Journal of Clinical Medicine Article Lipid Profile Rather Than the LCAT Mutation Explains Renal Disease in Familial LCAT Deficiency Itziar Lamiquiz-Moneo 1 , Fernando Civeira 1,2, * , Diego Gómez-Coronado 3 , Francisco Blanco-Vaca 4 , Hilda Mercedes Villafuerte-Ledesma 5 , Miriam Gil 6 , Nuria Amigó 6,7,8 , Rocío Mateo-Gallego 1,9 and Ana Cenarro 1 1 Lipid Unit, Hospital Universitario Miguel Servet, Instituto de Investigación Sanitaria Aragón (IIS Aragón), CIBER Cardiovascular (CIBERCV), 50009 Zaragoza, Spain; itziarlamiquiz@gmail.com (I.L.-M.); rmateo@unizar.es (R.M.-G.); ana.cenarro@gmail.com (A.C.) 2 Department of Internal Medicine, Psychiatry and Dermatology, Universidad de Zaragoza, 50009 Zaragoza, Spain 3 Department of Biochemistry-Research, Hospital Universitario Ramón y Cajal, IRYCIS, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), 28034 Madrid, Spain; diego.gomez@hrc.es 4 Hospital de la Santa Creu i Sant Pau, Servicio de Bioquímica—IIB Sant Pau, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Departament of Biochemistry and Molecular Biology, Universidad Autónoma de Barcelona, 08041 Barcelona, Spain; FBlancoVa@santpau.cat 5 Nephrology Department, Hospital Clínico Universitario Lozano Blesa, IIS Aragón, 50009 Zaragoza, Spain; hilda.villafuerte21@gmail.com 6 Biosfer Teslab, SL, 43201 Reus, Spain; mgil@biosferteslab.com (M.G.); nuriaamigo@gmail.com (N.A.) 7 Metabolomics Platform, Universidad Rovira i Virgili (URV), Instituto de Investigación Sanitaria Pere Virigili (IISPV), 43003 Tarragona, Spain 8 CIBER de diabetes y enfermedades metabólicas asociadas (CIBERDEM), 28029 Madrid, Spain 9 Department of Physiatry and Nursing, Universidad de Zaragoza, 50009 Zaragoza, Spain * Correspondence: civeira@unizar.es; Tel.: +34-976-765-500 (ext. 142895)  Received: 8 October 2019; Accepted: 30 October 2019; Published: 3 November 2019  Abstract: Renal complications are the major cause of morbidity and mortality in patients with familial lecithin–cholesterol acyltransferase (LCAT) deficiency (FLD). We report three FLD patients, two of them siblings—only one of whom developed renal disease—and the third case being a young man with early renal disease. The aim of this study was to analyze the clinical characteristics and possible mechanisms associated with renal disease in these patients. Plasma lipid levels, LCAT activity, lipoprotein particle profile by NMR and FPLC, free and esterified cholesterol, presence of lipoprotein X (LpX) and DNA sequencing in the three FLD patients have been determined. The three cases presented clinical characteristics of FLD, although only one of the siblings developed renal disease, at 45 years of age, while the other patient developed the disease in his youth. Genetic analysis revealed new missense homozygous mutations, p.(Ile202Thr) in both siblings and p.(Arg171Glu) in the other patient. Lipoprotein particle analysis showed that the two patients with renal disease presented higher numbers of small very low-density lipoprotein (VLDL) and a higher concentration of triglycerides in VLDL. This study reports three new cases of LCAT deficiency, not previously described. Renal disease is not only dependent on LCAT deficiency, and could be due to the presence of VLDL particles, which are rich in triglycerides, free cholesterol and LpX. Keywords: familial LCAT deficiency; fish eye disease; VLDL; NMR; FPLC; triglycerides; phospholipids J. Clin. Med. 2019, 8, 1860; doi:10.3390/jcm8111860 www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, 1860 2 of 14 1. Introduction Lecithin cholesterol acyltransferase (LCAT) is a lipoprotein-associated enzyme, which plays a central role in the esterification of free cholesterol (FC) in plasma, in the formation of mature high density-lipoprotein (HDL) particles, and in the intravascular stage of reverse cholesterol transport [1,2]. Human LCAT is a glycoprotein of 416 amino acids, encoded by a gene located on chromosome 16q21-22, which esterifies the majority of the free cholesterol located at the surface of lipoprotein particles, by catalyzing the translocation of fatty acid moiety in sn-2 position of lecithin to the free 3-OH group of cholesterol [3]. LCAT deficiency is a rare autosomal recessive disease, with prevalence below 1:1,000,000. Nearly 100 different mutations in the LCAT gene have been identified so far [4]. Mutations in both alleles of LCAT gene may lead to two syndromes, namely familial LCAT deficiency (FLD) and fish eye disease (FED). Both syndromes share several abnormalities in lipoproteins, including a decrease in HDL cholesterol level, apo A-I and apo A-II, a decrease in LDL cholesterol level and an increase in FC and apo E [2,5]. In FLD, the LCAT enzyme is either absent in plasma, or exhibits no catalytic activity on any lipoprotein [6]. However, FED is characterized by a partial enzyme deficiency, in which LCAT does not esterify cholesterol in HDL, but retains some activity on lipoproteins containing apo B (VLDL and LDL) [7]. Individuals with FED have very low levels of HDL cholesterol and corneal opacities [3,8]. In FLD, free cholesterol accumulates in all plasma lipoproteins, and patients present HDL cholesterol deficiency, corneal opacification, hemolytic anemia, hypertension, hypertriglyceridemia and proteinuria, frequently