Table 3.

PS3/BS3 functional study criteria specifications for LDLR

Criteria	LDLR Specification
Pathogenic
PS3 (level 1)	Study of the whole LDLR cycle (LDLR expression/biosynthesis, LDL binding, and LDL internalization) performed in heterologous cells (with no endogenous LDLR) transfected with a mutant plasmid. Assay result of <70% of wild-type activity in either LDLR expression/biosynthesis, LDL binding, or LDL internalization.
PS3_Moderate (level 2)	Study of only part of the LDLR cycle following level 1 methodology or whole or part of the LDLR cycle in true homozygous patient cells. Assay results of <70% of wild-type activity in either LDLR expression/biosynthesis, LDL binding, or LDL internalization. RNA studies, using RNA extracted from heterozygous or true homozygous patient cells, where aberrant transcript is confirmed by sequencing and is quantified as >25% of total transcript from heterozygous cells or 50% of total transcript from homozygous cells. Variants with 2 or more level 3 functional studies (must be different assays) or any level 3 functional study #1 to 4 performed by 2 or more independent labs with concordant results.
PS3_Supporting (level 3)	Study of LDLR cycle (whole or part) in heterozygous patient cells. Assay results of <85% of wild-type activity in either LDLR expression/biosynthesis, LDL binding, or LDL internalization. Luciferase studies with transcription levels of <50% compared with wild-type (applicable to 5′UTR/promoter variants). Minigene splicing assays with <10% of wild-type transcript present where an aberrant transcript from the candidate variant is confirmed by sequencing. High-throughput assays, which include alternative microscopy assays (eg, Thormaehlen et al., 201541), MAVE (eg, Weile and Roth, 201842), and deep mutational scanning assays, can be considered here, only if the assay has been validated with a minimum of 4 pathogenic and 4 benign variant controls in LDLR. Note: % activity thresholds will be defined by the FH VCEP as more data become available. RNA studies, using RNA extracted from heterozygous or homozygous patient cells, with aberrant transcript confirmed by sequencing (but without transcript quantification).
Benign
BS3 (level 1)	Study of the whole LDLR cycle (LDLR expression/biosynthesis, LDL binding, and LDL internalization) performed in heterologous cells (with no endogenous LDLR) transfected with a mutant plasmid. Assay result of >90% of wild-type activity in LDLR expression/biosynthesis, LDL binding, and LDL internalization. Note: studies of only part of the LDLR cycle are not eligible for BS3 or BS3_Supporting.
BS3_Supporting (level 3)	Study of the whole LDLR cycle in true homozygous patient cells with assay result of >90% of wild-type activity in LDLR expression/biosynthesis, LDL binding, and LDL internalization or in heterozygous patient cells with assay result of >95% of wild-type activity in LDLR expression/biosynthesis, LDL binding, and LDL internalization. Luciferase studies with transcription levels of >90% when compared with wild-type (applicable to 5′UTR/promoter variants). RNA studies, using RNA extracted from heterozygous or true homozygous patient cells with aberrant transcripts quantification, where aberrant transcript is <10% of total transcript, or without transcript quantification where no aberrant transcript is confirmed by sequencing. Minigene splicing assay where only wild-type transcript is present and confirmed by sequencing. High-throughput assays as defined earlier; only applicable when assay can indicate the whole LDLR cycle (LDLR expression/biosynthesis, LDL binding, and LDL internalization) is unaffected.

Functional assays performed in compound heterozygous patient cells are not considered applicable in PS3/BS3 criteria because it is difficult to delineate the individual effect of each variant.

FH, familial hypercholesterolemia; LDL, low-density lipoprotein; MAVE, Multiplex Assays of Variant Effect; UTR, untranslated region; VCEP, Variant Curation Expert Panel.