and non-pathogenic phenotypes did not disturb the critical region of LDLR. In addition, the root mean square deviation (RMSD) values of severe phenotype tended to be higher than the mild and non-pathogenic phenotypes, and the mean of solvent accessible surface area (ASA) of the residues in wild type structure for the severe phenotype was lower than mild and non-pathogenic phenotypes. These findings provide a better understanding in the structure-function relationships of LDLR mutations and may be useful in predicting FH severity based on future genotyping.

LRP5L-P36R Attenuating Matrix Metalloproteinase 9 Biological Function May Contribute to the Development of Cataract

Background: Congenital cataract is the leading cause of childhood blindness. Membranous cataract is a rare congenital cataract and the mechanism has not been understood. This study aimed to investigate the pathogenic mutation and its mechanism on the development of cataract in a four-generation Chinese congenital membranous cataract family. Methods: Whole exome sequencing was performed to validate the pathogenic mutation in a Chinese congenital membranous cataract family. Wild-type and mutant LDL receptor related protein 5 like (LRP5L) plasmids were transfected into human lens epithelial cells (HLE B-3). Reverse transcription-PCR, quantitative real-time PCR, and immunoblotting were used to investigate the contributions of mutant LRP5L to development of cataract. Tube formation and spheroid sprouting assays were used to evaluate the contributions of LRP5L to angiogenesis. Findings: We found a novel suspected pathogenic mutation in LRP5L (c.107C>G, p.P36R) in a Chinese congenital membranous cataract family. In HLE B-3, LRP5L and LRP5L-P36R increase matrix metalloproteinase 9 (MMP9) expressions by cooperation with MMP9. However, LRP5L may promote angiogenesis by increasing active MMP9, while LRP5L-P36R may inhibit angiogenesis via downregulating active MMP9 and laminin γ1. Exogenous transforming growth factor beta-1 recombinant protein could promote LRP5L expressions in HLE B-3 cells. Interpretation: Our data indicated that LRP5L-P36R may contribute to the development of cataract via attenuating biological function of MMP9. This study enriches our knowledge on LRP5L and its mutant, LRP5L-P36R, and will benefit the studies on pathogenesis of congenital membranous cataract. Funding Statement: This study was supported by the Heilongjiang Provincial Natural Science Foundation of China (H2017028); the Nature Science Foundation of China (81470618); and the Natural Science Foundation of Heilongjiang Province of China (H2016038 and H2016007). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Internal Review Board of Harbin Medical University and all the experiments were conducted in accordance with Declaration of Helsinki Principles. Written informed consents were obtained from all the participants or their guardians.

A xanthomatosis-susceptibility gene may exist in a Syrian family with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal-dominant inherited disorder characterized by high serum low-density lipoprotein (LDL)-cholesterol concentrations, xanthoma formation, and premature atherosclerosis. Homozygous individuals die of vascular disease as children or young adults; heterozygous persons are at high risk for premature cardiovascular death. Mutations in the LDL-receptor gene are responsible for FH. We studied 49 members of a consanguineous Syrian kindred containing 6 homozygous individuals from the same pedigree. Half of the homozygotes had giant xanthomas, while half did not, even though their LDL-cholesterol concentrations were elevated to similar degrees (> 14 mmol/l). Heterozygous FH individuals from this family were also clearly distinguishable with respect to xanthoma size. We performed DNA analysis and were successful in identifying a hitherto not described mutation in this family's LDL receptor. DNA sequence analysis of the LDL-receptor gene revealed a T to C substitution at nucleotide 1,999 in codon 646 of exon 14. We next conducted a segregation analysis, which suggests that a susceptibility gene may explain the formation of giant xanthomas in this family. We raise the hypothesis that the appearance of giant xanthomas in this FH family is controlled by a second gene acting in an autosomal-dominant or recessive fashion. Elucidation of this 'xanthoma' gene may shed additional light on LDL-cholesterol deposition.

Prevalence of familial hypercholesterolemia in patients with premature myocardial infarction.