progressing to end stage renal disease [5]. Renal involvement is the major cause of morbidity and mortality in FLD patients. It usually starts as proteinuria and progresses to renal disease. The cause of renal disease in FLD is not well understood, but has been attributed to the deposition of FC-rich lipoproteins, called Lipoprotein X (LpX), without hydrophobic core lipids particles, in the glomeruli and renal arterioles [9,10]. FC deposits appear as small vacuolations of various sizes on the epimembranous, intramembranous, and subendothelial spaces of the glomerular basement membrane and in the mesangium [5]. These deposits are suggested to be the main mechanism behind the development of renal disease [2,11]. We have had the opportunity to study three patients with FLD, two of them siblings sharing the same LCAT mutation. Interestingly, despite the identical genotype present in both siblings, only one developed renal disease. The third case studied was a young man with early renal disease. The aim of this study was to analyze the clinical characteristics of, and possible mechanisms associated with, the phenotype differences in these patients. 2. Materials and Methods 2.1. Subjects We present three cases, two of them siblings, with LCAT deficiency, attending our Lipid Unit, who were referred for extremely low levels of HDL cholesterol. The Lipid Unit is located in the Hospital Universitario Miguel Servet, which is the reference center for disorders of lipid metabolism in the Aragon region in Spain, which has approximately 1,300,000 habitants. 2.2. Lipid Analysis Lipid and lipoprotein analyses were performed on Ethylenediaminetetraacetic acid (EDTA) plasma samples collected after at least a 10 h overnight fast, without intake of lipid lowering drugs in at least 6 weeks. Total cholesterol and triglycerides (TG) levels were determined by standard enzymatic methods. High density lipoprotein cholesterol (HDL cholesterol) was measured by an enzymatic reaction using cholesterol oxidase (UniCel DxC 800; Beckman Coulter, Inc, Brea, CA). Lipoprotein(a), apo A-I, apo B, and C-reactive protein were determined by IMMAGE kinetic nephelometry (Beckman-Coulter, Inc). LDL cholesterol was calculated by Friedewald’s formula. All subjects signed an informed consent J. Clin. Med. 2019, 8, 1860 3 of 14 form, to a protocol previously approved by our local ethics committee (Comité Ético de Investigación Clínica of Aragon, PT13/0010/0017, Zaragoza). 2.3. Plasma LCAT Activity Plasma LCAT activity was determined using reassembled HDL, containing 1-palmitoyl-2-oleyl-sn-3-phosphocholine, [3 H] cholesterol, and apo A-I. Endogenous LCAT activity was measured using the lipoproteins of whole and apo B-depleted plasma as substrate, and expressed as fractional esterification rate (FER) [12]. 2.4. Genetic Analysis Whole blood genomic DNA was isolated using standard methods. Promoters, coding regions, and intron-exon boundaries of LCAT (NM_012108.3) were amplified by polymerase chain reaction and purified by ExoSap-IT (USB). Amplified fragments were sequenced by Sanger method, using the BigDye 3.1 sequencing kit (Applied Biosystems) in an automated ABI 3500xL sequencer (Applied Biosystems). DNA sequences were analyzed using Variant Reporter software (Applied Biosystems). The genetic analysis was extended to all available family members of the siblings (Figure S1). 2.5. Lipoprotein Particle Analysis Lipoprotein particle concentrations were measured by NMR, based on the LipoScale test ® . The lipid concentration (i.e., triglycerides and cholesterol) and the mean size of VLDL, LDL and HDL, as well as the particle number of nine subclasses (large, medium and small VLDL, LDL and HDL) were determined as previously reported [13]. Briefly, particle concentration and diffusion coefficients were obtained from the measured amplitudes, as well as attenuation of their spectroscopically distinct lipid methyl group NMR signals, using the 2D diffusion-ordered 1H NMR spectrometry (DSTE) pulse. The methyl signal was surface-fitted with nine lorentzian functions associated with each lipoprotein subclass—large, medium and small. The area of each lorentzian function was related to the lipid concentration of each lipoprotein subclass, and the size was calculated from their diffusion coefficient. Each subclass particle concentration was calculated by dividing the lipid volume by the particle volume of a given class. Lipid volumes were determined by using common conversion factors to convert concentration units into volume units. The weighted average of VLDL, LDL and HDL particle sizes were calculated from various subclass concentrations by summing the known diameter of each subclass, multiplied by its relative percentage of subclass particle number. To assess similarities and differences among LCAT deficiency patients in terms of their lipoprotein profile, we used a previously analyzed reference population from Biosfer Teslab data base, including 6000 subjects from the general population, of men and women of different ages (between 15 and 85 years old), and comparing the study cases with subjects with a normal profile of NMR lipoprotein and low HDL cholesterol (<35 mg/dL). Firstly, we applied a Principal Component Analysis (PCA) model, using the lipoprotein composition, size and the subclass’s particle number as input variables. Then, we applied a Partial Least Square Discriminant Analysis (PLS-DA) model (see below) to optimize the separation between LCAT deficiency patients and the general population, after a variable selection process, by using genetic algorithm methodologies [14]. 