Squalene Synthase Deficiency: Clinical, Biochemical, and Molecular Characterization of a Defect in Cholesterol Biosynthesis

Abstract

Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of cholesterol in embryogenesis and development. FDFT1 encodes for an evolutionarily conserved enzyme, squalene synthase (SS, farnesyl-pyrophosphate farnesyl-transferase 1), which catalyzes the first committed step in cholesterol biosynthesis. We report three individuals with profound developmental delay, brain abnormalities, 2-3 syndactyly of the toes, and facial dysmorphisms, resembling Smith-Lemli-Opitz syndrome, the most common cholesterol biogenesis defect. The metabolite profile in plasma and urine suggested that their defect was at the level of squalene synthase. Whole-exome sequencing was used to identify recessive disease-causing variants in FDFT1. Functional characterization of one variant demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis/uptake in steady state.

Keywords: FDFT1; cholesterol biosynthesis; dysmorphism; syndactyly.

Figure 1

Schematic Representation of the Isoprenoid/Cholesterol Biosynthesis Pathway Metabolites that accumulate as a result of squalene synthase deficiency are underlined (pathway re-drawn based on Tansey and Shechter¹³).

Figure 2

Facial Characteristics Individual 1 at 3 years of age (A) and 6 years of age (B) and individual 2 at 5 weeks of age (C) and 3 years of age (D). Dysmorphic features include depressed nasal bridge, low-set posteriorly rotated ears, square nasal tip, epicanthic folds, mild micrognathia, and retrognathia.

Figure 3

Urine Metabolite Profiles (A) Urine organic acid GC-MS profiles of individual 1 (top) and a control subject (bottom). Normal urine components are indicated in italics. Numbered peaks indicate abnormal metabolites: (1) methylsuccinic, (2) mevalonic lactone, (3) mesaconic acid, (4) 2-methylgluatic acid, (5) 3-methyladipic acid, (6) 3-methylhex-3-enedioic acid, (7) 3-methylhex-2-endioic acid, (8) 2,6-dimethylheptanedioic acid, (9) unknown, (10) 3-methylhex-2,4-dienedioic, (11) 2,6-dimethylhept-2-enedioic acid, (12) 3,7-dimethyloctanedioic, and (13) 3,7-dimethyl-2,6-dienedioic. Control values are given in Figure S1. (B) One-dimensional 500 MHz ¹H-NMR spectra of urine of individual 1 and age-matched control subject measured at pH 2.5 (regions 5.60–6.50 ppm and 1.50–2.25 ppm). The insert shows the structure of 3-methylhex-2,4-dienedioic acid. Proton assignments of 3-methylhex-3,4-dienedioic acid (A) and other farnesol-derived dicarboxylic acids (asterisk).

Figure 4

Schematic Representation of FDFT1, the Variants Identified in Individuals 1, 2, and 3, and Functional Follow-up (A) The maternal deletion identified in the sibship extends ∼50 kb more proximally, but no other genes are included. Genomic positions are based on hg19. Red horizontal lines indicate deleted sequence. For the paternal mutation in individuals 1 and 2, the wild-type protein amino acid sequence is provided in gray, whereas the predicted sequence of the splice mutation is shown in red. (B) Western blot analysis of individuals 1 (Ind 1) and 2 (Ind 2), using two different culturing conditions (fetal bovine serum [FBS] and lipid depleted [LD-] FBS), using an antibody to detect FDFT1 in fibroblast of individuals 1 and 2. Alpha-tubulin was used as loading control. FDFT1 reduction is more prominent when cells were cultured under lipid depletion. (C) Isoform-specific PCR using cDNA generated from EBV-transformed PBMCs of individual 3 (Ind 3) was used for the detection of FDFT1 isoforms. Three FDFT1 isoforms could not be detected in individual 3 whereas these were present in EBV-transformed PBMCs of a control individual. The addition of cycloheximide (CHX; - absent, + present) did not facilitate expression detection. Profile of all isoforms is presented in Figure S5. (D) The genomic segment containing the homozygous 16 bp deletion in individual 3 was tested for promoter activity using a luciferase assay, which showed significantly reduced activity when compared to the control construct containing the wild-type sequence (two-tailed t test, p = 2.9 × 10⁻⁶). ^∗∗∗p < 0.001. Error bars represent one SD.