Mutations in SREBF1, Encoding Sterol Regulatory Element Binding Transcription Factor 1, Cause Autosomal-Dominant IFAP Syndrome

Abstract

IFAP syndrome is a rare genetic disorder characterized by ichthyosis follicularis, atrichia, and photophobia. Previous research found that mutations in MBTPS2, encoding site-2-protease (S2P), underlie X-linked IFAP syndrome. The present report describes the identification via whole-exome sequencing of three heterozygous mutations in SREBF1 in 11 unrelated, ethnically diverse individuals with autosomal-dominant IFAP syndrome. SREBF1 encodes sterol regulatory element-binding protein 1 (SREBP1), which promotes the transcription of lipogenes involved in the biosynthesis of fatty acids and cholesterols. This process requires cleavage of SREBP1 by site-1-protease (S1P) and S2P and subsequent translocation into the nucleus where it binds to sterol regulatory elements (SRE). The three detected SREBF1 mutations caused substitution or deletion of residues 527, 528, and 530, which are crucial for S1P cleavage. In vitro investigation of SREBP1 variants demonstrated impaired S1P cleavage, which prohibited nuclear translocation of the transcriptionally active form of SREBP1. As a result, SREBP1 variants exhibited significantly lower transcriptional activity compared to the wild-type, as demonstrated via luciferase reporter assay. RNA sequencing of the scalp skin from IFAP-affected individuals revealed a dramatic reduction in transcript levels of low-density lipoprotein receptor (LDLR) and of keratin genes known to be expressed in the outer root sheath of hair follicles. An increased rate of in situ keratinocyte apoptosis, which might contribute to skin hyperkeratosis and hypotrichosis, was also detected in scalp samples from affected individuals. Together with previous research, the present findings suggest that SREBP signaling plays an essential role in epidermal differentiation, skin barrier formation, hair growth, and eye function.

Keywords: MBTPS2; SREBF1; atrichia; ichthyosis follicularis; photophobia; sterol biosynthesis.

Figure 1

Pedigrees and Clinical Manifestations of the Present IFAP-Affected Cases (A) Pedigrees for the two Chinese families presented in this study. (B) Atrichia or hypotrichosis in Fam2: II-7 and affected individual 10. Sparsity of hair involving scalp, eyebrows, and eyelashes in affected individual 4. Loss of axillary hair in Fam 1: III-3. (C) Hyperkeratosis of the eyelid (Fam2: III-2) and periorificial region (individual 11). Multiple nail dystrophy in individual 5. (D) Ophthalmologic examination showing inspissated Meibomian glands with thickened meibum, decreased number of Meibomian glands, and pediatric cataract in Fam2: III-2.

Figure 2

Cleavage Mechanism, Amino Acid Conservation Status, and Sanger Sequencing Results (A) Schematic illustration of SREBP-1 function. When sterol is sufficient, SREBP-1 is retained in the ER via formation of a complex with SCAP and Insig. In sterol-deficient cells, the SREBP-1 attached to SCAP is transported by forming a complex with SCAP to the Golgi apparatus where it is cleaved by S1P and S2P sequentially; the cleaved form is then translocated to the nucleus where it binds to SRE. (B) The arginine-X-X-leucine motif is recognized and cleaved by S1P. Residues arginine 527 and leucine 530 are highly conserved among species. (C) Three different mutations in SREBF1 in individuals with IFAP shown by Sanger sequencing traces.

Figure 3

SREBP-1 Variants Are Transcriptionally Inactive due to Failed Cleavage by S1P (A) Immunoblot analysis of FLAG-tagged SREBP-1 from HEK293 cells transfected with wild-type and the mutant SREBF1 cDNA construct. Histone-H3 and GAPDH were used as loading controls of nuclear extract and cytoplasm fractions, respectively. A 71-kD band corresponding to mature form of SREBP-1 was detected only in the sterol-deprived cells transfected with pTK-FLAG-SREBF1. P, precursor form; m, mature form. (B) Confocal images showing the subcellular localization of SREBP-1 in transfected HEK293 cells. Scale bars represent 10 μm. (C) HEK293 cells cotransfected with a construct expressing wild-type SREBF1 and a luciferase reporter gene under transcriptional control of sterol responsive elements (SRE) showed restored sterol responsiveness in the sterol-deficient medium, while the IFAP variants all exhibit a reduced response to the sterol-deprived condition. Transfection efficiency is normalized to cotransfected Renilla (Ren) luciferase. The results are representative of three independent transfection experiments. Error bars represent SEM. ^∗∗p < 0.01.

Figure 4

Volcano Plot of Differentially Expressed Genes in Scalp Skin of Affected Individuals Gene expression was compared using RNA-sequencing in scalp skin biopsies obtained from four affected individuals (Fam1: III-3, Fam1: IV-1, Fam2: II-7, and Fam2: III-2) and four age-matched control subjects. Significantly up- or downregulated genes with a log₂ fold change outside the range of −1 and 1 are given as dark gray dots. Genes with an FDR < 0.05 are given as red dots, with those most promising candidates highlighted with black outlines.

Figure 5

Increased Apoptosis in the Epidermis of IFAP-Affected Individuals (A) TUNEL in situ staining of the scalp skin biopsy samples from a normal control subject and two affected individuals. Dotted lines indicate the boundary of epidermis and dermis. Scale bars represent 50 μm. (B) Apoptosis rate of the keratinocytes in the epidermis. ^∗∗∗p < 0.001. Error bars represent SEM.