2.6. Gel Filtration Fast Protein Liquid Chromatography (FPLC) Two hundred and fifty µl of fresh plasma were injected into a Superose 6 HR 10/30 column (Pharmacia) equilibrated with TBS containing 0.02% NaN3 . Chromatography was performed at a flow rate of 0.3 mL/min and, after elution of the first 5 mL, fractions of 0.5 mL were collected. Cholesterol, triglyceride and choline-containing phospholipid concentrations were measured in the collected fractions by enzymatic methods. A portion of selected fractions was concentrated three-fold by using appropriate centrifugal concentrators (MW cut off 10000 Ultrafree-MC, Millipore). Equal aliquots of J. Clin. Med. 2019, 8, 1860 4 of 14 the concentrates were subjected to agarose gel electrophoresis and staining for lipids or, alternatively, anti-apo A-I or anti-apo A-II immunoblotting. 2.7. LpX Determination Presence of LpX was analyzed as reported by Freeman et al [15]. Briefly, 10 µL of fresh EDTA-plasma (not frozen) or 10 µL synthetic LpX were loaded on Sebia Hydragel 7 lipoprotein(e) gels (#4134; Sebia, Inc., Norcross, GA). Syntethic LpX was prepared by mixing 6.1 mg L-α-lecithin (Calbiochem, Millipore) with 1.06 mg cholesterol (Sigma–Aldrich, Spain) from their stock solutions in chloroform, and then drying the lipid mixtures under nitrogen. Two mL of normal saline were added, and the mixture was vortexed for 10 min and sonicated for 10 min to generate multilamellar particles enriched in free cholesterol and phospholipid, in the size range of LpX. Sequential dilutions of LpX were loaded onto the gel. Electrophoresis was for 30 min at 100 V in a Hydrasys 2 System (Sebia). Gels were fixed with 10% trichloroacetic acid and incubated with Filipin Stain to detect free cholesterol. Filipin Stain was prepared fresh each time by dissolving 12.5 mg Filipin (Sigma–Aldrich, Spain) in 0.5 mL N, N’ dimethyl formamide, and then adding 50 mL 1XPBS containing 0.1% sodium azide and mixing thoroughly. Gels were incubated overnight in 50 mL Filipin Stain at 4 ◦ C with gentle rocking, shielded from light, and then washed for 20 min at room temp with 50 mL 1XPBS. Imaging was carried out with a UV transilluminator. We estimated LpX concentration through the measurement of total and free cholesterol. Lipids were extracted from five serum samples (three controls and two LCAT deficiency patients) using a slightly modified BUME method [16], replacing heptane with diisopropyl ether. Dried lipid extracts were reconstituted with CDCl3 -CD3 OD-D2 O (16:7:1), containing 1.18 mM tetramethylsilane (TMS) and transferred to 5 mm NMR glass tubes for analysis. One dimensional 1 H-NMR spectra were obtained, using a 90◦ pulse with pre-saturation sequence (zgpr). Spectra were phased, baseline corrected, and referenced (TMS 0 ppm), and then selected peaks (free and esterified cholesterol) were deconvoluted using LipSpin software [17]. 2.8. Statistical Analysis Multivariate statistical analyses were computed in MATLAB (Eigenvector Research Inc., Manson, WA, USA). PLS-DA was used as a supervised classification method for LCAT deficiency identification among (i) a general population and ii) individuals with low HDL cholesterol levels. PLS-DA relates the X matrix (experimental data) and the Y matrix (classes of samples), in order to find the maximum discrimination between classes and the maximum covariance between the X and Y matrices simultaneously [18]. 3. Results The first patient was referred to our Lipid Unit for an extremely low plasma concentration of HDL cholesterol, corneal opacities and progressive renal disease when he was 65 years old. He reported low plasma levels of total cholesterol, HDL cholesterol, moderately elevated TG and proteinuria since his mid-forties. His lipid values at that time are reported in Table 1. He developed progressive renal disease requiring hemodialysis at the age of 75 and he died 12 years later. The second patient, sister of the first patient, was a 78-year-old female, that was also referred to our Lipid Unit for an extremely low plasma concentration of HDL cholesterol in a routine analysis. This patient reported that she had suffered from corneal opacity during the last 30 years. Currently, she is 84 years old, asymptomatic, and her renal function remains within normal values, including urinalysis without proteinuria. Physical examination showed corneal opacity (Figure 1A). Her blood lipid values confirmed severe hypoalphalipoproteinemia (Table 1). The third patient was a 38-year-old male who had suffered from corneal opacity, low levels of HDL cholesterol and abnormal results of urinalysis from the age of 20. Physical examination showed corneal opacity (Figure 1B). Urinalysis revealed proteinuria, and blood analysis showed mild impaired renal function. The renal biopsy specimen revealed a thickening J. Clin. Med. 2019, 8, 1860 5 of 14 of capillary walls and the presence of foamy lipid deposits at the capillary and mesangial levels (Figure 