Association of USF1 and APOA5 polymorphisms with familial combined hyperlipidemia in an Italian population.

Familial combined hyperlipidemia (FCH) is a polygenic and multifactorial disease characterized by a variable phenotype showing increased levels of triglycerides and/or cholesterol. The aim of this study was to identify single nucleotides (SNPs) in lipid-related genes associated with FCH. METHODS AND RESULTS: Twenty SNPs in lipid-related genes were studied in 142 control subjects and 165 FCH patients after excluding patients with mutations in the LDLR gene and patients with the E2/E2 genotype of APOE. In particular, we studied the 9996G > A (rs2073658) and 11235C > T (rs3737787) variants in the Upstream Stimulatory Factor 1 gene (USF1), and the -1131T > C (rs662799) and S19W (rs3135506) variants in the Apolipoprotein A-V gene (APOA5). We found that the frequencies of these variants differed between patients and controls and that are associated with different lipid profiles. At multivariate logistic regression SNP S19W in APOA5 remained significantly associated with FCH independently of age, sex, BMI, cholesterol and triglycerides. CONCLUSIONS: Our results show that the USF1 and APOA5 polymorphisms are associated with FCH and that the S19W SNP in the APOA5 gene is associated to the disease independently of total cholesterol, triglycerides and BMI. However, more extensive studies including other SNPs such as rs2516839 in USF1, are required

Rituximab retention rate in systemic sclerosis: a long term real-life multicenter study

eywords: B-cells; real-life data; retention rate; rituximab; systemic sclerosis

MOLECULAR CHARACTERIZATION AND SPECTRUM OF MUTATIONS ASSOCIATED WITH AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIA

Introduction- Familial Hypercholesterolemia (FH) is a common genetic disease that is inherited in an autosomal dominant manner. FH can be caused by mutations in the low-density lipoprotein receptor gene (LDLR) in 80% of cases, in its main ligand apolipoprotein B gene (ApoB-100) in 5% of cases, in the propotein convertase subtilin/kexin type 9 gene (PCSK9), an enzyme involved in LDLR degradation, in 1-2% of cases, while the genetic alteration is still unidentified in 15% of cases. FH leads to premature coronary heart disease(CHD), namely myocardial infarction and angina pectoris, due to elevated plasma low-density lipoprotein cholesterol (LDL-C) levels and high levels of total cholesterol (TC). About 1 in 500 individuals in the general population is affected, making it the most common monogenic form of hypercholesterolemia. Patients with homozygous alleles for defective LDLR are very rare, the frequency is about 1:106. Three scientific groups have developed diagnostic tools for FH: The US MedPed Program (Make Early Diagnosis, Prevent Early Death), the Simon Broome Register Group in the United Kingdom , and the Dutch Lipid Clinic Network (DLCNC). The major difference between these systems is the use of different cut-offs for premature CHD. The US MedPed Program based on validated age and sex adjusted for serum cholesterol cut-off points. The Simon Broome criteria take into account that TC and LDL-C levels differ for adults and children. The criteria also take into account of evidence of dominant transmission and the age of onset of coronary disease in the kindred. Using this approach, cases are categorized as ‘definite’ and ‘possible’. The DLCNC score is based on a family history of hypercholesterolemia, premature CHD, clinical features, LDL-C levels and DNA analysis. It uses a numeric score for each criterion, and classifies individuals as “definite”, “probable” or “possible” FH. A diagnosis is considered definite if the score is greater than 8, considered probable if the score is between 6 and 8 points, and considered possible when the score is between 3 and 5 points. Aims- To determine the frequency and spectrum of mutations causing FH in patients attending the different Italian lipid clinics. To investigate genotype-phenotype correlations in FH carrying different mutations of the LDL-R gene. To identify the presence of a likely polygenic cause due to the inheritance of LDL-C-raising SNPs. In addition, to evaluate which of diagnostic criteria DCLN or Simon Broome is more accurate to detect patients with mutations in our population. Results- Mutations were found in 214/322 subjects with a mutation rate of 66%. Out of 214 mutated subjects about 95.3% were carriers of LDLR mutations, 2.8% of APOB mutations and 1.8% of PCSK9 mutations. In the LDLR gene we found 58 different mutations of which 5 were novels (c.102C>A; c.892A>G; c.1277T>G; c.694+1G>C c.1070_1071dupAG). Regarding the APOB gene we identified four different mutations of which two novels (p.Val3306Ile and p.Trp3633Arg ). In the PCSK9 gene we found four different variations of which two novels (p.Pro331Ala, p.Arg499Hys). The gradually increase of the TC and LDL-C among patients with different types of mutation shows that the type of the LDLR mutation influences the lipid profile. Patients with radical mutations show a worse lipid profile than missense carriers allowing a prognostic evaluation for physicians. In a proportion of patients with the FH phenotype but without mutations in the main candidate genes, there is a likely polygenic cause due to the inheritance of LDL-C-raising SNPs which increases LDL-C concentration in patients. In addition, we examined the reliability of the Dutch Lipid Clinic Network (DCLN) score and Simon Broome criteria, to identify patients with a high or low probability of carrying an FH-causing mutation. The DCLN criteria was more accurate than Simon Broom showing very high sensitivity and specificity to detect patients with mutations. Conclusions- The results of this study represent an update of FH genetic background in an Italian population of patients from southern regions. These data enlarge the spectrum of mutations causing FH. The correlation between mutation types and lipid profile underlines importance of genetic screening as a prognostic tool. The comparison of the Dutch Lipid Clinic Network (DCLN) score and Simon Broome revealed that genetic screening is also useful to confirm the diagnosis, especially in patients with an uncertain phenotype

