An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations.

The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.

FBS (Lonza) and 10% DMSO in liquid nitrogen until required for use.
EBV-transformed cell lines were generated by exposing PBMCs to free EBV particles, produced by an EBV-infected marmoset cell line (B95. 8). After EBV infection, transformed PBMCs were cultured in an incubator at 37°C in a 5% CO 2 atmosphere in Iscove's Modifi ed Dulbecco's Medium (IMDM, Sigma-Aldrich) supplemented with 20% FBS and 2% Ultraglutamine (Lonza). The continuous cell lines bearing the LDLR mutations were also analyzed to verify the presence of DNA variations. In order to upregulate LDLR, the cells were incubated in a medium containing a lipoprotein deprived serum (LPDS, Sigma-Aldrich) for 48 h before being analyzed.

LDL uptake assay by fl uorescent-activated cell sorter fl ow cytometry
Residual LDLR activity was evaluated by measuring the binding and uptake of a fl uorescently labeled LDL, namely 1,19-dioctadecyl-3,3,3 ′ ,3 ′ -tetramethylindocarbocyanine perchlorate-conjugated LDL (DiI-LDL, Invitrogen), in EBV-transformed B lymphocytes and mitogen-stimulated T-lymphocytes. Briefl y, cells were collected and diluted at 1 × 10 6 cells/ml in DMEM:F12 (Sigma-Aldrich) without phenol red and LPDS. Two aliquots of cells for each sample analyzed were then incubated for 3 h at 37°C and 5% CO 2 as follows: a ) with 30 mg/L unlabeled LDL (Sigma-Aldrich), to measure background fl uorescence; and b ) with 10 mg/L DiI-LDL to estimate the maximum LDL binding and uptake. In preliminary experiments we performed an additional incubation with 30 mg/L unlabeled LDL plus 10 mg/L DiI-LDL (ratio 3:1) to evaluate the displacement of DiI-LDL and in this condition, we observed a reduction of cell fl uorescence indicating the specifi c binding of DiI-LDL (supplementary The results are expressed as the median of fl uorescence and as the ratio between the median fl uorescence intensity of cells from FH patients and the mean of the median fl uorescence intensity of control cells. This ratio represents residual LDLR activity. All measurements were performed in duplicate.

Statistical analysis
The normality of variable distributions was evaluated with Kolmogorov-Smirnov test. Continuous variables were indicated as mean ± SD. Differences between the continuous variables were evaluated with t -test. A value of P < 0.05 was considered statistically signifi cant. Statistical analyses were carried out using the ( 3,4 ) or leukocytes ( 5,6 ). The Epstein-Barr virus (EBV)transformed B-lymphocytes show high LDLR levels and allow an unlimited supply of cells without being infl uenced by the patient's diet and drug treatment ( 7 ). The main problem of these functional assays was the bad separation between residual LDLR activity from FH patients and controls. The separation of different leukocyte populations obtains more accurate results ( 8 ); the selection of viable lymphocytes by stimulation with a mitogen or by fl ow cytometry gating improves the discrimination between patients and controls although it does not allow complete discrimination of FH heterozygote patients from healthy controls ( 9,10 ).
The aim of this study was to improve the method of T-lymphocyte stimulation for LDLR activity evaluation through the comparison of different mitogen combinations. We also aimed to compare continuous EBV-transformed-lymphocytes and mitogen stimulated T-lymphocytes obtained from peripheral blood samples in order to identify the best method to detect LDLR activity by fl uorescently-labeled LDL. In addition, we characterized both novel and known mutations in the LDLR gene.

FH patients and control subjects
Patients with clinically diagnosed FH were enrolled at the Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Napoli Federico II and at the AORN Cardarelli, Napoli. The FH diagnosis was based on the criteria established by the Società Italiana per lo Studio della Arteriosclerosi for the identifi cation and treatment of dyslipidemias. Twenty-four patients bearing new and known mutations were selected for functional characterization. As healthy controls, 24 unrelated subjects were selected from voluntary donors of the Centro Trasfusionale of the AORN Cardarelli, Napoli, on the basis of normal lipid levels and absence of LDLR mutations. The supplementary table reports characteristics of FH patients and controls. The study was performed according to the current version of the Helsinki Declaration. Informed consent was obtained for each patient.

Mutation screening
Molecular analysis for the identifi cation of LDLR mutations was performed as previously described ( 11 ). Briefl y, the promoter and the 18 exons of the LDLR gene were amplifi ed by PCR and directly sequenced. In the case of novel mutations, we verifi ed the absence in 150 chromosomes from normocholesterolemic individuals of the same ethnic group, the segregation of the sequence variant in the relatives available, and the conservation of substituted amino acid residues across homologous proteins by BLASTP.