2).The cholesterol esterification rate in serum, as a measure of endogenous LCAT activity, in all cases, was nearly absent, as compared with control values (1.70 nmol/h*mL, 0.05 nmol/h*mL and 1.35 nmol/h*mL, respectively). All patients reported null endogenous LCAT activity, with an FER of 1.40%, 1.5% and 1.9% per hour, respectively, below the minimum reference value (value reference: >3.5% by hour) (Table 1). Table 1. Lipid profile and lecithin cholesterol acyltransferase (LCAT) activity in all cases. Reference First Case Second Case Third Case Values Currently age, years 88 84 39 NA Age at diagnosis, years 65 78 38 NA BMI, kg/m2 25.3 24.7 24.2 18.5–25 Total cholesterol, mg/dL 119 147 187 150–219 Triglycerides, mg/dL 245 158 181 30–149 Non-HDL cholesterol, mg/dL 112 133 174 100–160 HDL cholesterol mg/dL 7 14 13 40–60 Apoliprotein J. Clin. Med. A1, mg/dL 2019, 8, 1860 66 51.3 30 119–155 3 of 14 Apolipoprotein B, mg/dL 96 131 35 73–109 Plasma LCAT activity was determined using reassembled HDL, containing 1-palmitoyl-2-oleyl- Creatinine, mg/dL 5.35 0.88 2.07 0.51–0.95 sn-3-phosphocholine, [3H] cholesterol, and apo A-I. Endogenous LCAT activity was measured using Proteinuria, g/L 1.95 Negative 0.77 0.0–0.15 the lipoproteins of whole and apo B-depleted plasma as substrate, and expressed as fractional Renal disease esterification rate (FER) [12]. Yes No Yes NA c.605T > C c.605T > C c.512G > A LCAT genetic analysis NA 2.4. Genetic Analysis p.(Ile202Thr) p.(Ile202Thr) p.(Arg171Glu) Exogenous LCAT activity Whole blood genomic DNA was isolated 0.05 using standard 1.7 methods. Promoters, 1.35 coding regions, (23.0–57.0) (nmol/h*mL) and intron-exon boundaries of LCAT (NM_012108.3) were amplified by polymerase chain reaction Endogenous LCAT activity and purified by ExoSap-IT (USB). Amplified 1.50 fragments were1.40 sequenced by1.90 >3.50% Sanger method, using the (Fractional esterification rate, %/h) BigDye 3.1 sequencing kit (Applied Biosystems) in an automated ABI 3500xL sequencer (Applied BMI: Body Mass Index; NA: Not Applicable. Biosystems). DNA sequences were analyzed using Variant Reporter software (Applied Biosystems). The genetic analysis was extended to all available family members of the siblings (Figure S1). Figure 1. Figure Cornealopacity 1. Corneal opacityininFLD FLDpatients. patients.Corneal Corneal opacity opacity from from second second casecase (A) (A) andand third third casecase (B) (B) are are shown. shown. 2.5. Lipoprotein Particle Analysis Lipoprotein particle concentrations were measured by NMR, based on the LipoScale test ®. The lipid concentration (i.e., triglycerides and cholesterol) and the mean size of VLDL, LDL and HDL, as well as the particle number of nine subclasses (large, medium and small VLDL, LDL and HDL) were determined as previously reported [13]. Briefly, particle concentration and diffusion coefficients were obtained from the measured amplitudes, as well as attenuation of their spectroscopically distinct lipid methyl group NMR signals, using the 2D diffusion-ordered 1H NMR spectrometry (DSTE) Renal disease Yes No Yes NA c.605T > C c.605T > C c.512G > A LCAT genetic analysis NA p.(Ile202Thr) p.(Ile202Thr) p.(Arg171Glu) Exogenous LCAT activity (nmol/h*mL) 0.05 1.7 1.35 (23.0–57.0) Endogenous LCAT activity (Fractional 1.50 1.40 1.90 >3.50% esterification rate, %/h) J. Clin. Med. 2019, 8, 1860 6 of 14 BMI: Body Mass Index; NA: Not Applicable. Figure 2. Histological analysis of renal biopsy. (A) shows panoramic of the renal tissue cylinder studied; (B) shows detail of glomerular involvement, with expansion of the mesangium; (C and D) show details of thickening of capillary walls, and presence of foamy lipid deposits at capillary and mesangial level. Genetic analysis of both siblings revealed a homozygous mutation c.605T>C, which is predicted to cause a substitution of isoleucine by threonine at amino acid 202 of LCAT, p.(Ile202Thr). Genetic analysis in the third case showed a homozygous mutation c.512G>A, which is predicted to cause a substitution of arginine by glutamic acid at amino acid 171 of LCAT, p.(Arg171Glu). Both mutations have been classified as pathogenic by bioinformatic analysis (Polyphen-2 [19], MutationTaster [20] and Predict SNP [21]). The first mutation, p.(Ile202Thr), has been previously associated with hypoalphalipoproteinemia in the heterozygosity state by our group [22] (Table 1). Plasma agarose gel electrophoresis revealed several disturbances in the lipid profile of the patients, in comparison with the other members of their family. As shown in Supplementary Figure S1, both siblings were deficient in α-migrating lipoproteins. Moreover, pre-β-migrating particles were virtually absent. An accumulation of lipids, with a mobility intermediate between those of β- and pre-β-migrating particles, appeared in the first patient. As regards the plasma β-migrating particles, those from the first and second case showed a slightly slower mobility than in non-affected family members (Figure S1). Lipid composition of plasma lipoproteins was analyzed by FPLC, as well as the subsequent determination of lipid levels in eluted fractions (Figure 3). As expected, HDL contained scarce cholesterol, and only substantial amounts of phospholipids were detected in the corresponding