BackgroundFamilial hypercholesterolemia (FH) is a genetic cause of premature myocardial infarction (PMI). Early diagnosis of FH is critical for prognosis.HypothesisTo investigate the prevalence of FH among a cohort of Chinese patients with PMI using genetic testing, and to evaluate different diagnostic criteria.MethodsA total of 225 consecutive PMI patients were recruited. Low‐density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin‐kexin type 9 (PCSK9) and low‐density lipoprotein receptor adaptor protein 1 (LDLRAP1) genes were detected by Sanger sequencing. FH was diagnosed using the Dutch Lipid Clinic Network (DLCN) criteria and modified DLCN criteria, respectively. The prevalence and clinical features of FH were analyzed.ResultsIn all PMI patients, pathogenic mutations of LDLR, APOB, PCSK9 and LDLRAP1 genes were found in 10 of 225 patients. Among all mutations, four mutations (LDLR c.129G>C, LDLR c.1867A>T, LDLRAP1 c.65G>C, and LDLRAP1 c.274G>A) were newly discovered. The prevalence of FH diagnosed by genetic testing was 4.4%. The prevalence of definite/probable FH diagnosed by DLCN and modified DLCN criteria reached 8.0% and 23.6%, respectively, and the mutation rates were 33.3% and 12.2%, respectively. The low‐density lipo‐protein cholesterol (LDL‐C) levels in PMI patients with FH were far from goal attainment. Only one of the FH patients had LDL‐C <2.5 mmol/L, and none of them had LDL‐C <1.8 mmol/L.ConclusionsThe prevalence of FH among Chinese patients with PMI appeared relatively common. Underdiagnosis and undertreatment of FH are still a big problem, which should arouse a widespread concern.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays.

ObjectiveMutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a.MethodsBioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR.ResultsGel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = −0.656, p<0.05).ConclusionLDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

New Horizons in the Pathogenesis, Pathophysiology and Treatment of Familial Hypercholesterolaemia.

Familial Hypercholesterolaemia (FH) is an autosomal-dominant genetic disease and represents the most common genetic disorder: heterozygous 1/250 births, homozygous 1/300, 000 births. FH is characterized by high to very high low-density lipoprotein cholesterol (LDL-C), which is the main cause of increased incidence of premature atherosclerotic Cardiovascular Disease (CVD) or aortic stenosis. The aim of the review was to investigate the pathogenesis and the pathophysiology of FH. The most common (60-80%) FH cause is mutations of the LDL Receptor (LDLR) protein (6 classes with a different number of receptors and functionality). Moreover, mutations in apolipoprotein B (APOB) (<5%) and gain-of-function mutations of proprotein convertase subtilisin/kexin type 9 genes (PCSK9) (<1%) contribute to its pathogenesis. An Autosomal Recessive Hypercholesterolaemia (ARH) is another cause, very rare (1/2.500 births), mainly in Sardinia. The remaining patients with a clinical diagnosis of monogenic hypercholesterolaemia do not present any known genetic cause. Since FH is a significant public health problem, early diagnosis and treatment are of utmost importance. Recent studies demonstrated the influence of the LDLR mutation type in the FH phenotype, associating a more severe clinical phenotype and worse advanced CVD in patients with null mutation than those with receptor-defective mutations. This analysis completes the adequate clinical diagnosis. Both homozygous and heterozygous FH are related to mutations of LDLR (mainly), APOB, PCSK9, while other rare forms exist. All aberrations lead to the impaired removal of LDL-C from the blood leading to its accumulation and subsequent CVD earlier than in the general population.

Abstract 17183: Genetically Engineered Calcification Increases Surface Roughness and Changes the Distribution of Atherosclerotic Plaques in Mice With Atherosclerosis

