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Case Reports
. 2019 Jul;7(7):e00733.
doi: 10.1002/mgg3.733. Epub 2019 May 8.

A mutation in Site-1 Protease is associated with a complex phenotype that includes episodic hyperCKemia and focal myoedema

George G Schweitzer  1 , Connie Gan  1 , Robert C Bucelli  2 , Daniel Wegner  3 , Robert E Schmidt  4 , Marwan Shinawi  3   5 , Brian N Finck  1 , Rita T Brookheart  1
Affiliations
Case Reports

A mutation in Site-1 Protease is associated with a complex phenotype that includes episodic hyperCKemia and focal myoedema

George G Schweitzer et al. Mol Genet Genomic Med. 2019 Jul.

Abstract

Background: Site-1 Protease (S1P) is a Golgi-resident protein required for the activation of regulatory proteins that drive key cellular functions, including, the unfolded protein response (UPR) and lipid and cholesterol biosynthesis. While disruptions in S1P function have been widely characterized in animal models, to date, the implications of disrupted S1P function in human disease states are not completely known.

Methods: The patient and both parents underwent whole exome and mitochondrial DNA sequencing, and Sanger sequencing was used to confirm the mutation. Western blotting and immunofluorescence studies were performed on either proband-derived fibroblasts or on an established cell line to assess protein expression and cellular localization of the mutated S1P protein. Quantitative real-time PCR and luciferase reporter assays were used to examine activation of S1P target pathways in the context of the S1P mutation.

Results: We describe a female patient with a de novo heterozygous missense mutation in the transmembrane domain of S1P (p. Pro1003Ser). The patient presented to our neuromuscular clinic with episodic, activity-induced, focal myoedema and myalgias with hyperCKemia. Her clinical phenotype was complex and included gastrointestinal hypomotility, ocular migraines, and polycystic ovary syndrome. Molecular analysis using proband-derived fibroblasts and cell lines harboring the Pro1003Ser mutation demonstrated increased activation of UPR and lipid and cholesterol regulatory pathways and localization of S1P Pro1003Ser in the Golgi.

Conclusion: These findings suggest a critical function for S1P in several human organ systems and implicate an important role for S1P in various human disease states.

Keywords: ER stress; MBTPS1; PCOS; SREBP; Site-1 Protease; hyperCKemia; myoedema.

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Conflict of interest statement

Rita Brookheart and Brian Finck hold a provisional patent related to this work (provisional patent application #62/638,531).

Figures

Figure 1
Figure 1
Genetic and functional analysis of S1P Pro1003Ser. (a) Schematic of S1P protein with the Pro1003Ser mutation and S1P protein domains indicated, TM, transmembrane domain. (b) Multispecies alignment of S1P amino acid sequences demonstrating the mutated proline 1003 residue is conserved (shown in bold). (c) mRNA expression levels of the MBTPS1 transcript in cultured control‐ and patient‐derived skin fibroblasts. n = 3. (d) Sanger sequencing trace files of patient genomic DNA (top) and cDNA (bottom)‐derived PCR products at the MBTPS1 c.3007C>T locus. Intron–exon boundary (genomic) and exon–exon junction (cDNA) are indicated. Arrows denote the variant location. (e) Western blot of whole‐cell lysates (60μg) from SRD‐12B cells transiently transfected with mock, WT S1P, or S1P Pro1003Ser plasmids after 24 hr. S1P‐A, B, and C forms are indicated. Blots were probed with S1P and α‐tubulin (loading control) antibodies. n = 3. (f) SRD‐12B cells were transfected as in (e) and grown either in medium supplemented with lipids and cholesterol (left column) or in lipid‐ and cholesterol‐free medium (right column) for 7 days followed by fixation in methanol and crystal violet staining, as reported previously (Rawson et al., 1998). Images are representative of three independent experiments
Figure 2
Figure 2
S1P Pro1003Ser has enhanced activity and accumulates in the Golgi. mRNA expression levels in cultured control‐ and patient‐derived skin fibroblasts of (a) SREBP1 and 2 target genes after 4 hr of treatment with the indicated concentrations of mevastatin and (b) UPR and ATF6 target genes following treatment with 1 µg/ml of tunicamycin for 4, 8, and 12 hr. (c) SRD‐12B cells transiently transfected with FAS‐Luc, Renilla, and WT S1P or S1P Pro1003Ser plasmids as indicated, after 10 hr, cells were treated with DMEM/F12 for 16 hr. Luciferase activities were measured and the ratio between firefly and Renilla luciferase activities was determined. (d) Immunofluorescence of S1P, ER marker KDEL, and Golgi marker GM130 in S1P Pro1003Ser patient and control fibroblasts. Cells were transfected with FLAG‐tagged WT S1P or S1P Pro1003Ser as indicated. Representative images are shown. All data are expressed as mean S.E. of three to five independent experiments, *p < 0.05
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