fractions in the first and second cases. Moreover, HDL phospholipids reached their maximum concentration later, as compared with those from the control, suggesting the predominance of smaller HDL particles in the two patients. Although VLDL levels were higher in the first case than in his sister, both subjects had phospholipid and cholesterol-rich VLDL levels that were higher than the control’s. In addition, both siblings had lower LDL-cholesterol levels, but higher LDL-triglyceride levels, than the control (Figure 3). Agarose gel electrophoresis and immunoblotting for apo A-I and apo A-II of HDL-containing FPLC fractions showed a substantial reduction in the levels of both apolipoproteins in the two patients (Figure 3). sister, both subjects had phospholipid and cholesterol-rich VLDL levels that were higher than the control’s. In addition, both siblings had lower LDL-cholesterol levels, but higher LDL-triglyceride levels, than the control (Figure 3). Agarose gel electrophoresis and immunoblotting for apo A-I and apo A-II of HDL-containing FPLC fractions showed a substantial reduction in the levels of both J.apolipoproteins in the two patients (Figure 3). Clin. Med. 2019, 8, 1860 7 of 14 Figure 3. Plasma Gel Filtration Fast Protein Liquid Chromatography (FPLC) analysis. Plasmas were Figure 3. Plasma subjected to FPLC,Gel asFiltration describedFast Protein Liquid in Methods, Chromatography and the concentrations (FPLC) analysis. of cholesterol (C),Plasmas were triglyceride subjected to FPLC, as described in Methods, and the concentrations of cholesterol (C), (TG) and choline-containing phospholipids (PL) were determined in the indicated fractions. Inserts:triglyceride (TG)indicated the and choline-containing phospholipids fractions corresponding (PL) were to HDL weredetermined in the concentrated, andindicated fractions. subjected to agarose Inserts: gel the indicated fractions corresponding to HDL were concentrated, and subjected to agarose electrophoresis and immunoblotting for apo A-I and apo A-II. The profiles corresponding to the first gel electrophoresis and immunoblotting for apo A-I and apo A-II. case (A), second case (B) and control subjects (C) are presented. The profiles corresponding to the first case (A), second case (B) and control subjects (C) are presented. The NMR-Liposcale test, performed in serum from the second and third patients, revealed a distinctive lipoprotein profile, with null value of small HDL particles, under the limit of detection (<0.01µmol/L) and 0.01 µmol/L, respectively, compared with the control population (average small HDL-P = 21.52 ± 5.24 µmol/L), without blatant differences in medium or large HDL particles. Both patients presented higher values of VLDL (large, medium and small), especially in the third case, compared with the control population (Table 2). Figure 4a shows the score plot of the first PLS-DA model, considering the first two latent variables (LVs), designed to discriminate patients with LCAT deficiency among the general population. We applied a second PLS-DA model to discriminate between patients with LCAT deficiency and patients with low HDL cholesterol levels (HDL cholesterol <35 mg/dL), which represent 2.5% of the studied population (Figure 4b). The PLS-DA score plot showed a clear separation between patients with LCAT deficiency and the rest of the population, excepting one individual, whose position in the variance plot was extreme. This individual was later diagnosed with extreme HDL deficiency, and his lipoprotein pattern was even more severe, since it did not present small HDL particles, neither medium nor large HDL particles. J. Clin. Med. 2019, 8, 1860 8 of 14 Table 2. Results of Liposcale analysis. Large Medium Small Large Medium Small Large Medium Small VLDL-C LDL-C HDL-C VLDL-TG LDL-TG HDL-TG VLDL-P VLDL-P VLDL-P LDL-P LDL-P LDL-P HDL-P HDL-P HDL-P (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (nmol/L) (nmol/L) (nmol/L) (nmol/L) (nmol/L) (nmol/L) (µmol/L) (µmol/L) (µmol/L) Second 0.73 3.83 22.6 62.3 407 606.17 0.33 8.35 0.01 7.01 107.44 5.97 40.19 44.43 27.95 case Third 1.60 10.6 55.3 51.1 53.6 145.91 0.25 8.11 0.38 15.20 26.25 22.61 108.23 13.39 11.69 case Control 0.69 ± 2.61 ± 20.1 ± 81.7 ± 183 ± 346 ± 0.24 ± 8.76 ± 24.4 ± 9.71 ± 81.5 ± 59.8 ± 29.2 ± 11.2 ± 16.3 ± population 0.23 0.67 5.45 19.4 65.8 78.4 0.06 1.97 7.38 4.04 22.9 15.1 7.02 2.73 7.61 VLDL-P: Very Low-density lipoprotein particles; LDL-P: Low-density lipoprotein particles; HDL-P: High-density lipoprotein particles; VLDL-C: Very Low-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol; VLDL-TG: Very low-density lipoprotein-triglyceride; LDL-TG: Low-density lipoprotein-triglyceride; HDL-TG: High-density lipoprotein-triglyceride. J. Clin. Med. 2019, 8, 1860 9 of 14 J. Clin. Med. 2019, 8, 1860 9 of 14 J. Clin. Med. 2019, 8, 1860 9 of 14 Figure Figure 4. Partial 4. Partial least square least square discriminant discriminant analysis models analysis (PLS-DA) (PLS-DA) models for LCAT for LCAT deficiency deficiency classification, classification, Figure based on4.NMR based Partial on NMR least Lipoprotein square Lipoprotein profile discriminant data. (A) profile data. plot(A) analysis Scores Scores (PLS-DA) from plot a PLS-DA from models a PLS-DA modelfordesigned LCAT modelfor