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

doi:10.1016/j.ajhg.2018.05.004

. 2018 Jul 5;103(1):125-130.

doi: 10.1016/j.ajhg.2018.05.004. Epub 2018 Jun 14.

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

David Coman¹, Lisenka E L M Vissers², Lisa G Riley³, Michael P Kwint², Roxanna Hauck⁴, Janet Koster⁵, Sinje Geuer², Sarah Hopkins⁶, Barbra Hallinan⁷, Larry Sweetman⁸, Udo F H Engelke⁹, T Andrew Burrow¹⁰, John Cardinal¹¹, James McGill¹², Anita Inwood¹³, Christine Gurnsey¹⁴, Hans R Waterham⁵, John Christodoulou¹⁵, Ron A Wevers⁹, James Pitt¹⁶

Affiliations

¹ Department of Metabolic Medicine, Lady Cilento Children's Hospital, Brisbane, QLD 4101, Australia; Department of Paediatrics, Wesley Hospital, Brisbane, QLD 4066, Australia; School of Medicine, University of Queensland, Brisbane, QLD 4067, Australia; School of Medicine, Griffith University, Gold Coast, QLD 4222, Australia. Electronic address: david.coman@health.qld.gov.au.
² Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands.
³ Genetic Metabolic Disorders Research Group, Sydney Children's Hospital Network, Sydney, NSW 2145, Australia; Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia.
⁴ Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia.
⁵ Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, 105 AZ Amsterdam, the Netherlands.
⁶ Division of Neurology, Children's Hospital, Philadelphia, PA 19104, USA.
⁷ Division of Neurology, Cincinnati Children's Medical Centre, Cincinnati, OH 45229, USA.
⁸ Institute of Metabolic Disease, Baylor Scott & White Research Institute, Baylor University Medical Center, Dallas, TX 75204, USA.
⁹ Translational Metabolic Laboratory - 830 TML, Department of Laboratory Medicine, Radboud University Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, the Netherlands.
¹⁰ University of Arkansas for Medical Sciences College of Medicine, Department of Pediatrics Little Rock, Arkansas, AR 72205, USA.
¹¹ Department of Paediatrics, Wesley Hospital, Brisbane, QLD 4066, Australia.
¹² Department of Metabolic Medicine, Lady Cilento Children's Hospital, Brisbane, QLD 4101, Australia; Department of Chemical Pathology, Pathology Queensland, Royal Brisbane & Women's Hospital, Brisbane, QLD 4006, Australia; School of Medicine, University of Queensland, Brisbane, QLD 4067, Australia.
¹³ Department of Metabolic Medicine, Lady Cilento Children's Hospital, Brisbane, QLD 4101, Australia.
¹⁴ Department of Chemical Pathology, Pathology Queensland, Royal Brisbane & Women's Hospital, Brisbane, QLD 4006, Australia.
¹⁵ Genetic Metabolic Disorders Research Group, Sydney Children's Hospital Network, Sydney, NSW 2145, Australia; Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia; Neurodevelopmental Genomics Research Group, Murdoch Children's Research Institute, Melbourne VIC 3052, Australia; Genetic Metabolic Disorders Research Group, Sydney Children's Hospital Network, Sydney NSW 2145, Australia; Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, VIC 3052, Australia.
¹⁶ Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia. Electronic address: james.pitt@vcgs.org.au.

PMID: 29909962
PMCID: PMC6037199
DOI: 10.1016/j.ajhg.2018.05.004

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

David Coman et al. Am J Hum Genet. 2018.

. 2018 Jul 5;103(1):125-130.

doi: 10.1016/j.ajhg.2018.05.004. Epub 2018 Jun 14.