Mononuclear cell isolation and generation of EBV-transformed B-lymphocytes
Peripheral blood mononuclear cells (PBMCs) were isolated from 9 ml of EDTA blood collected from each subject enrolled in this study. The sample was diluted 1 in 2 with PBS without Ca 2+ and Mg 2+ pH 7.4, and then layered on Ficoll-Paque (Lymphocyte Separation Medium, MP Biomedicals). After centrifugation at 1,500 rpm for 30 min at 4°C, mononuclear cells were recovered at interface, washed three times in PBS and stored in 90% 87.3%, respectively. As regards LDL-R expression evaluated by the uptake of DiI-LDL, in case of PHA stimulation, the uptake of DiI-LDL was lower than that observed with PHA plus PMA and ionomycin plus PMA (supplementary Fig. I).
Due to the higher yield of live T-lymphocytes obtained with ionomycin plus PMA with respect to PHA plus PMA and considering the LDLR upregulation in T-lymphocytes described by Makar et al. ( 12 ) using ionomycin plus PMA, we selected this method for further experiments.

Functional characterization of LDLR activity
The median of fl uorescence intensity and the residual LDLR activity evaluated in EBV-transformed B-lymphocytes and in ionomycin plus PMA stimulated T-lymphocytes from healthy subjects with normal lipid profi le and no mutations in LDLR are reported in Table 1 . A representative set of data from fl ow cytometry analysis is shown in supplementary Fig. III. The residual LDLR activity was calculated using the mean of median values of all analyzed controls as areference (1,752 for EBV-transformed B-lymphocytes and 1,011 for stimulated T-lymphocytes). As for controls, this calculation represents a data normalization allowing the comparison between the two methods used. The minimum and maximum values of residual LDLR activity evaluated on EBV-transformed B-lymphocytes from controls were 71% and 132%, respectively, whereas the minimum and maximum values of residual LDLR activity evaluated on stimulated T-lymphocytes were 79% and 133%, respectively. The median of fl uorescence intensity and the residual LDLR activity in EBV-transformed B-lymphocytes and in stimulated T-lymphocytes from FH patients with mutations in LDLR gene are reported in Table 2 . The residual LDLR activities were statistically different between control subjects and FH patients analyzed by both methodologies ( P < 0.0001 both). Regarding the functional characterization of new mutations, we observed low residual LDLR activities consistent with a heterozygous status of FH ( Table 2 ). Figure 1 shows a graphical representation of residual LDLR activity of each control and FH patient evaluated by the two methods. We observed that, using EBV-transformed B-lymphocytes, there is an overlap of residual LDLR activities between controls and patients in the range 71-75%, whereas, using stimulated T-lymphocytes, no value in the range 65-79% was observed for controls nor for FH patients. The use of stimulated T-lymphocytes allows a complete discrimination between controls and FH patients.

Analysis of ROC curves
The ROC curves are constructed using the residual LDLR activity evaluated in EBV-transformed B-lymphocytes and in stimulated T-lymphocytes as a discriminator parameter between FH patients and healthy controls ( Fig. 2 ). The AUC of the methods based on EBV-transformed B-lymphocytes and on stimulated T-lymphocytes are 0.984 [95% confi dence interval (CI) 0.880-1.000] and 1.000 (95% CI 0.907-1.000), respectively ( P < 0.0001 both). The comparison of the AUC of the two methods does not reveal any statistically signifi cant difference ( P = 0.41).
statistical Predictive Analytics Software version 18.0 (SPSS Inc.). Analysis of receiver operating characteristic (ROC) curves and dot diagrams was performed with MedCalc Version 11.5.1. The statistical signifi cance of the area under the ROC curves (AUC) was calculated against the null hypothesis AUC = 0.5 as recommended by DeLong et al. ( 13 ). The threshold values were determined by the farthest point from the bisector of the ROC curve.

Mutation screening
We identifi ed four new mutations absent in 150 chromosomes from normal individuals ( Table 1 ). These alterations are novel missense mutations (c.440C>T; c.974G>A; c.1130G>T; c.1739C>T) that result in a single amino acid substitution in LDL receptor protein (p.Thr147Ile; p.Cys325Tyr; p.Cys377Phe; p.Ser580Phe). The novel mutations cause changes of amino acid residues conserved across fourteen species examined during our study.