designed deficiency LCAT for LCAT classification, deficiency deficiency based on NMR identification identification among Lipoproteinamong the general the profile general population. data. (A) The population. Scores The individual plot fromprojection individual a PLS-DA on projection model on the designed the first two first for LCAT latent two latent deficiency variables variables is indicated identification is indicated with among with a black a dottheblack dot for general for individuals population. individuals with HDL-c with The ≥ HDL-c individual ≥ 35 and small 35 and projection small HDL on the HDL particle particle concentration concentration first two latent>variables > 7umol/L,iswith7umol/L, indicated with a red with dot fora red dot a black LCAT dotfor LCAT deficiency for individuals deficiency with patients, patients, HDL-c with with ≥ 35 dot a green a andforgreen small HDL-CHDLfor dot >35 HDL-C particle mg/dL, >35 mg/dL, a blueand concentration and a blue >circle if thecircle 7umol/L, with smallifaHDL the redsmall dot HDL for particleLCATparticle concentration deficiency concentration is with < 7umol/dL. is patients, < 7umol/dL.Biplot(B) a green (B) of Biplot dot for the of PLS-DA the HDL-C >35 PLS-DA model mg/dL, model and afor designed designed blue for LCAT circledeficiency LCAT if the small deficiency identification HDL particleamong identification among concentration patients patientsiswith < 7umol/dL. with very low(B) very Biplot levels low of levels of HDL of thecholesterol. HDL PLS-DA model cholesterol. The designed for The individual individual projection LCAT deficiency projection on the first on the firstidentification two latent variables among patients two latent variables is indicatedis indicated withwith verya redwith low a red levels triangle triangle offor HDL for low lowcholesterol. HDL-C HDL-C The patients (npatients individual (n = 150on = 150 individuals), projection individuals), the first and withtwo and latent a green with a green variables square for is two square indicated for with individuals two a individuals (2ndredandtriangle (2nd 3rd cases)for and low 3rd with cases) HDL-C LCAT with LCAT patients deficiency. (n =deficiency. 150 individuals), and with a green square for two individuals (2nd and 3rd cases) with LCAT deficiency. TheThe study study of of thethe distribution distribution of of free free cholesterol cholesterol byby agarose agarose gelgel electrophoresis electrophoresis showed showed a large a large amount amount of Theofstudy free cholesterol free cholesterol in Patient in Patientof of the distribution 3, 3, and and free the the presence presenceby cholesterol of particles of agarose particles gel migrating migrating toward toward the electrophoresis the cathode showed cathode with a large with reverse reverse amount electrophoretic electrophoretic mobility of free cholesterol mobility compared in Patient compared 3, and tothe other to other lipoproteins, lipoproteins, presence which of particles whichtoward correspond migrating correspond to LpX to LpX the particle cathode particle (Figure with 5). (Figure reverse 5). electrophoretic mobility compared to other lipoproteins, which correspond to LpX particle (Figure 5). Figure 5. Filipin Staining of synthetic Lp-X and EDTA-plasma after agarose gel electrophoresis. 10 µL of sequential dilutions of synthetic Lp-X were loaded into lanes 1–3, as follows. 1: 0.530 mg/mL Figure 5. Filipin Staining of synthetic Lp-X and EDTA-plasma after agarose gel electrophoresis. 10 μL cholesterol; 2: 0.265 mg/mL cholesterol; 3: 0.1325 mg/mL cholesterol. 10 µL of fresh EDTA-plasma were of sequential Figure 5. Filipin dilutions Staining ofofsynthetic syntheticLp-X Lp-X were and loaded intoafter EDTA-plasma lanes 1–3, as agarose gelfollows. 1: 0.530 10 electrophoresis. mg/mL μL loaded into lanes 4–7, as follows: 4, FLD patient 2 (without renal disease); 5, FLD patient 3 (with renal of cholesterol; 2: 0.265 mg/mL sequential dilutions cholesterol; of synthetic 3: 0.1325 Lp-X were mg/mL loaded into cholesterol. 10 follows. lanes 1–3, as μL of fresh EDTA-plasma 1: 0.530 mg/mL disease); 6 and 7, normolipidemic controls. Origin: application point. cholesterol; 2: 0.265 mg/mL cholesterol; 3: 0.1325 mg/mL cholesterol. 10 μL of fresh EDTA-plasma J. Clin. Med. 2019, 8, 1860 10 of 14 1 H-NMR analysis revealed an abnormal lipid profile in the serum of LCAT deficiency patients, compared to controls (Table 3). As expected, mean cholesterol composition in control serum was 28% free and 72% esterified. In contrast, serum from LCAT deficiency patients was highly enriched in free cholesterol. Cholesterol composition in the second case was 67% free and 33% esterified, suggesting the presence of some LpX. Interestingly, the third case had 98% free and 2% esterified cholesterol composition, confirming the presence of high levels of LpX. Supplementary Figure S2 shows the 1 H-NMR spectra of serum lipids, highlighting the signals of free and esterified cholesterol. Table 3. Percentages of esterified and free cholesterol in the serum of 2 LCAT deficiency samples and 3 controls. Patient % Esterified Cholesterol % Free Cholesterol Second case 32.9 67.1 Third Case 1.6 98.4 Control 75.3 24.4 Control 67.4 32.6 Control 74.1 25.9 4. Discussion In the present study, we report three cases with FLD with corneal opacities, due to two homozygous mutations (c.605T>C, c.512G>A) not previously described in patients with LCAT deficiency. The mutation, c.605T>C, which produces a substitution of isoleucine by threonine at amino acid 202 of LCAT, p.