Background: Atherosclerosis is an inflammatory disease that contributes significantly to cardiovascular mortality. Arterial stiffening due to calcification can coexist with or directly influence pathogenesis of atherosclerosis. We hypothesize that arteriosclerosis caused by genetically engineered calcification in mice redistributes shear stress on the endothelial cells, which in turn leads to further accelerated rate of plaque formation. Methods: All of the mice had an LDL receptor mutation and were fed pro-atherogenic diet (1.25% cholesterol). The case group overexpressed tissue-nonspecific alkaline phosphatase in endothelial cells (eTNAP). The luminal surfaces of descending thoracic aorta (TA) and suprarenal abdominal aorta (AA) were scanned using Sensofar S Neox optical confocal microscope at 1 μm vertical resolution. The surface topographies per sample, each measuring 500 x 700 microns, were analyzed for ISO 25178-2 areal roughness parameters using SensoMap. Parameters were compared by t test (n=5 eTNAP, n=6-7 control). Results: We observed a significant difference in the incidence as well as the morphology of the plaques in the eTNAP group compared to control mice. There was a 4.8 fold increase in the number of topological motifs in the thoracic aorta of the eTNAP vs. controls (p &lt; 0.01). Similarly, the number of motifs was increase in the abdominal aorta of the eTNAP mice compared to controls (2.8 fold; p=0.06). The average area of the motifs was significantly larger in the controls compared to the eTNAP group in both segments of the aorta (p=0.06 TA; p &lt; 0.05 AA). The maximum height of the lesions in the TA was not different between the groups (p=0.62), however increased by 74% in the abdominal aorta of the eTNAP compared to controls (p&lt;0.05). Finally, the surface developed interfacial area ratio (Sdr, a parameter of texture) was increased in the eTNAP group in both the TA (p &lt; 0.05) and AA regions (p &lt; 0.01). Conclusion: Arteriosclerosis increases surface roughness. In our model, the abdominal region had a high incidence of taller, irregular lesions whereas those in the thoracic aorta were flatter and more spaced. The high resolution scans will be further used to study the hemodynamic disturbances to the endothelial cells using flow dynamics simulations.

Use of plasma metabolomics to analyze phenotype-genotype relationships in young hypercholesterolemic females

Hypercholesterolemia is characterized by high plasma LDL cholesterol and often caused by genetic mutations in LDL receptor (LDLR), APOB, or proprotein convertase subtilisin/kexin type 9 (PCSK9). However, a substantial proportion of hypercholesterolemic subjects do not have any mutations in these canonical genes, leaving the underlying pathobiology to be determined. In this study, we investigated to determine whether combining plasma metabolomics with genetic information increases insight in the biology of hypercholesterolemia. For this proof of concept study, we combined plasma metabolites from 119 hypercholesterolemic females with genetic information on the LDL canonical genes. Using hierarchical clustering, we identified four subtypes of hypercholesterolemia, which could be distinguished along two axes represented by triglyceride and large LDL particle concentration. Subjects with mutations in LDLR or APOB preferentially clustered together, suggesting that patients with defects in the LDLR pathway show a distinctive metabolomics profile. In conclusion, we show the potential of using metabolomics to segregate hypercholesterolemic subjects into different clusters, which may help in targeting genetic analysis.

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Autosomal dominant hypercholesterolemia in Catalonia: Correspondence between clinical-biochemical and genetic diagnostics in 967 patients studied in a multicenter clinical setting

Autosomal dominant hypercholesterolemia (ADH) is associated with mutations in the low-density lipoprotein (LDL) receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes, and it is estimated to be greatly underdiagnosed. The most cost-effective strategy for increasing ADH diagnosis is a cascade screening from mutation-positive probands. The objective of this study was to evaluate the results from 2008 to 2016 of ADH genetic analysis performed in our clinical laboratory, serving most lipid units of Catalonia, a Spanish region with approximately 7.5 million inhabitants. After the application of the Dutch Lipid Clinic Network (DLCN) clinical diagnostic score for ADH, this information and blood or saliva from 23 different lipid clinic units were investigated in our laboratory. DNA was screened for mutations in LDLR, APOB, and PCSK9, using the DNA-array LIPOchip, the next-generation sequencing SEQPRO LIPO RS platform, and multiplex ligation-dependent probe amplification (MLPA). The Simon Broome Register Group (SBRG) criteria was calculated and analyzed for comparative purposes. A total of 967 unrelated samples were analyzed. From this, 158 pathogenic variants were detected in 356 patients. The main components of the DLCN criteria associated with the presence of mutation were plasma LDL cholesterol (LDLc), age, and the presence of tendinous xanthomata. The contribution of family history to the diagnosis was lower than in other studies. DLCN and SBRG were similarly useful for predicting the presence of mutation. In a real clinical practice, multicenter setting in Catalonia, the percentage of positive genetic diagnosis in patients potentially affected by ADH was 38.6%. The DLCN showed a relatively low capacity to predict mutation detection but a higher one for ruling out mutation.

LDL receptor mutation in a familial hypercholesterolemia cohort-results of a single centre study from Singapore

Aim: Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease characterized by the presence of high plasma low density lipoproteins cholesterol (LDL-c). The aim of the study was to assess the heterogeneity, association of LDL Receptor mutations in patients, clinically diagnosed with FH in Singapore.

