FH Patients Research Articles

Clinical reality and challenges with familial hypercholesterolemia patients management. 2024 results from the Regional Center for Rare Diseases (RCRD) Registry in Poland

Abstract Background HeFH is one of the most prevalent genetic diseases significantly increasing the risk of CVD events and mortality. Despite new achievements with earlier diagnosis and new effective drugs, most of heFH patients do not achieve LDL-C goal and are still on residual CVD risk. Purpose We aimed to present the most recent data from the regional FH registry in Poland to show the challenges and reality of everyday clinical management with FH patients. Methods The registry is conducted at the RCRD in the 2nd largest, supraregional hospital in Poland. The main inclusion criteria were being phenotypically or genetically diagnosed with FH. Nonadherence was investigated based on detailed interview, monitoring the treatment’s effects and purchasing prescribed medications. For this analysis we investigated only adult FH patients from the registry. Due to small number of cases (n=5), patients treated with inclisiran were not included in this analysis. Results We included 142 consecutive heFH patients at mean age 45,8±14,9 years (min/max 18-75 years; 60% women). Mean BMI was 29.6±5.52 kg/m2, mean baseline TC was 282.9±80 mg/dl (the highest observed was 600 mg/dl), LDL-C 204.2±72.5 mg/dL (the highest observed -762 mg/dl), TG - 151.1±123.4 mg/dl, Lp(a) 37.6±41.5 mg/dl and HDL-C - 52.3±14.2 mg/dl. Most patients were diagnosed with LDLR (72%) or APOB (24%) mutations. High intensity statin therapy was used in 74.3% of patents, ezetimibe (mostly as a part of combination LLT) in 47.2%, and PCSK9 inhibitors in all those that met the reimbursement criteria – 25.6% of FH patients. Non-adherence to statin therapy and ezetimibe in the follow up of 2 years was 27%. In patients treated with statin therapy and ezetimibe mean achieved LDL-C was 153.6±82.82 mg/dl and on triple therapy - 64.5±43.73 mg/dl. Mean achieved LDL-C in the investigated population was 87.9±55.5 mg/dl. LDL-C goal was achieved in 39% of patients – in those treated with triple therapy with PCSK9 inhibitors it was 57.14%, and in those on statins and ezetimibe only 27.1%. Combination therapy of statins and ezetimibe did not significantly change baseline Lp(a) level (p=0.109), but when PCSK9 inhibitors were introduced the mean Lp(a) reduction by 34.2% (p=0.049) was observed. Based on the multivariable regression analysis, the following factors played the most important role for FH patients to be on LDL-C target: DLCN total score (OR 1.21, 95%CI: 1.04-1.40; p=0.013), DLCN category (10.39, 1.43-75.29; p=0.021), ezetimibe use (4.78, 1.15-19.92; p=0.031), and PCSK9 inhibitors use (4.56; 1.40-14.86; p=0.012). Conclusions FH patients in Poland are diagnosed definitely too late (45 years) and most of them are still not on LDL-C goal. Double and especially triple therapy significantly, even 5 times, increase the chance of achieving LDL-C goal, therefore PCSK9 inhibitors should be available for all FH patients, without the limitations existing currently e.g., within B101 drug program in Poland.

Clinical reality and challenges with familial hypercholesterolemia patients' management. 2024 results from the Regional Center for Rare Diseases (RCRD) Registry in Poland