(Ile202Thr), had been previously described by our group in heterozygosity, in patients with hypoalphalipoproteinemia [22]. The mutation c.512 G>A, which is predicted to cause the substitution of arginine with glutamic acid at amino acid 171 of LCAT, p.(Arg171Glu), has not been previously reported, although Taramelli et al. described another mutation at the same position, which produces a substitution of arginine with threonine at the same amino, and also causes LCAT deficiency [6]. The current study shows that the main cause of mortality in LCAT deficiency does not depend only on LCAT mutation, because two of our cases are siblings. Despite sharing the same mutation, just one of them developed renal disease. LCAT adopts an α/β hydrolase fold, with a predominately parallel β-sheet sandwiched on both sides by α-helices [23]. The mutation p.(Arg171Glu) is located close to Ser181, which seems to be responsible, together with Cys31 and Leu182, for the possible oxyanion hole amino acid residues. The mutation p.(Ile202Thr) is located close to the second subdomain, formed by the insertions between β6/β7 and β7/αE, which is in much closer proximity to the catalytic site [23,24]. Our data are in agreement with the fact that previously reported LCAT mutations causing FED are located in the same domains of LCAT gene as those causing FLD, such as, for example, the mutations p.(Pro34Gln) [25] and p.(Pro34Leu) [26], reported in FED and FLD, respectively, although both are located in the same amino acid and in close proximity to the oxyanion hole amino acid 31 [23]. One of the most striking discoveries of our study, when analyzing the differences between the lipoprotein distribution by different methods, between the two FLD patients with renal disease and the FLD patient without renal disease, was the presence of abnormal lipoprotein profile in FPLC with higher concentrations of small–medium VLDL in NMR. Similar data have been reported by other studies [7,27]. LpX was confirmed by agarose gel electrophoresis and filipin-staining, and estimated by the distribution of FC and CE by NMR. Some studies reported that LpX is a nephrotoxic particle, that induces all the histological and functional renal hallmarks of FLD [9,10]. Besides, Vaisman et al. have reported on the close relationship between LCAT and LpX, as intravenous treatment with recombinant LCAT in micelcat-/- restored the normal lipoprotein profile, eliminated LpX in plasma and markedly decreased proteinuria [15]. J. Clin. Med. 2019, 8, 1860 11 of 14 It seems obvious that the LCAT genotype, which mostly determines the activity of the LCAT enzyme, is not uniquely responsible for renal disease in patients. Differences between FLD and FED are based on the cholesterol esterification in VLDL [8]. It is also evident that, in cases of LCAT deficiency, the complete metabolism of lipoproteins would be altered, presenting larger VLDL with a high concentration of free cholesterol and of phospholipids in plasma and glomeruli, which all may be factors in the deterioration of kidneys in patients with a familial LCAT deficiency [28]. We have analyzed different biochemical and clinical parameters and we have found that the main differences do not reside in the HDL particle. FPLC and NMR detect both free and esterified cholesterol; besides, FPLC has been previously used in patients with biliary cirrhosis, who showed an abnormal distribution pattern of lipoproteins, with a large accumulation of phospholipids and LpX [29]. In the current study, we confirm that the difference between subjects with renal disease resides in medium size VLDL with high triglyceride and cholesterol content, and the presence of LpX. To date, renal disease has been attributed to the cholesterol esterification of lipoproteins containing apolipoprotein B (VLDL and LDL) [7,8], but we report here two siblings with LCAT deficiency, who share the same LCAT mutation and negligible LCAT activity, only one of whom developed renal disease, which suggests that other genetic or environmental factors can influence the development of renal disease. Hence, the presence of renal disease could be explained by gene–gene or environment–gene interactions, that would favor the presence and amount of LpX. According with this, Rial-Crestelo D et al. [30] reported the case of one family, in which one member was diagnosed as FLD, while their sister was found to have FED, carrying the same LCAT mutation. However, the FED subject was too young to discard the possibility of future renal disease. In our study, FLD patients with renal disease showed higher numbers of small and medium VLDL particles, and higher concentrations of triglyceride and cholesterol in VLDL by FPLC and by NMR, than in the patient who has not developed renal disease. The substantial concentration of phospholipids in the HDL fraction by FPLC is consistent with LCAT deficiency, as previously described [7]. Advanced NMR testing determined no presence of small HDL particles in the serum