Validation of LDLr Activity as a Tool to Improve Genetic Diagnosis of Familial Hypercholesterolemia: A Retrospective on Functional Characterization of LDLr Variants.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by high blood-cholesterol levels mostly caused by mutations in the low-density lipoprotein receptor (LDLr). With a prevalence as high as 1/200 in some populations, genetic screening for pathogenic LDLr mutations is a cost-effective approach in families classified as ‘definite’ or ‘probable’ FH and can help to early diagnosis. However, with over 2000 LDLr variants identified, distinguishing pathogenic mutations from benign mutations is a long-standing challenge in the field. In 1998, the World Health Organization (WHO) highlighted the importance of improving the diagnosis and prognosis of FH patients thus, identifying LDLr pathogenic variants is a longstanding challenge to provide an accurate genetic diagnosis and personalized treatments. In recent years, accessible methodologies have been developed to assess LDLr activity in vitro, providing experimental reproducibility between laboratories all over the world that ensures rigorous analysis of all functional studies. In this review we present a broad spectrum of functionally characterized missense LDLr variants identified in patients with FH, which is mandatory for a definite diagnosis of FH.

Genetically confirmed familial hypercholesterolemia in outpatients with hypercholesterolemia.

BackgroundFamilial hypercholesterolemia (FH) is an autosomal dominant disorder of lipoprotein metabolism which can lead to premature coronary heart disease (pCHD). There are about 3.8 million potential FH patients in China, whereas the clinical and genetic data of FH are limited.MethodsDutch Lipid Clinic Network (DLCN) criteria was used to diagnose FH in outpatients with hypercholesterolemia. Resequencing chip analysis combined with Sanger sequencing validation were used to identify mutations in the definite FH patients according to DLCN criteria. In silico analysis was conducted in mutations with previously unknown pathogenicity. Then, the novel mutant receptors were transfected into human embryo kidney 293 (HEK-293) cells. The binding and the internalization activities of the mutant receptors were analyzed by flow cytometry.ResultsThe prevalence of definite FH in outpatients with hypercholesterolemia in this study is 3.2%. Using genetic testing, one homozygous FH (HoFH), one heterozygous FH (HeFH) and three compound heterozygous FH patients were confirmed. Eight mutations in low-density lipoprotein receptor (LDLR) gene were identified, in which c.357delG was a novel mutation and co-segregated with the FH phenotype. Bioinformatic analysis confirmed that c.357delG was a pathogenic mutation. Furthermore, when compared with the wild-type LDLRs by flow cytometry analysis, the binding and internalization activities of c.357delG mutant LDLRs were reduced by 35% and 49%, respectively.ConclusionsThis study identified eight LDLR gene mutations in five patients with definite FH, in which c.357delG is a novel pathogenic mutation. These findings increase our understanding of the genetic spectrum of FH in China.

Spectrum of low-density lipoprotein receptor (LDLR) mutations in a cohort of Sri Lankan patients with familial hypercholesterolemia \u2013 a preliminary report

BackgroundHypercholesterolemia is a major determinant of cardiovascular disease-associated morbidity and mortality. Mutations in the LDL-receptor (LDLR) gene are implicated in the majority of the cases with familial hypercholesterolemia (FH). However, the spectrum of mutations in the LDLR gene in Sri Lankan patients has not been investigated. The objective of this study was to report the frequency and spectrum of variants in LDLR in a cohort of Sri Lankan patients with FH.MethodsA series of consecutive patients with FH, diagnosed according to Modified Simon Broome criteria or Dutch Lipid Clinic Network criteria at the University Medical Unit, Colombo, were recruited. Clinical data was recorded. DNA was extracted from peripheral blood samples. The LDLR gene was screened for genetic variants by Sanger sequencing.ResultsA total of 27 patients [13 (48%) males, 14 (52%) females; age range 24–73 years] were tested. Clinical features found among these 27 patients were: xanthelasma in 5 (18.5%), corneal arcus in 1 (3.7%), coronary artery disease (CAD) in 10 (37%), and a family history of hypercholesterolemia and/or CAD in 24 (88.9%) patients. In the entire cohort, mean total cholesterol was 356.8 mg/dl (±66.4) and mean LDL-cholesterol was 250.3 mg/dl (±67.7). Sanger sequencing of the 27 patients resulted in the identification of known pathogenic missense mutations in 5 (18.5%) patients. Four were heterozygotes for 1 mutation each. They were c.682G > C in 2 patients, c.1720C > A in 1 patient, and c.1855 T > A in 1 patient. One patient with severe FH phenotypes was a compound heterozygote for one known mutation, c.2289G > T, and another missense variant, c.1670C > G (p.Thr557Ser), with unknown functional impact. This latter variant has not been reported in any other population previously.ConclusionsThe frequency of known mutations in the LDLR gene in this cohort of patients was markedly low compared to frequencies reported in other populations. This highlights the likelihood of a complex, polygenic inheritance of FH in Sri Lankan patients, indicating the need for a comprehensive genetic evaluation that includes the screening for mutations in other genes that cause FH, such as APOB, PCSK9, and LDLRAP1.