BackgroundDespite advancements in early diagnosis and effective medications in last decade, most heterozygous familial hypercholesterolemia (heFH) patients still fail to achieve their low-density lipoprotein cholesterol (LDL-C) goals and remain at residual cardiovascular disease risk. We present recent data from the regional FH registry in Poland, highlighting the challenges and real-life clinical management of FH patients. MethodsThe registry is held at the Regional Centre for Rare Diseases, founded in 2016, at the 2nd largest, supraregional hospital in Poland, where >80 different rare diseases in patients from all over Poland are diagnosed and treated, including phenotypically or genetically diagnosed FH patients. Our analysis focused on both children and adult FH patients, excluding those treated with inclisiran due to a small sample size (n = 5). ResultsWe studied 173 consecutive heFH patients, median age for adult population was 40 years (range: 27–57), of whom 56.14 % were women. Among the population, 82.1 % were adults (n = 142), and 31 were children (17.92 %; median age 9 (8–13), females 58.16 %). Children exhibited lower total cholesterol and triglyceride levels compared to adults, with no significant differences in LDL-C and high-density lipoprotein cholesterol (HDL-C) levels. Molecular diagnosis in the whole population revealed that 76.6 % had an LDL receptor (LDLR) mutation, while 23.4 % had an apolipoprotein B (APOB) mutation. Risk assessment categorized patients into high (70.7 %), very high (22.1 %), and extremely high (7.1 %) risk groups. Triple therapy achieved treatment goals in 61.76 % of adults and 70.97 % of children. At baseline, 36.62 % of adult patients were not using statins. High-intensity statin therapy combined with ezetimibe was initiated for the remaining patients. Only 3.33 % of patients avoided statins due to complete intolerance. Ezetimibe was used in 57.27 % of patients (mostly in combination therapy), and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors were prescribed for 28.17 % FH patients. In adults receiving statin and ezetimibe therapy, median achieved LDL-C was 141 mg/dl (107–184). For triple therapy, median achieved LDL-C was 52.5 mg/dL (32–86.5). Overall median achieved LDL-C in the study population was 99.5 mg/dl (57.5–145.4). PCSK9 inhibitors reduced LDL-C by 165.6 mg/dl. Combination therapy did not significantly alter baseline lipoprotein(a) (Lp(a)) levels (p = 0.134), and PCSK9 inhibitors led to a mean Lp(a) reduction of 18.66 mg/dl (45 % reduction; p = 0.013). Multivariable regression analysis identified key factors for achieving LDL-C targets in FH patients: DLCN total score, DLCN category, ezetimibe use, and PCSK9 inhibitors. ConclusionsIn Poland, FH patients are often diagnosed too late (usually over 40 years of age), and many still do not reach their LDL-C goals. Combination LLT double or triple therapy significantly increases the likelihood of achieving LDL-C targets – even up to fivefold. Therefore, unrestricted access to PCSK9 inhibitors for all FH patients is crucial, without the current limitations imposed by drug reimbursement programs like B101.

Cascade screening for FH patients with VUS to improve pathogenicity classification

Cascade screening for FH patients with VUS to improve pathogenicity classification

LDLR c.89_92dup: a novel frameshift variation in familial hypercholesterolemia

BackgroundFamilial hypercholesterolemia (FH) is a common inherited metabolic disease that causes premature atherosclerosis, cardiovascular disease, and even death at a young age. Approximately 95% of FH-causing genetic variants that have been identified are in the LDLR gene. However, only 10% of the FH population worldwide has been diagnosed and adequately treated, due to the existence of numerous unidentified variants, uncertainties in the pathogenicity scoring of many variants, and a substantial number of individuals lacking access to genetic testing.ObjectiveThe aim of this study was to identify a novel variant in the LDLR gene that causes FH in a Chinese family, thereby expanding the spectrum of FH-causing variants.MethodsPatients were recruited from Beijing Anzhen Hospital, Capital Medical University. FH diagnosis was made according to the Dutch Lipid Clinical Network (DLCN) criteria. Whole-exome sequencing (WES) was conducted to identify the FH-causing variant in the proband, and amplicon sequencing was used to verify the variant in his family members.ResultsA three-generation Chinese family was recruited, and two FH patients were clinically diagnosed, both without known FH-causing variants. These two FH patients and another possible patient carried a novel variant, NC_000019.9(NM_000527.5):c.89_92dup (NP_000518.1:p.Phe32Argfs*21), in the ligand-binding domain of the low-density lipoprotein (LDL) receptor that led to a frameshift. The FH adults in the family showed severe clinical symptoms and statin therapy resistance.ConclusionThis study identified a novel pathogenic LDLR variant, c.89_92dup, associated with severe FH clinical manifestations and statin therapy resistance.

LDLR variant classification for improved cardiovascular risk prediction in familial hypercholesterolemia

Background and aimsFamilial hypercholesterolemia (FH) is a genetic disorder marked by high LDL cholesterol and an increased premature coronary artery disease (CAD) risk. Current dichotomous classification of LDL receptor gene (LDLR) variants may inadequately capture patient variability in LDL cholesterol levels and CAD risk. This study assessed a novel approach for determining LDLR variant severity using variant-specific LDL cholesterol percentiles. MethodsParticipants of the Dutch FH cascade screening program were screened for 456 LDLR variants. For each LDLR variant carrier, a sex- and age-specific LDL cholesterol percentile was derived from the LDL cholesterol level measured at study entry, i.e. generally from the blood drawn for DNA analysis. These percentiles were used to calculate the mean LDL cholesterol percentile for each variant. Based on the variant-specific LDL cholesterol percentiles, carriers were grouped into the following LDL cholesterol strata: <75th, 75th-88th, 88th-92nd, 92nd-96.5th, 96.5th-98th, and ≥98th percentile. Additionally, variants were categorized into class 1 (LDLR deficient) and non-class 1 (often LDLR defective) variants. CAD risk between carriers in the different LDL cholesterol strata and non-carriers was compared using a Cox proportional hazard model. ResultsOut of 35,067 participants, 12,485 (36 %) LDLR variant carriers (mean age 38.0 ± 20.0 years, 47.7 % male) were identified. Carriers had a 5-fold higher CAD risk compared with non-carriers. Hazard ratios for CAD increased gradually from 2.2 (95%CI 0.97–5.0) to 12.0 (95%CI 5.5–24.8) across the LDL cholesterol strata. A 7.3-fold and 3.9-fold increased CAD risk was observed in carriers of class 1 and non-class 1 LDLR variants, respectively. ConclusionsThis study presents a refined approach for classifying LDLR variants based on their impact on LDL cholesterol levels, allowing for more precise, genotype-specific CAD risk estimation in FH patients compared with traditional methods.