Functional characterization of mutant genes associated with autosomal dominant familial hypercholesterolemia: Integration and evolution of genetic diagnosis

Familial Hypercholesterolemia (FH) is one of the most frequent dyslipidemias, the autosomal dominant form of which is primarily caused by mutations in the LDL receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes, although in around 20% of patients the genetic cause remains unidentified. Genetic testing has notably improved the identification of patients suffering from FH, the most frequent cause of which is the presence of mutations in the LDLR gene. Although more than 1200 different mutations have been identified in this gene, about 80% are recognized to be pathogenic. We aim to overview the current methods used to perform the functional characterization of mutations causing FH and to highlight the conditions requiring a functional characterization of the variant in order to obtain a diagnostic report

Identification and functional characterization of LDLR mutations in familial hypercholesterolemia patients from Southern Italy

OBJECTIVE: Autosomal dominant hypercholesterolemias are due to defects in the LDL receptor (LDLR) gene, in the apolipoprotein B-100 gene or in the proprotein convertase subtilisin/kexin type 9 gene. The aim of this study was to identify and functionally characterize mutations in the LDLR gene that account for most cases of familial hypercholesterolemia (FH). METHODS: We enrolled 56 unrelated patients from Southern Italy with a clinical diagnosis of FH. The mutation screening was performed by direct sequencing of the promoter and the 18 exons of the LDLR gene and by multiplex ligation-dependent probe amplification (MLPA) analysis to search for large rearrangements. RESULTS AND CONCLUSION: We found 5 new mutations, the causative role of which was demonstrated by functional characterization performed by quantification of fluorescent LDL uptake in EBV-transformed B lymphocytes. These results enlarge the spectrum of FH-causative LDLR mutations. Lastly, screening for large rearrangements is highly recommended for the genetic diagnosis of FH

Refinement of Variant Selection for the LDL Cholesterol Genetic Risk Score in the Diagnosis of the Polygenic Form of Clinical Familial Hypercholesterolemia and Replication in Samples from 6 Countries.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12-single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples. METHODS: We used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M-), those with a mutation (FH/M+), and controls from a UK population sample (WHII). RESULTS: Increasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M- and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M- cohorts showed a consistently higher score in comparison to the WHII population (P 95% likelihood of a polygenic explanation of their increased LDL-C. CONCLUSIONS: A 6-SNP LDL-C score consistently distinguishes FH/M- patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis