Study Group Characteristics
Two patients with familial LCAT deficiency (FLD) and 2 patients with fish-eye disease (FED) participated. One patient with FED was homozygous for the p.T123I mutation34
(which is now annotated p.T147I, including the 24-aa leader sequence, following the guidelines of the Human Genome Variation Society to describe mutations), the other compound heterozygous for p.T147I and p.V333M.35
The patients with FLD were homozygous for either p.C337Y36
In addition, 63 heterozygous carriers of LCAT gene mutations and 63 unaffected family controls participated in the study. Twenty heterozygotes carried a mutation known to cause FLD when present on both alleles; 39 heterozygotes carried a mutation known to cause FED when present on both alleles; finally, 4 heterozygotes carried point mutations of which it is unknown whether they cause FED or FLD when present on both alleles (no homozygous patients have been described to help in this regard). Table I in the online-only Data Supplement
gives an overview of the exact molecular LCAT defects in the heterozygotes.
summarizes the demographic, lifestyle, and clinical characteristics of cases and controls. Age, body mass index, alcohol use, and the percentage of smokers were similar in heterozygotes and family controls. There were more men among the heterozygotes, but this did not reach statistical significance. Heterozygotes experienced more cardiovascular disease (P<0.01) concordant with a more frequent use of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) compared with controls (P<0.001). HDL-c and apoA-I were gene-dose–dependently decreased in carriers of LCAT mutations, whereas levels of LDL-c, apoB, and tri-glycerides did not differ between the groups. The gene-dose–dependent decrease in total cholesterol could therefore be attributed to the decrease in HDL-c. Carriers of 2 defective LCAT alleles presented with severe HDL-c deficiency and severely reduced Lp(a).
Clinical Characteristics, Lipids, and Lipoproteins
LCAT, PON1, and PAF-AH Activities
To study the role of LCAT in lipid oxidation in humans, we studied all HDL-related enzymes with an antioxidative function comparable with LCAT (ie, hydrolysis of OxPL). LCAT7,16,17
are well known in this respect; for PON1, this has been strongly suggested.38,39
shows a highly significant gene-dose–dependent decrease of plasma LCAT and HDL-associated PAF-AH activities in carriers of LCAT mutations (for both, P for ANOVA <0.001). HDL-associated PON1 activity levels were similar in heterozygotes and controls, whereas carriers of 2 defective LCAT alleles showed a 85% reduction in PON1 activity (P<0.001).
LCAT, PAF-AH, and PON1 Activities
LysoPC Molecular Species
LCAT, via its PLA2 activity, hydrolyzes phospholipids removing sn-2 fatty acyl groups for subsequent esterification (via acyltransferase activity) to cholesterol and releasing lysoPC. As a measure of LCAT PLA2 activity, we quantified individual molecular species of lysoPC. shows slightly lower concentrations of all molecular species of lysoPC in the heterozygotes compared with controls, but this did not reach significance for any of the individual lysoPC species. In the homozygotes/compound heterozygotes however, all molecular species of lysoPC were significantly reduced (average reduction 58%) compared with controls.
Lysophosphatidylcholine Molecular Species
AA and LA and Their Oxidized Derivatives
Loss of LCAT activity was associated with decreased total levels of essential fatty acids (free plus esterified; ): total plasma levels of AA were gene-dependently decreased in carriers of LCAT mutations (P for ANOVA=0.049). Total plasma levels of LA were also significantly decreased in heterozygotes compared with controls (P=0.007; P=0.02 after adjustment for age and sex). Carriers of 2 defective alleles, however, showed similar levels of LA as the heterozygotes.
Arachidonic and Linoleic Acids and Their Oxidized Derivatives
Measuring oxidation products of AA, we found that all HETEs (ie, 5-,8-,9-, 11-,12-,15-HETEs) were significantly reduced in carriers of 2 defective LCAT alleles compared with controls. The 8-, 9-, 11-, and 12-HETEs levels were also significantly reduced when comparing heterozygotes and controls, and for these parameters we also observed a gene-dose effect (ANOVA: P=0.008, P=0.024, P=0.006, P=0.005, respectively). Among the multiple LA oxidation products monitored, we found no significant differences in the LA oxidation products 9- and 13-HODEs when comparing the carriers of LCAT mutations and controls.
Because absolute fatty acid levels were decreased, we calculated the ratio of oxidized to total fatty acids. Table II in the online-only Data Supplement
shows that ratios of HETEs/ AA and HODEs/LA are not different in heterozygotes and controls. In carriers of 2 defective LCAT alleles, 9-, 11-, and 12-HETEs/AA ratios were significantly lower compared with controls.
OxPL/ApoB and OxPL/Apo(a), ApoB-Immune Complexes, and Autoantibodies Against MDA-LDL
OxPL/apoB were significantly increased in heterozygotes compared with family controls (P=0.01; ) and remained significantly increased (P=0.017) after correction for age and sex. This difference also retained significance after exclusion of participants who had experienced atherosclerotic cardiovascular disease (7 heterozygotes and 1 control; P=0.030). In a linear regression model with only PAF-AH activity as a potential confounder, the difference also retained significance (P=0.049). However, significance was lost in a linear regression model including age, sex, and PAF-AH activity (P=0.125). In carriers of 2 defective LCAT alleles, OxPL/apoB were similar to controls.
(Auto-)Antibodies to Oxidized Phospholipids and Apolipoproteins
In heterozygotes, OxPL/apo(a) were similar to controls (P=0.68) but were markedly decreased in carriers of 2 defective LCAT alleles (87% reduction; P<0.001).
We also measured IgG and IgM immune complexes on apoB-containing lipoproteins and IgG and IgM titers against MDA-LDL (). Of these 4 parameters, 1 parameter was significantly different among the groups: IgM immune complexes on apoB-containing lipoproteins were significantly decreased in heterozygotes compared with controls (P=0.03).
Antioxidant Capacity of HDL
The capacity of HDL to inhibit oxidized LDL from forming DCF (cell-free assay)22
was gene-dose–dependently decreased. The fluorescence intensity reflecting the potential of HDL to inhibit the action of OxLDL in this assay was 1.3×105
arbitrary units in carriers of 2 mutant alleles, 3.0×104
arbitrary units in heterozygotes, and 2.3×104
arbitrary units in controls (; P
<0.001 for all comparisons including ANOVA). Multivariate regression analysis shows that this difference retained significance after adjustments for age, sex, body mass index, HDL-c, and statin use (P
<0.001). Additional statistical corrections for PON1 and PAF-AH activities in HDL did not change this result either (P
<0.001). After exclusion of participants who had experienced atherosclerotic cardiovascular disease, the antioxidative capacity of HDL also remained significantly lower in heterozygous carriers of LCAT gene mutations compared with controls.
Antioxidative Capacity of HDL