Consensus document on diagnosis and management of familial hypercholesterolemia from the Italian Society for the Study of Atherosclerosis (SISA)

AimsFamilial Hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism that causes an increased risk of premature atherosclerotic cardiovascular disease (ASCVD). Although early diagnosis and treatment of FH can significantly improve the cardiovascular prognosis, this disorder is underdiagnosed and undertreated. For these reasons the Italian Society for the Study of Atherosclerosis (SISA) assembled a Consensus Panel with the task to provide guidelines for FH diagnosis and treatment. Data synthesisOur guidelines include: i) an overview of the genetic complexity of FH and the role of candidate genes involved in LDL metabolism; ii) the prevalence of FH in the population; iii) the clinical criteria adopted for the diagnosis of FH; iv) the screening for ASCVD and the role of cardiovascular imaging techniques; v) the role of molecular diagnosis in establishing the genetic bases of the disorder; vi) the current therapeutic options in both heterozygous and homozygous FH. Treatment strategies and targets are currently based on low-density lipoprotein cholesterol (LDL-C) levels, as the prognosis of FH largely depends on the magnitude of LDL-C reduction achieved by lipid-lowering therapies. Statins with or without ezetimibe are the mainstay of treatment. Addition of novel medications like PCSK9 inhibitors, ANGPTL3 inhibitors or lomitapide in homozygous FH results in a further reduction of LDL-C levels. LDL apheresis is indicated in FH patients with inadequate response to cholesterol-lowering therapies. ConclusionFH is a common, treatable genetic disorder and, although our understanding of this disease has improved, many challenges still remain with regard to its identification and management.

LDLR gene variant rs688 TT genotype; genetic association with obesity in Familial Hypercholesterolemia patients

The predominant genetic risk factor for familial hypercholesterolemia along with obesity is LDLR allele status. These receptors maintain cellular cholesterol homeostasis. For instance, a common SNP rs688 in different populations has been reported to be associated with elevatedplasma LDL, resulting in hypercholesterolemia. The potential role of variant rs688 with obesity in FH patients was observed. This is a case-control study with 120 blood samples of clinically diagnosed obese familial hypercholesterolemia patients by physicians and 120 healthy individuals’ blood samples were recruited for the study. Genomic DNA was extracted from blood samples. By using Tetra ARMS PCR, genotyping of LDLR rs688 (C&gt;T) exon 12 gene variation was performed. Moreover, BMI, BP, and BSL of obese FH patients were alsoobtained; a chi-square test was performed on the obtained data to evaluate the association between rs688 and obese FH patients. Genotype CC, CT, and TT frequencies reported in the obese FH patients’ group were 0.33, 0.41, and 0.25, while in healthy individuals were 0.37, 0.41, and 0.21 respectively. Chi–square values showed a significant association with BMI, BP, and BSL. It was assessed that there is a 4% increased susceptibility of FH development along with obesity in individuals with TT genotype. So, for obese familial hypercholesterolemia, the LDLR rs688 C&gt;T gene variation can be used as a predisposing genetic marker.

Enhanced identification of familial hypercholesterolemia using central laboratory algorithms

Background and aimsFamilial hypercholesterolemia (FH) is a highly prevalent genetic disorder resulting in markedly elevated LDL cholesterol levels and premature coronary artery disease. FH underdiagnosis and undertreatment require novel detection methods. This study evaluated the effectiveness of using an LDL cholesterol cut-off ≥99.5th percentile (sex- and age-adjusted) to identify clinical and genetic FH, and investigated underutilization of genetic testing and undertreatment in FH patients. MethodsIndividuals with at least one prior LDL cholesterol level ≥99.5th percentile were selected from a laboratory database containing lipid profiles of 590,067 individuals. The study comprised three phases: biochemical validation of hypercholesterolemia, clinical identification of FH, and genetic determination of FH. ResultsOf 5614 selected subjects, 2088 underwent lipid profile reassessment, of whom 1103 completed the questionnaire (mean age 64.2 ± 12.7 years, 48% male). In these 1103 subjects, mean LDL cholesterol was 4.0 ± 1.4 mmol/l and 722 (65%) received lipid-lowering therapy. FH clinical diagnostic criteria were met by 282 (26%) individuals, of whom 85% had not received guideline-recommended genetic testing and 97% failed to attain LDL cholesterol targets. Of 459 individuals consenting to genetic validation, 13% carried an FH-causing variant, which increased to 19% in clinically diagnosed FH patients. ConclusionsThe identification of a substantial number of previously undiagnosed and un(der)treated clinical and genetic FH patients within a central laboratory database highlights the feasibility and clinical potential of this targeted screening strategy; both in identifying new FH patients and in improving treatment in this high-risk population.

