Mutation Report for: L294S_human-LIPA

Hydrolysis of cholesteryl esters and triglycerides in the lysosome is performed by lysosomal acid lipase (LAL). In this study we have investigated how 23 previously identified missense mutations in the LAL gene (LIPA) (OMIM# 613497) affect the structure of the protein and thereby disrupt LAL activity. Moreover, we have performed transfection studies to study intracellular transport of the 23 mutants. Our main finding was that most pathogenic mutations result in defective enzyme activity by affecting the normal folding of LAL. Whereas, most of the mutations leading to reduced stability of the cap domain did not alter intracellular transport, nearly all mutations that affect the stability of the core domain gave rise to a protein that was not efficiently transported from the endoplasmic reticulum (ER) to the Golgi apparatus. As a consequence, ER stress was generated that is assumed to result in ER-associated degradation of the mutant proteins. The two LAL mutants Q85K and S289C were selected to study whether secretion-defective mutants could be rescued from ER-associated degradation by the use of chemical chaperones. Of the five chemical chaperones tested, only the proteasomal inhibitor MG132 markedly increased the amount of mutant LAL secreted. However, essentially no increased enzymatic activity was observed in the media. These data indicate that the use of chemical chaperones to promote the exit of folding-defective LAL mutants from the ER, may not have a great therapeutic potential as long as these mutants appear to remain enzymatically inactive.

Lysosomal acid lipase hydrolyzes cholesteryl esters and triglycerides contained in low density lipoprotein. Patients who are homozygous or compound heterozygous for mutations in the lysosomal acid lipase gene (LIPA), and have some residual enzymatic activity, have cholesteryl ester storage disease. One of the clinical features of this disease is hypercholesterolemia. Thus, patients with hypercholesterolemia who do not carry a mutation as a cause of autosomal dominant hypercholesterolemia, may actually have cholesteryl ester storage disease. In this study we have performed DNA sequencing of LIPA in 3027 hypercholesterolemic patients who did not carry a mutation as a cause of autosomal dominant hypercholesterolemia. Functional analyses of possibly pathogenic mutations and of all mutations in LIPA listed in The Human Genome Mutation Database were performed to determine the pathogenicity of these mutations. For these studies, HeLa T-REx cells were transiently transfected with mutant LIPA plasmids and Western blot analysis of cell lysates was performed to determine if the mutants were synthesized in a normal fashion. The enzymatic activity of the mutants was determined in lysates of the transfected cells using 4-methylumbelliferone-palmitate as the substrate. A total of 41 mutations in LIPA were studied, of which 32 mutations were considered pathogenic by having an enzymatic activity <10% of normal. However, none of the 3027 hypercholesterolemic patients were homozygous or compound heterozygous for a pathogenic mutation. Thus, cholesteryl ester storage disease must be a very rare cause of hypercholesterolemia in Norway.

BACKGROUND AND AIMS: Childhood/Adult-onset Lysosomal Acid Lipase Deficiency (LAL-D) is a recessive disorder due to loss of function variants of LAL, the enzyme which hydrolyses cholesteryl esters, derived from internalized apoB containing lipoproteins. The disease is characterized by multi-organ involvement including the liver, spleen, intestine and cardiovascular system. The aim of this study was the clinical and molecular characterization of 14 (13 unrelated) previously unreported patients with childhood-onset LAL-D.
METHODS: Data collected included clinical and laboratory investigations, liver imaging, liver biopsy and LIPA gene analysis. The response to lipid-lowering medications, liver transplantation and enzyme replacement therapy (ERT) was reported for some patients.
RESULTS: LAL-D was suspected at 4.4 +/- 3.3 years of age for the presence of hepatomegaly, elevated serum transaminases and hypercholesterolemia, and was confirmed by liver biopsy/imaging and LAL assay. The follow up period ranged from 3 to 40 years (mean 7.8 +/- 4.0 years in 13 cases). Patients treated with statins with or without ezetimibe showed 28% reduction of plasma LDL-cholesterol without a tangible effect on liver enzymes; some patients receiving ERT showed normalized lipoprotein profile and transaminase levels. The common c.894G > A variant was observed in homozygosity or compound heterozygosity in 10 patients. We found seven previously reported variants: p.(Trp140*), p.(Arg218*), p.(Gly266*), p.(Thr288Ile), p.(Leu294Ser), p.(His295Tyr) and p.(Gly342Arg) and two novel variants: p.(Asp345Asn), affecting the LAL catalytic triad, and c.229+3A > C, affecting splicing. Homozygosity for p.(Thr288Ile) or c.229+3A > C was associated with a severe phenotype.
CONCLUSIONS: This study provides additional data on the features of childhood-onset LAL-D and describes two novel pathogenic variants of the LIPA gene.

Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G3' splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (delta 205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5' splice site mutation (G to A) that leads to skipping of exon 8 (delta 254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an Xbal restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and delta 254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.