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. 2020 Feb 3;10(1):1727.
doi: 10.1038/s41598-020-58734-9.

Mutation type classification and pathogenicity assignment of sixteen missense variants located in the EGF-precursor homology domain of the LDLR

Unai Galicia-Garcia #  1 , Asier Benito-Vicente #  2 , Kepa B Uribe  2 , Shifa Jebari  2 , Asier Larrea-Sebal  2 , Rocio Alonso-Estrada  1 , Joseba Aguilo-Arce  2 , Helena Ostolaza  2 , Lourdes Palacios  3 , Cesar Martin  4
Affiliations

Mutation type classification and pathogenicity assignment of sixteen missense variants located in the EGF-precursor homology domain of the LDLR

Unai Galicia-Garcia et al. Sci Rep. .

Abstract

The primary genetic cause of familial hypercholesterolemia (FH) is related to mutations in the LDLR gene encoding the Low-density Lipoprotein Receptor. LDLR structure is organized in 5 different domains, including an EGF-precursor homology domain that plays a pivotal role in lipoprotein release and receptor recycling. Mutations in this domain constitute 51.7% of the total missense variants described in LDLR. The aim of the present work was to analyse how clinically significant variants in the EGF-precursor homology domain impact LDLR. The activity of sixteen LDLR variants was functionally characterized by determining LDLR expression by Western blot and LDLR expression, LDL binding capacity and uptake, and LDLR recycling activity by flow cytometry in transfected CHO-ldlA7 cells. Of the analysed variants, we found six non-pathogenic LDLR variants and ten pathogenic variants distributed as follow: three class 3 variants; four class 2 variants; and three class 5 variants. These results can be incorporated into clinical management of patients by helping guide the appropriate level of treatment intensity depending on the extent of loss of LDLR activity. This data can also contribute to cascade-screening for pathogenic FH variants.

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Conflict of interest statement

Progenika Biopharma SA, is an Spanish biotech company founded in 2000 with headquarters in Derio, Bizkaia (SPAIN). Progenika Biopharma SA did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors’ salary to L.P.A. U.G.-G., A.B.-V., K.B.U., S.J., A.L.-.S., R.A.-E., J.A.-A., H.O. and C.M. declare no competing interest.

Figures

Figure 1
Figure 1
Location and frequency of LDLR variants within the EGF-precursor homology domain according to ClinVar database. Colours represent the number of described variants at a given amino acid.
Figure 2
Figure 2
Expression of wt and sixteen LDLR variants in CHO-ldlA7 transfected cells. Expression of LDLR was determined 48 h post-transfection with the plasmids carrying the different LDLR variants by Western blot and flow cytometry. (A,B,E,F) Western blot analysis of LDLR expression. (C,G) Quantification of the intensities of the bands obtained by Western blot by densitometry and, (D,H) LDLR expression determined by flow cytometry. A representative blot is shown in panel A,B,E and F. C and G represent the mean of band quantification of three independent Western blots. The values in D and H represent the mean of triplicate determinations (n = 3); error bars represent ± SD. *P < 0.001 compared to wt using a Student’s t-test.
Figure 3
Figure 3
LDLR activity of wt and p.(Gln378Pro), p.(Ala399Thr), p.(Thr413Met), p.(His656Asn), p.(Thr659Asn) and p.(Ala606Ser) LDLR variants. (A) LDL-LDLR binding and (B) FITC-LDL uptake activity. Assays were performed as described in Materials and Methods. Data show the mean of three independent experiments; error bars represent ± SD. *P < 0.001 compared to wt using a Student’s t-test.
Figure 4
Figure 4
LDLR activity of wt and p.(Ser326Cys), p.(Cys338Phe), p.(Cys368Tyr), p.(Ser584Pro), p.(Asp622Gly), p.(Cys698Tyr) and p.(Asp707Tyr) LDLR variants. (A,C) LDL- LDLR binding and (B,D) FITC-LDL uptake activity. Assays were performed as described in Materials and Methods. Data show the mean of three independent experiments; error bars represent ± SD. *P < 0.001 compared to wt using a Student’s t-test.
Figure 5
Figure 5
LDLR activity of wt and p.(Asp492Asn), p.(Arg633Cys), and p.(Asp700Gly) LDLR variants. (A) LDL- LDLR binding, (B) FITC-LDL uptake activity and (C) LDL binding at different pH. Assays were performed as described in Materials and Methods. Data show the mean of three independent experiments; error bars represent ± SD. *P < 0.001 compared to wt using a Student’s t-test.
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