Prevalence, clinical features and prognosis of familial hypercholesterolemia in Chinese Han patients with acute coronary syndrome after a coronary event: a retrospective observational study

BackgroundFamilial hypercholesterolemia (FH) is an autosomal semi-dominant disease, characterized by markedly elevated levels of low-density lipoprotein cholesterol (LDL-c) from conception and accelerated atherosclerotic cardiovascular disease, often resulting in early death. The aim of this study was to evaluate the prevalence of clinically defined FH in Chinese Han patients with acute coronary syndrome (ACS) and compare the long-term prognosis of ACS patients with and without FH receiving lipid-lowering therapy containing statins after a coronary event.MethodsAll ACS patients were screened at the Second Affiliated Hospital of Xi’an Jiaotong University between Jan 2019 and Sep 2020, and 531 participants were enrolled. All were examined for FH under the Dutch Lipid Clinical Network (DLCN) criteria, and those patients were divided into definite/probable FH, possible FH and unlikely FH. The severity of coronary artery disease was evaluated by the Gensini scoring system. Plasma levels of total cholesterol (TC), triacylglycerol (TG), HDL-cholesterol (HDL-c), LDL-cholesterol (LDL-c), very low-density lipoproteins-cholesterol (VLDL-c), apolipoprotein A1 (apoA1), apolipoprotein B (apoB) and lipoprotein (a) (Lp(a)) were determined centrally at baseline and the last follow-up visit in the fasting state. The non-high-density lipoprotein cholesterol (non-HDL-c) concentration, the TC/HDL-c and apoB/apoA1 ratios were calculated. After FH patients received lipid-lowering treatment containing statin, the target LDL-c levels recommended by the guidelines (LDL-c < 1.8 mmol/L or < 1.4 mmol/L and a reduction > 50% from baseline) were evaluated, and the occurrence of major adverse cardiovascular and cerebrovascular events (MACCE) during the 12-month follow-up was recorded.ResultsThe prevalence of clinically definite or probable FH was 4.3%, and the prevalence of possible FH was 10.6%. Compared with the unlikely FH patients with ACS, the FH patients had higher levels of TC, LDL-c, apoB, Lp(a), non-HDL-c, TC/HDL-c and apoB/apoA1 ratio, more severe coronary artery diseases and greater prevalence of left main and triple or multiple vessel lesions. After lipid-lowering therapy containing statins, a minority of FH patients reached the target LDL-c levels defined by the guidelines (χ2 = 33.527, P < 0.001). During the 12-month follow-up, a total of 72 patients experienced MACCE. The survival curve in patients in the FH group was significantly lower than that in the unlikely FH group (HR = 1.530, log-rank test: P < 0.05). Furthermore, the survival curve in patients with high LDL-c (≥ 1.8 mmol/L) was significantly lower than that in patients with low LDL-c (< 1.8 mmol/L) at the 12-month follow-up visit (HR = 1.394, log-rank test: P < 0.05). No significant difference was observed between patients with LDL-c levels ≥ 1.4 mmol/L and with < 1.4 mmol/L at the 12-month follow-up visit by using Kaplan–Meier survival analysis (HR = 1.282, log-rank test: P > 0.05).ConclusionsFH was an independent risk factor for MACCE in adult patients after a coronary event during long-term follow-up. However, there was inadequate high-intensity statins prescriptions for high-risk patients in this current study. It is important for FH patients to optimize lipid-lowering treatment strategies to reach the target LDL-c level to improve the long-term prognosis of clinical outcomes.

LDLR gene rearrangements in Czech FH patients likely arise from one mutational event

BackgroundLarge deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements.MethodsThe breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing.ResultsThe breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1–4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred.ConclusionsThe most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.

Genetic heterogeneity of familial hypercholesterolaemia in two populations from two different countries

BackgroundFamilial hypercholesterolemia (FH) is a genetically determined monogenic disorder of predominantly autosomal dominant inheritance. A number of studies on differences in the genetic profile of patients with FH have demonstrated the importance of a more substantive evaluation of genetic features. The aim of this study was to evaluate the genetic profile of patients with clinical FH among Italian and Russian patients. MethodsWe included 144 Italian and 79 Russian FH patients; clinical diagnosis was based on the same criteria. Patients were divided in: positive to genetic test (one causative variant), inconclusive (only variants of uncertain clinical significance [VUS]), and negative (with likely benign/benign variants, heterozygous variants in LDLRAP1 gene, or without causative variants). ResultsThe genetic test was positive in 76.4 % of the Italian patients and in 49.4 % of the Russian patients. The presence of VUS alone was detected in 7.6 % and in 19.0 % (p < 0.001), respectively. Among patients with positive genetic diagnosis, pre-treatment LDL-C levels were higher in the Russian cohort (353.5 ± 111.3 vs. 302.7 ± 52.1 mg/dL, p = 0.009), as well as the percentage of treated patients (53.8 % vs. 14.5 %, p < 0.001) and the prevalence of premature coronary heart disease (12.8 % vs. 3.6 %, p = 0.039). Among patients carrying only VUS, mean pre-treatment LDL-C levels were similar between the cohorts (299.5 ± 68.1 vs. 295.3 ± 46.8 mg/dL, p = 0.863). Among pathogenic/likely pathogenic variants and VUS, only 5 % and 4 % was shared between the two cohorts, respectively. ConclusionThe genetic background of patients clinically diagnosed with FH in two different countries is characterized by high variability.

Analysis of the Correlation between FH and PCSK9 and APOB Gene Mutations in Han and Mongolian Populations of the Hulunbuir.

The goal of the study was to provide an individual and precise genetic and molecular biological basis for the early prevention, diagnosis, and treatment of local FH by analyzing the risk factors for the development of FH in Han and Mongolian patients in the Hulunbuir, comparing the lipid levels of FH patients of the two ethnicities, and assessing differences in mutations to two genes between the two ethnic groups. Twenty cases each of Han Chinese and Mongolian healthy controls and fifty patients who each met the inclusion criteria from November 2021 to December 2022 in five general hospitals in Hulunbuir were selected. Multifactor logistic analysis was used to analyze the risk factors associated with the development of FH. We used t-tests to analyze statistical differences in lipid levels between the groups, and Sanger sequencing to detect the dis-tribution of common mutation sites of PCSK9 and APOB in all study subjects. The mutation rates and differences between regions and ethnic groups were summarized and compared. 1) Gender, age, alcohol consumption, dietary status, and a family history of FH were risk factors associated with the development of FH. 2) TC, LDL-C, and APOB were significantly higher in Mongolian cases than Han cases (p < 0.05). sdLDL-C was not statistically different between the two ethnicities (p > 0.05). 3) We detected four (8%) heterozygous mutations at the PCSK9 gene E670G mutation site in the Han case group and a total of nine (18%) mutations at this site in the Mongolian cases, including one (2%) homozygous and eight (16%) heterozygous mutations. One case of a heterozygous mutation was detected in the Mongolian control group. We detected a total of ten (20%) mutations at the APOB gene rs1367117 mutation site in the Han case group, including eight (16%) heterozygous and two (4%) homozygous mutations, 11 cases (22%) of heterozygous mutations in the Mongolian case group, two cases of heterozygous mutations in the Han control group, and one case of a heterozygous mutation in the Mongolian control group. 4) The D374Y and S127R mutation sites of PCSK9 and the R3500Q mutation site of APOB were not detected in any of the study subjects. The mutation sites of the PCSK9 and APOB genes in FH patients in Hulunbuir are different from other regions, and the mutation rate is higher than in other regions. Therefore, we recommend that the mutation sites of the PCSK9 and APOB genes described herein be used as clinical detection indicators to assist the diagnosis of FH in this region.

Detection of hidden cases of familial hypercholesterolemia in the Netherlands: a central laboratory data-driven strategy

Abstract Introduction Familial hypercholesterolemia (FH) is a common genetic disorder in which patients are at high risk for premature coronary artery disease (CAD) due to lifelong exposure to low-density lipoprotein cholesterol (LDL-C). Although early diagnosis and lipid lowering therapy (LLT) are essential to prevent adverse cardiovascular outcomes, FH patients remain globally underdiagnosed and undertreated, warranting novel screening methods to identify these patients. In the present study we aimed to evaluate whether existing central laboratory data can be utilized as a new method to detect undiagnosed FH patients. Additionally, we investigated the extent of underdiagnosis and undertreatment among patients with severe hypercholesterolemia in the Netherlands. Methods In this single-center, cross-sectional study, we selected individuals with at least one previously measured LDL-C plasma level ≥99.5th percentile for age and sex, from a central laboratory database containing lipid profiles of approximately 600.000 primary care patients. First, a blood sample was obtained to get more recent lipid values. Participating patients were subsequently asked to fill out a detailed questionnaire on medical and family history. Using these data, we calculated the Dutch Lipid Clinic Network (DLCN) criteria for each patient for the clinical diagnosis of FH. Results We identified a total of 5,100 patients with severe hypercholesterolemia (at least one LDL-C ≥99.5th percentile for age and sex) from the central laboratory database. Out of these, 1,206 patients (24%) participated in this study. Mean ± SD LDL-C corrected for LLT was 5.72 ± 1.83 mmol/L, 785 patients (65.1%) used LLT, and 150 patients (12.4%) had CVD in their medical history. In total, 131 patients (11%) had a persistent LDL-C ≥99.5th percentile for age and sex. DLCN criteria for phenotypic FH (DLCN score ≥6) were met by 302 patients (25%) of whom 39 patients (12.9%) had undergone prior genetic testing according to clinical guidelines. Of the 302 clinically diagnosed FH patients only 1 (0.3%) met the European Society of Cardiology (ESC) guideline recommending LDL-C targets &amp;lt;1.8 mmol/L in FH patients. Conclusion These data highlight the significant underdiagnosis and undertreatment of FH in the Netherlands, despite the fact that this country has a relatively high FH identification rate. We demonstrate that the use of central laboratory data is an effective strategy for identifying previously undetected cases of FH and eagerly await the genetic confirmation of the newly identified FH patients in this study, which will provide valuable insights into the genetic basis of this disorder and help further improve patient care.

B-252 In Vitro Expression Analysis of Variants in the Upstream Region of Genes Related to Familial Hypercholesterolemia

Abstract Background Familial hypercholesterolemia (FH) is commonly described as an autosomal dominant disorder caused mainly by variants in genes LDLR, APOB, and PCSK9, resulting in severe hypercholesterolemia. However, the substantial number of individuals who are clinically diagnosed but negative for known FH-causing variants indicates that relevant variants outside of frequently analyzed regions might exist. Considering the increasing number of promoter variants reported as likely to cause FH and the relevance of in vitro expression assays in the pathogenicity evaluation of these variants, this work aimed to examine the functionality of variants LDLR rs36218923-T and APOB rs934197-T, identified in a Brazilian FH cohort. Methods Upstream fragments of LDLR (-861 bp to +85 bp) and APOB (-1460 bp to +127 bp) were PCR amplified from genomic DNA of two FH patients carrying LDLR rs36218923-T and APOB rs934197-T in heterozygosis and homozygosis, respectively. After linearization and PGK promoter removal of the pGL4.53 vector using Kpn I and Hind III restriction enzymes, fragments were cloned upstream of the Firefly Luciferase (luc2) coding region using the NEBuilder HiFi DNA Assembly Cloning Kit. Site-directed mutagenesis was also performed using partially overlapping primers with 3´-overhangs and Platinum SuperFi II DNA Polymerase to produce different constructs, including one containing LDLR rs879254375-G, which is a known FH-causing variant. All constructs were confirmed by Sanger sequencing. pGL4.53 constructs containing either wild type or variant alleles and pNL1.1.TK (control of transfection variation, expressing NanoLuc Luciferase) were co-transfected into HepG2 cells. Wild type constructs were considered positive controls, while promoterless pGL4.53 was considered negative control. Luciferase assays were carried out 48 h after transfection using the Nano-Glo Dual-Luciferase Reporter Assay System. After normalization of luminescence units of each assay (Firefly/Nanoluc), luciferase activity was determined as the mean of four transfections with the assay performed in triplicate. Results The construct carrying LDLR rs36218923-T variant caused a significant mean reduction of luciferase activity (75,4% ± 2,9% SEM) over the promoterless construct relative to LDLR wild type construct. However, the construct harboring the LDLR rs879254375-G variant caused a more substantial reduction of luciferase activity relative to the wild type construct (91,2% ± 0,9% SEM). This reduction was consistent with shown by the previous study that reported LDLR rs879254375-G as a functional variant, which corroborates the methods adopted in this study. APOB rs934197-T was previously reported as functional based on a CAT assay using constructs of two tandemly arranged 30 bp fragments of APOB promoter, but in our study, the construct carrying the entire APOB promoter sequence and harboring rs934197-T variant did not cause a significant difference in mean luciferase activity compared to APOB wild type construct (P = 0.564). It is important to mention that our APOB upstream constructs had the additional variants rs617314-G, rs1560357-T, rs1625764-A, and rs1800481-G, which are considered benign because they occur at high frequency in the general population (0.82-1). Conclusion Our results suggest that LDLR rs36218923-T is likely to contribute to FH phenotype and emphasize the importance of performing in vitro expression assays in determining relevant upstream variants for FH molecular characterization.