Abstract 214: Functional Analysis of the Low Density Lipoprotein Receptor Using Saturation Mutagenesis Strategies

Low density lipoprotein receptor (LDLR) genetic mutations are well-documented to cause familial hypercholesterolemia (FH) in patients. These mutations affect a broad range of LDLR functions, including LDL binding and receptor trafficking, and five classes exist to describe their functional consequences. Although over 1,000 LDLR mutants have been identified, an additional 15,000+ LDLR mutants are possible by only a single amino acid substitution in the ~160kDa protein. Since cataloging mutants would undoubtedly be of therapeutic benefit, we are systematically classifying the effect of every single amino acid substitution on LDLR function using saturation mutagenesis. LDLR was knocked out of HEK293 cells using standard CRISPR/Cas9 practices and decreased protein levels were assessed by flow cytometry and immunoblot staining. In order to introduce each individual mutation into the genomic DNA sequence for high-throughput, functional analyses, the PiggyBac transposon system was used to insert a serine-integrase attachment site into a single allele in the LDLR locus, whereby efficient knock in of LDLR mutants from a pooled cDNA library can be achieved. Mutant LDLR libraries containing ~800 single amino acid substitutions were generated using commercial oligo pools and Gibson cloning. Of particular therapeutic interest is the identification of mutations that alter LDL’s ability to bind to the receptor. In our first set of experiments, fluorescently labeled LDL was incubated with cells containing pooled single amino acid substitutions in LDLR, and cells were sorted using flow cytometry. Sorted groups ranging from fluorescent negative to positive were sent for next generation sequencing in order to identify severe vs benign mutants on receptor function. As some of these mutations may decrease LDL binding activity due to inhibited receptor trafficking, we also stained pooled cells with an antibody directed against LDLR, sorted by flow cytometry and analyzed using next generation sequencing with similar strategies. Ultimately, cataloging LDLR genetic mutations should be of great therapeutic benefit, as it will allow better diagnosis and treatment of patients suffering from FH.

Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene.

Paraoxonase 1 (PON1) is a serum enzyme associated with high density lipoprotein (HDL) regulation through its paraoxonase and arylesterase activity. PON1 inhibits the oxidation of HDL and low density lipoprotein (LDL), and is involved in the pathogenesis of a variety of diseases including atherosclerosis. Conversely, mutations in the low density lipoprotein receptor (LDLR) result in failure of receptor mediated endocytosis of LDL leading to its elevated plasma levels and onset of familial hypercholesterolemia (FH). In the current study we investigated the role of PON1 polymorphisms rs662; c.575A > G (p.Gln192Arg) and rs854560; c.163T > A (p.Leu55Met) in a large family having FH patients harboring a functional mutation in LDLR. Genotypes were revealed by RFLP, followed by confirmation through Sanger sequencing. PON1 activity was measure by spectrophotometry. Our results show significantly reduced serum paraoxonase and arylesterase activities in FH patients compared with the healthy individuals of the family (p < 0.05). PON1 QQ192 genotype showed a significantly higher association with FH (p=0.0002). PON1 Q192 isoform was associated with reduced serum paraoxonase activity by in silico analysis and PON1 R192 exhibited higher serum paraoxonase and arylesterase activity than the other polymorphs. Our results highlight that the combination of LDLR mutations and PON1 MMQQ genotypes may lead to severe cardiac events.