of LCAT deficiency samples. Nascent preβ-HDL particles were not detected by the Liposcale test, as these particles do not carry either triglycerides or cholesterol within their core. Therefore, levels of small HDL particles determined by Liposcale were below the limit of detection of the NMR-derived particle size. The weighted HDL particle size is calculated from the subclass particle number multiplied by its associated diameter. In cases of LCAT deficiency, small HDL particles are not detected, and, therefore, size distribution is shifted towards larger particles. However, comparing NMR data from LCAT deficiency patients and subjects with low HDL cholesterol levels, there were differences in some lipoprotein variables, which identified a distinctive lipoprotein profile in patients with LCAT deficiency. FLD patients showed an increased particle-weighted HDL mean size (>9.10 nm), but a normal to elevated number of medium HDL particles (>11 µmol/L). Our data demonstrated that NMR can not only unequivocally detect this lipoprotein abnormality, but also identify the abnormal VLDL accumulation associated with renal disease. Therefore, this rapid procedure may be helpful in the screening and management of this rare disease. Our study has some limitations. We report analysis of only three cases, but this disease has an extremely low frequency in the general population and, besides, our results seem to be concordant with other studies [7,9,31]. The NMR used by the LipoScale test is not able to quantify small HDL particles, but has been able to identify a distinctive lipoprotein pattern, which allows the clear identification of patients with LCAT deficiency. The first case reported developed renal disease, and, although this could have been caused by other diseases, the patient demonstrated progressive proteinuria, without other pathologies, such as diabetes or hypertension, which could have conditioned the development of nephropathy. Finally, the increase in lipoprotein particles could be the consequence of renal disease, but we had access to the entire clinical records of the third case, and could check that the lipid disorders preceded renal disease. J. Clin. Med. 2019, 8, 1860 12 of 14 In conclusion, the current study presents three new cases of LCAT deficiency, which have not been previously described. Two of the three cases are siblings, but only one developed renal disease, although they had the same LCAT genotype and the same LCAT activity, which suggests that renal disease is not only dependent on LCAT deficiency. The classical separation between FED and FLD could be due to the presence of VLDL particles, rich in triglyceride, free cholesterol and LpX, which are possibly responsible for renal disease. Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/8/11/1860/s1, Figure S1: Agarose gel electrophoresis and lipid staining of the plasmas of the family kindred of the first (I.1) and the second (I.3) cases, Figure S2: 1 H-NMR spectra region of serum lipids highlighting the signals of free cholesterol (FC) and esterified cholesterol (EC). Author Contributions: Conceptualization, F.C., D.G.-C. and A.C.; Methodology, I.L.-M., N.A., M.G. and A.C.; Software N.A. and M.G.; Validation, D.G.-C., I.L.-M. and A.C.; Formal Analysis, I.L.-M. and F.B.C.; Investigation, F.B.-V., R.M.-G., and A.C.; Resources, F.C., F.B.-V.; Data Curation, N.A., M.G. and I.L.-M.; Writing–Original Draft Preparation, I.L.-M. and R.M.-G.; Writing–Review and Editing, F.C., A.C., D.G.-C., F.B.C., H.M.V.-L.; Visualization, D.G.-C. and F.B.C.; Supervision, F.C. and A.C.; Project Administration, F.C. and A.C.; Funding Acquisition, F.C. and A.C. Funding: This work was supported by grants from the Spanish Ministry of Economy and Competitiveness PI15/01983, PI18/01777, CIBERCV, CIBERDEM and CIBEROBN. These projects are co-financed by Instituto de Salud Carlos III and the European Regional Development Fund (ERDF) of the European Union “A way to make Europe”. CIBERCV, CIBERDEM and CIBEROBN are project of Instituto de Salud Carlos III. Acknowledgments: The authors thank Elkie Suñén for her English editorial assistance. All authors have read and approved the final manuscript. Conflicts of Interest: N.A. is a stock owner of Biosfer Teslab, and has a patent for an NMR method for lipoprotein characterization. The authors declare no conflict of interest. References 1. Calabresi, L.; Simonelli, S.; Gomaraschi, M.; Franceschini, G. Genetic lecithin: cholesterol acyltransferase deficiency and cardiovascular disease. Atherosclerosis 2012, 222, 299–306. [CrossRef] [PubMed] 2. Saeedi, R.; Li, M.; Frohlich, J. A review on lecithin: cholesterol acyltransferase deficiency. Clin. Biochem. 2015, 48, 472–475. [CrossRef] [PubMed] 3. McIntyre, N. Familial LCAT deficiency and fish-eye disease. J. Inherit. Metab. Dis. 1988, 11 (Suppl. 1), 45–56. [CrossRef] [PubMed] 4. HGMD® . Available online: http://www.hgmd.cf.ac.uk/ac/index.php (accessed on 18 July 2018). 5. Hirashio, S.; Ueno, T.; Naito, T.; Masaki, T. 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Co-existence of classic familial lecithin-cholesterol acyl transferase deficiency and fish eye disease in the same family. Indian J. Nephrol. 2015, 25, 362–365. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).