Long-Term LDL-Apheresis Treatment and Dynamics of Circulating miRNAs in Patients with Severe Familial Hypercholesterolemia.

Lipoprotein apheresis (LA) is a therapeutic option for patients with severe hypercholesterolemia who have persistently elevated LDL-C levels despite attempts at drug therapy. MicroRNAs (miRNAs), important posttranscriptional gene regulators, are involved in the pathogenesis of atherosclerosis. Our study aimed to monitor the dynamics of twenty preselected circulating miRNAs in patients under long-term apheresis treatment. Plasma samples from 12 FH patients (men = 50%, age = 55.3 ± 12.2 years; mean LA overall treatment time = 13.1 ± 7.8 years) were collected before each apheresis therapy every sixth month over the course of four years of treatment. Eight complete follow-up (FU) samples were measured in each patient. Dynamic changes in the relative quantity of 6 miRNAs (miR-92a, miR-21, miR-126, miR-122, miR-26a, and miR-185; all p < 0.04) during FU were identified. Overall apheresis treatment time influenced circulating miR-146a levels (p < 0.04). In LDLR mutation homozygotes (N = 5), compared to heterozygotes (N = 7), we found higher plasma levels of miR-181, miR-126, miR-155, and miR-92a (all p < 0.03). Treatment with PCSK9 inhibitors (N = 6) affected the plasma levels of 7 miRNAs (miR-126, miR-122, miR-26a, miR-155, miR-125a, miR-92a, and miR-27a; all p < 0.04). Long-term monitoring has shown that LA in patients with severe familial hypercholesterolemia influences plasma circulating miRNAs involved in endothelial dysfunction, cholesterol homeostasis, inflammation, and plaque development. The longer the treatment using LA, the better the miRNA milieu depicting the potential cardiovascular risk.

Dual therapy with inclisiran and evolocumab in a heterozygous FH patient while on maximum statin and ezetimibe dose to achieve LDL targets

Dual therapy with inclisiran and evolocumab in a heterozygous FH patient while on maximum statin and ezetimibe dose to achieve LDL targets

In vitro assessment of the pathogenicity of the LDLR c.2160delC variant in familial hypercholesterolemia

BackgroundFamilial hypercholesterolemia (FH) is an inherited disorder with markedly elevated low-density lipoprotein cholesterol (LDL-C) and premature atherosclerotic cardiovascular disease. Although many mutations have been reported in FH, only a few have been identified as pathogenic mutations. This study aimed to confirm the pathogenicity of the LDL receptor (LDLR) c.2160delC variant in FH.MethodsIn this study, the proband and her family members were systematically investigated, and a pedigree map was drawn. High-throughput whole-exome sequencing was used to explore the variants in this family. Next, quantitative polymerase chain reaction (qPCR), western blot (WB) assays, and flow cytometry were conducted to detect the effect of the LDLR c.2160delC variant on its expression. The LDL uptake capacity and cell localization of LDLR variants were analyzed by confocal microscopy.ResultsAccording to Dutch Lipid Clinic Network (DLCN) diagnostic criteria, three FH patients were identified with the LDLR c.2160delC variant in this family. An in-silico analysis suggested that the deletion mutation at the 2160 site of LDLR causes a termination mutation. The results of qPCR and WB verified that the LDLR c.2160delC variant led to early termination of LDLR gene transcription. Furthermore, the LDLR c.2160delC variant caused LDLR to accumulate in the endoplasmic reticulum, preventing it from reaching the cell surface and internalizing LDL.ConclusionsThe LDLR c.2160delC variant is a terminating mutation that plays a pathogenic role in FH.

#4013 LIPIDOMIC ANALYSIS OF PFSGS PATIENTS' SERUM TO IDENTIFY POTENTIAL CANDIDATES FOR THE CIRCULATING PERMEABILITY FACTOR(S) IN THE LIPID DOMAIN

