Familial Hypercholesterolemia (FH) is a monogenic autosomal dominant disease also known as Autosomal Dominant Hypercholesterolemia - ADH) that leads to dramatically increased levels of Low Density Lipoprotein (LDL) and total cholesterol associated to tendon xanthomas, xanthelasma, corneal arcus, premature atherosclerosis and to an increased risk of coronary artery disease (CAD) and myocardial infarction.

FH is mainly caused by mutations in genes encoding for proteins affecting hepatic LDL cholesterol uptake including the LDL receptor (LDLR) gene or the gene encoding the only apolipoprotein of LDL, the apolipoprotein B (APOB), or the gene encoding a protease regulating LDLR levels on the cell membrane Lysosomal Acid Lipase A (LIPA) gene encode for Lysosomal acid lipase (LAL) enzyme responsible for hydrolyzing cholesterol esters and triglycerides that are delivered to lysosomes. Mutations in LIPA that completely inactivate LAL are the molecular cause of Wolman disease, a rapidly lethal disease of infancy while mutations in LIPA that result in residual enzymatic activity of LAL are responsible of a disorder characterized by a less severe phenotype known as cholesterol ester storage disease (CESD). Patients with CESD usually show a phenotype characterized by hepatic disease and mixed hyperlipidemia with elevated levels of LDL-C and triglycerides (TG) and decreased HDL-C levels.

A broader phenotypic presentation for loss of function mutations in LIPA suggests that LIPA mutations may be considered in patients with apparently monogenic FH in whom mutations in the known candidate genes are not detectable.

The project is aimed to evaluate the prevalence and the mutation rate of LIPA gene in subjects with a clinical diagnosis of FH and already genetically characterized in whom pathogenic mutations in the known candidate genes have not been identified. The analysis will be performed in about 250 FH pediatric subjects and putative causal mutations will be also tested for co-segregation in available families in affected and unaffected members.

Lysosomal acid lipase (LAL) is encoded by LIPA gene located on chromosome 10q23.3-q23 and consists of 10 exons. LIPA mRNA (messenger RiboNucleic Acid) (GenBank accession number NM_000235) is 2782 bp long and encodes a mature protein of 375 residues (GenBank accession number NP_000226). The sequencing of all 10 exons of LIPA gene will consist of 10 PCR (Polymerase Chain Reaction) amplification reactions (for the 10 exons and the proximal promoter) followed by 20 sequence reactions (forward and reverse sequencing) with appropriate primers designed to include the intron-exon boundaries. This analysis will be performed in about 250 FH pediatric subjects as specified in project description.

The sequencing work will be performed taking advantage of 2 automated 8 capillaries automated DNA Sequencer (3500 Genetic Analyzer, Thermo Fisher Scientific, Monza, Italy) currently available in the laboratory of the Units involved in the project.

In case of identification of unreported sequence variants, the presence of these mutations will be assessed in a sample of at least 100 normolipidemic subjects of the population, in order to define whether the nucleotide changes are rare sequence variations (with a putative functional effect) or represent common polymorphisms. In case of finding of rare variants in the coding regions, an in silico analysis will be performed by using two different softwares (Polyphen, http://genetics.bwh.harvard.edu/pph/ and Panther, http://www.pantherdb.org/) to predict the putative damaging role of the mutations on the protein. In case of intronic variants, the specifically designed software Automated Splice Site Analysis will be applied (https://www.splice.uwo.ca/).

Putative causal mutations will be also tested for co-segregation in available families in affected and unaffected members.

In order to test the effect of variants on enzyme activity LAL-activity will be assayed with dried blood spot (DBS) technique using the inhibitors Lalistat 2 in carriers and non carriers of these mutations belonging to available kindred.

Inclusion Criteria:

• Pediatric subjects (<18 years old) with a clinical diagnosis of FH and without identified pathogenic mutations in the known candidate genes.

Exclusion Criteria:

• Subjects with a clinical diagnosis of FH with identified pathogenic mutations in the known candidate genes.

Clinically diagnosed FH pediatric patients (age <18 years) included in the Lipid TransPort Disorders italian Genetic Network (LIPIGEN) database, already genetically characterized.

Upon preventive request for a scientific collaboration

For three years from the end of the study

Upon preventive request for a scientific collaboration