T-lymphocytes mitogen selection
In order to select the best way to stimulate T-lymphocytes able to express LDLR, we compared three known procedures for in vitro growth stimulation of T-cells. The fl ow cytometry analysis showed that the amount of viable T-lymphocytes in cells treated with 1 ) PHA, 2 ) PHA plus PMA, and 3 ) ionomycin plus PMA was 76.4%, 83.8%, and Missing data are relative to unavailable samples. CV%, coeffi cient of variation. because in our experimental conditions, it gives the best results. In other studies, functional assays were conducted on T-lymphocytes using only PHA ( 21,22 ) although the comparison between different mitogens was not performed. For the fi rst time, we compared two cell culture techniques useful in detecting residual LDLR activity in FH patients: EBV-transformed B-lymphocytes and mitogen stimulated T-lymphocytes both treated with fl uorescentlylabeled LDL. In controls, we observed a higher median of fl uorescent intensity in EBV-transformed B-lymphocytes than in stimulated T-lymphocytes, suggesting the highest LDLR expression in the fi rst cell type. The residual LDLR activity evaluated on stimulated T-lymphocytes of controls shows a more limited range of values, suggesting a minor LDLR expression variability.
The minimum value of residual LDLR activity in controls is higher for stimulated T-lymphocytes than for EBVtransformed B-lymphocytes (79% and 71%, respectively) suggesting that the T-lymphocytes based method has a better capacity to detect functional activity than the other one. With ionomycin plus PMA stimulated T-lymphocytes, no value was observed in the range 65-79% (discriminating range) for controls nor for FH patients, whereas using EBV-transformed B-lymphocytes, we observed an overlap in residual LDLR activities between controls and patients in the range 71-75% (absence of a discriminating range). Then, using stimulated T-lymphocytes, we obtained a complete discrimination between FH patients and normocholesterolemic By the analysis of ROC curves, the suggested threshold of residual LDLR activity for discrimination between controls and patients is 67.7% for EBV-transformed B-lymphocytes (86.96% sensitivity and 100% specifi city) and 64.6% for stimulated T-lymphocytes (100% sensitivity and 100% specifi city).

DISCUSSION
The functional characterization of LDLR mutations by use of EBV-transformed B-lymphocytes provides the advantages of continuous cell lines that undergo rapid proliferation combined with a long lifespan. These are easily reestablished in culture after being frozen and are useful in the creation of cell banks for future molecular studies. These advantages enable a reliable characterization of novel and known mutations in LDLR gene and other genetic disorders ( 20 ). However, EBV-transformed B-lymphocytes must be used carefully because of decreased genetic stability and possible changes in cellular processes induced by viral transformation ( 20 ). The generation of EBV-transformed B-lymphocytes is wasteful, time-consuming, and often results tare unproductive depending on the quantity of leukocytes isolated from each subject.
In order to overcome these problems, we have improved an assay to evaluate LDLR activity in peripheral-blood lymphocytes using ionomycin plus PMA-induced proliferating T-lymphocytes. We selected this mitogen combination simple and fast and improves the detection of LDL receptor activity defects, allowing complete discrimination between controls and FH patients. Furthermore, the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.
subjects, although we observed reduced LDLR activities in FH patients with both methods, which is in agreement with the presence of LDLR mutations. The technical advancement reached with our method consists in the complete discrimination between FH patients and controls, a feature that could allow use of this functional assay for screening purposes. The analysis of ROC curves in stimulated T-lymphocytes suggests a threshold value of residual LDLR activity equal to 64.6% with a 100% sensitivity and a 100% specifi city, although the use of a higher threshold in the discriminating range (65-79%) could improve the diagnostic sensitivity of the method.
To date, many techniques have been used to diagnose FH disease, including serum lipid profi le and molecular methods such as DNA sequencing for the screening of point mutations, multiplex ligation-dependent probe amplifi cation to identify large rearrangements, and RNA analysis for splicing variants ( 2,(23)(24)(25). The residual LDLR activity evaluated on stimulated T-lymphocytes (ionomycin plus PMA used as mitogen stimulators) may be used as a screening method for identifi cation of LDLR defects in patients with a clinical suspect of FH. In addition, our method is relatively easy to handle and appears less laborious and time-consuming than other described techniques such as the use of EBV-transformed B-lymphocytes whose generation requires to work in class 1 biosafety level laboratories.
In conclusion, our method based on the DiI-LDL uptake in ionomycin plus PMA-stimulated T-lymphocytes is