Abstract Background and Aims Patients with primary focal segmental glomerulosclerosis (pFSGS) present with nephrotic syndrome. Whereas the exact pathogenesis is largely unknown, there is strong evidence that one or more circulating permeability factors in the circulation induce podocyte injury. Podocyte injury eventually leads to foot process effacement, massive proteinuria and kidney function loss. Historically, research into the cause of pFSGS has focused primarily on identifying the circulating factor in the protein domain, but reports of successful treatment with lipid apheresis suggest that it could also be a lipid compound. Here, we set out to investigate the lipidome of pFSGS patients’ serum to identify potential candidates for the circulating permeability factor(s) in the lipid domain. Method For this lipidomics study, we included 5 patients with pFSGS in whom serum had been collected at the time they presented at our center with active nephrotic syndrome. We compared the serum lipidome of the pFSGS patients with age matched healthy controls (n = 5), and with age matched patients with familial hypercholesterolemia (n = 5) who, like pFSGS patients, have significantly elevated lipid values. Serum was analyzed using the Shotgun Lipidomics platform by Lipotype GmbH (Dresden, Germany), as previously described [1]. The amount of lipids were normalized to the total amount of lipids present within the sample. Results The lipidomic profile of patients with pFSGS was markedly different from the profile of healthy controls and patients with hypercholesterolemia. We found differences in various lipid classes in pFSGS patients, including cholesterol esters (CE), lyso-phophatidylcholine (LPC), phophatidylethanolamine (PE), diacylglycerol (DAG) and triacylglycerol (TAG) that were significantly changes compared to both healthy controls and FH patients. Diacylglycerols (DAG) were identified as the most significantly increased lipid species in pFSGS. By analyzing the subspecies we discovered that DAGs with carbon chains greater than 38 carbon atoms in both chains combined (long carbon chain DAGs, lccDAGs) were exclusively present in patients with pFSGS. Conclusion Our lipidomics data show that patients with pFSGS have a substantially different lipid profile than healthy controls and patients with familial hypercholesterolemia. We identified lccDAGs as unique lipids in patients with pFSGS, that were not present in controls. This is an interesting finding since the DAG-analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) is known to be able to induce podocyte injury in vitro [2]. In further research, we will focus on confirming whether lccDAGs are unique to pFSGS patients, whether it is suitable as a biomarker, and whether it plays a causal role in the disease process.

The German CaRe high registry for familial hypercholesterolemia – Sex differences, treatment strategies, and target value attainment

Background and aimsFamilial hypercholesterolemia (FH) is among the most common genetic disorders in primary care. However, only 15% or less of patients are diagnosed, and few achieve the goals for low-density lipoprotein cholesterol (LDL-C). In this analysis of the German Cascade Screening and Registry for High Cholesterol (CaRe High), we examined the status of lipid management, treatment strategies, and LDL-C goal attainment according to the ESC/EAS dyslipidemia guidelines. MethodsWe evaluated consolidated datasets from 1501 FH patients diagnosed clinically and seen either by lipid specialists or general practitioners and internists. We conducted a questionnaire survey of both the recruiting physicians and patients. ResultsAmong the 1501 patients, 86% regularly received lipid-lowering drugs. LDL-C goals were achieved by 26% and 10% of patients with atherosclerotic cardiovascular disease (ASCVD) according to the 2016 and 2019 ESC/EAS dyslipidemia guidelines, respectively. High intensity lipid-lowering was administered more often in men than in women, in patients with ASCVD, at higher LDL-C and in patients with a genetic diagnosis of FH. ConclusionsFH is under-treated in Germany compared to guideline recommendations. Male gender, genetic proof of FH, treatment by a specialist, and presence of ASCVD appear to be associated with increased treatment intensity. Achieving the LDL-C goals of the 2019 ESC/EAS dyslipidemia guidelines remains challenging if pre-treatment LDL-C is very high.

Impact of High-Density Lipoprotein Function, Rather Than High-Density Lipoprotein Cholesterol Level, on Cardiovascular Disease Among Patients With Familial Hypercholesterolemia.

Recently, the function of high-density lipoprotein (HDL), rather than the HDL cholesterol (HDL-C) level, has been attracting more attention in risk prediction for coronary artery disease (CAD). Patients with clinically diagnosed familial hypercholesterolemia (FH; n=108; male/female, 51/57) were assessed cross-sectionally. Serum cholesterol uptake capacity (CUC) levels were determined using our original cell-free assay. Linear regression was used to determine associations between CUC and clinical variables, including low-density lipoprotein cholesterol and the carotid plaque score. Multivariable logistic regression analysis was used to test factors associated with the presence of CAD. Among the 108 FH patients, 30 had CAD. CUC levels were significantly lower among patients with than without CAD (median [interquartile range] 119 [92-139] vs. 142 [121-165] arbitrary units [AU]; P=0.0004). In addition, CUC was significantly lower in patients with Achilles tendon thickness ≥9.0 mm than in those without Achilles tendon thickening (133 [110-157] vs. 142 [123-174] AU; P=0.047). Serum CUC levels were negatively correlated with the carotid plaque score (Spearman's r=0.37; P=0.00018). Serum CUC levels were significantly associated with CAD, after adjusting for other clinical variables (odds ratio=0.86, 95% CI=0.76-0.96, P=0.033), whereas HDL-C was not. HDL function, assessed by serum CUC level, rather than HDL-C level, adds risk stratification information among FH patients.