Mutation Report for: G342R_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.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Mechanisms producing the divergent phenotypes, Wolman disease (WD) and cholesterol ester storage disease (CESD), associated with the genetic deficiency of human lysosomal acid lipase/cholesterol ester hydrolase (hLAL) function were investigated with the determination of HLAL activity levels, mRNA and protein expression, and defects in structural gene sequences in cells from three WD and five CESD patients. Measured with natural substrates, HLAL activities were all below 2% of normal, regardless of phenotype. Immunoblotting showed a lack of detectable hLAL protein in all mutant fibroblasts. Four CESD, but no WD genomes contained at least one allele with a specific exon 8 splice junction mutation, c.894 G>A, that encodes a shortened form of hLAL mRNA. Other CESD mutations were identical in type to the WD defects: nucleotide deletions (positions 397, 684, 980), insertions (594), or substitutions (193, 347) that result in premature terminations precluding any function. The only exception was a substitution at nucleotide 866 in the CESD case without an exon 8 splicing mutation; expression of the predicted S289C change in a transfection assay produced a low, but clearly measurable, level of acid esterase activity. Although it is not easily demonstrated in conventional assays, CESD is distinct from WD in that at least one mutant allele has the potential to produce enough residual enzymatic function to ameliorate the phenotype; in the majority of CESD cases this may come from a single, easily detected, splicing mutation in one allele.