Authors

Affiliations

¹ Department of Metabolic Medicine, Lady Cilento Children's Hospital, Brisbane, QLD 4101, Australia; Department of Paediatrics, Wesley Hospital, Brisbane, QLD 4066, Australia; School of Medicine, University of Queensland, Brisbane, QLD 4067, Australia; School of Medicine, Griffith University, Gold Coast, QLD 4222, Australia. Electronic address: david.coman@health.qld.gov.au.
² Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands.
³ Genetic Metabolic Disorders Research Group, Sydney Children's Hospital Network, Sydney, NSW 2145, Australia; Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia.
⁴ Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia.
⁵ Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, 105 AZ Amsterdam, the Netherlands.
⁶ Division of Neurology, Children's Hospital, Philadelphia, PA 19104, USA.
⁷ Division of Neurology, Cincinnati Children's Medical Centre, Cincinnati, OH 45229, USA.
⁸ Institute of Metabolic Disease, Baylor Scott & White Research Institute, Baylor University Medical Center, Dallas, TX 75204, USA.
⁹ Translational Metabolic Laboratory - 830 TML, Department of Laboratory Medicine, Radboud University Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, the Netherlands.
¹⁰ University of Arkansas for Medical Sciences College of Medicine, Department of Pediatrics Little Rock, Arkansas, AR 72205, USA.
¹¹ Department of Paediatrics, Wesley Hospital, Brisbane, QLD 4066, Australia.
¹² Department of Metabolic Medicine, Lady Cilento Children's Hospital, Brisbane, QLD 4101, Australia; Department of Chemical Pathology, Pathology Queensland, Royal Brisbane & Women's Hospital, Brisbane, QLD 4006, Australia; School of Medicine, University of Queensland, Brisbane, QLD 4067, Australia.
¹³ Department of Metabolic Medicine, Lady Cilento Children's Hospital, Brisbane, QLD 4101, Australia.
¹⁴ Department of Chemical Pathology, Pathology Queensland, Royal Brisbane & Women's Hospital, Brisbane, QLD 4006, Australia.
¹⁵ Genetic Metabolic Disorders Research Group, Sydney Children's Hospital Network, Sydney, NSW 2145, Australia; Discipline of Child & Adolescent Health, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia; Neurodevelopmental Genomics Research Group, Murdoch Children's Research Institute, Melbourne VIC 3052, Australia; Genetic Metabolic Disorders Research Group, Sydney Children's Hospital Network, Sydney NSW 2145, Australia; Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, VIC 3052, Australia.
¹⁶ Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia. Electronic address: james.pitt@vcgs.org.au.

PMID: 29909962
PMCID: PMC6037199
DOI: 10.1016/j.ajhg.2018.05.004

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.

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Figures

**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.

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References

1. Waterham H.R. Defects of cholesterol biosynthesis. FEBS Lett. 2006;580:5442–5449. - PubMed
1. Schechter I., Conrad D.G., Hart I., Berger R.C., McKenzie T.L., Bleskan J., Patterson D. Localization of the squalene synthase gene (FDFT1) to human chromosome 8p22-p23.1. Genomics. 1994;20:116–118. - PubMed
1. Do R., Kiss R.S., Gaudet D., Engert J.C. Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathway. Clin. Genet. 2009;75:19–29. - PubMed
1. Jemal M., Ouyang Z. Gas chromatography-mass spectrometric method for quantitative determination in human urine of dicarboxylic (dioic) acids produced in the body as a consequence of cholesterol biosynthesis inhibition. J. Chromatogr. B Biomed. Sci. Appl. 1998;709:233–241. - PubMed
1. Vaidya S., Bostedor R., Kurtz M.M., Bergstrom J.D., Bansal V.S. Massive production of farnesol-derived dicarboxylic acids in mice treated with the squalene synthase inhibitor zaragozic acid A. Arch. Biochem. Biophys. 1998;355:84–92. - PubMed

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[1] Waterham H.R. Defects of cholesterol biosynthesis. FEBS Lett. 2006;580:5442–5449. - PubMed

[2] Waterham H.R. Defects of cholesterol biosynthesis. FEBS Lett. 2006;580:5442–5449. - PubMed

[3] Schechter I., Conrad D.G., Hart I., Berger R.C., McKenzie T.L., Bleskan J., Patterson D. Localization of the squalene synthase gene (FDFT1) to human chromosome 8p22-p23.1. Genomics. 1994;20:116–118. - PubMed

[4] Schechter I., Conrad D.G., Hart I., Berger R.C., McKenzie T.L., Bleskan J., Patterson D. Localization of the squalene synthase gene (FDFT1) to human chromosome 8p22-p23.1. Genomics. 1994;20:116–118. - PubMed

[5] Do R., Kiss R.S., Gaudet D., Engert J.C. Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathway. Clin. Genet. 2009;75:19–29. - PubMed

[6] Do R., Kiss R.S., Gaudet D., Engert J.C. Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathway. Clin. Genet. 2009;75:19–29. - PubMed

[7] Jemal M., Ouyang Z. Gas chromatography-mass spectrometric method for quantitative determination in human urine of dicarboxylic (dioic) acids produced in the body as a consequence of cholesterol biosynthesis inhibition. J. Chromatogr. B Biomed. Sci. Appl. 1998;709:233–241. - PubMed

[8] Jemal M., Ouyang Z. Gas chromatography-mass spectrometric method for quantitative determination in human urine of dicarboxylic (dioic) acids produced in the body as a consequence of cholesterol biosynthesis inhibition. J. Chromatogr. B Biomed. Sci. Appl. 1998;709:233–241. - PubMed

[9] Vaidya S., Bostedor R., Kurtz M.M., Bergstrom J.D., Bansal V.S. Massive production of farnesol-derived dicarboxylic acids in mice treated with the squalene synthase inhibitor zaragozic acid A. Arch. Biochem. Biophys. 1998;355:84–92. - PubMed

[10] Vaidya S., Bostedor R., Kurtz M.M., Bergstrom J.D., Bansal V.S. Massive production of farnesol-derived dicarboxylic acids in mice treated with the squalene synthase inhibitor zaragozic acid A. Arch. Biochem. Biophys. 1998;355:84–92. - PubMed

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

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

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