Fig 2: FLAD1 Variants and Gene and Protein Structure(A) Pedigrees of the investigated families (F1–F7) with recessively inherited FLAD1 variants. Affected individuals are indicated by closed symbols.(B) Gene structure with exons and introns shows the localization of the investigated gene variations (homozygous variants are underlined). Met residues located upstream of the FADS domain are presented in yellow with their corresponding protein positions with respect to isoform 1 (GenBank: NM_025207.4). Isoforms 1 and 2 are reported in the UCSC Genome Browser as transcripts possessing an intact and active FADS domain. The protein structure highlights the MPTb domain in violet and the FADS domain in orange. Protein consequences of the identified FLAD1 mutations include the frameshift variants located in the MPTb domain and the two amino acid changes in a region of the FADS domain, which is highly conserved among eukaryotic species. Amino acid residues that are conserved across all species are highlighted in dark blue.

Fig 3: Alternative FLAD1 Transcripts and Their Predicted Products(A) Analysis of transcriptome data for FLAD1 from two unrelated fibroblast control samples (C1 and C3) and one human blood sample (C2) revealed several possible transcripts, summarized as Sashimi plots (bottom). Schematic diagram shows isoform transcript structures with MPTb and FADS domains marked in violet and orange, respectively (top). The most abundant transcript (isoform 2) corresponds to the sequence reported in RefSeq (GenBank: NM_201398.2) but has an alternative intron in exon 1 and lacks the predicted MTS. Isoforms 5 and 6 represent transcripts without exons 1b and/or 2, where the identified FLAD1 frameshift variants are located, but both are able to express an active FADS domain. Isoform 5 can use the Met marked as M-X in the very beginning of the transcript, whereas isoform 6 utilizes Met268. In RNA from fibroblasts or blood, we did not identify a single transcript resulting in isoforms 1 (GenBank: NM_025207.4), 3 (GenBank: NM_00114891), or 4 (GenBank: NM_001184892) as reported in RefSeq. Isoforms 3 and 4 are not shown.(B) PCR products of cDNA from control subjects and subject S4a confirmed the presence of non-degraded FLAD1 mRNA (all details are provided in Figure S1). The primer pair is able to amplify either isoform 2 or isoform 6 of the transcript, resulting in 307 or 84 bp products, respectively.(C) Immunoblot analysis of the different FADS isoforms (Met98, Met268, and Met355) overproduced in fibroblasts derived from a healthy control individual shows their stability and detectability by anti-FADS antibody. A control fibroblast sample (C) is shown for comparison.

Fig 4: Analysis of FADS Proteins(A and B) Protein extract from cultured fibroblasts (A) or a liver biopsy (B) were separated by SDS-PAGE and immunoblotted with polyclonal antibodies against the C-terminal end of the human FADS. Subjects (S1a, S2, S4a, and S5) and healthy control individuals (C1, C2, C3, and C) are indicated on top of the gels. Purified human FADS2 is indicated by “hF.” Trihalo staining or GAPDH were used as loading controls. The numbers (P1–P4) on the right side of the immunoblot in (A) correspond to FADS peptides detected by mass spectrometry (illustrated in C). Bands marked with an asterisk represent non-specific anti-FADS antibody bindings as defined by mass spectrometry.(C) The amino acid sequence of human FADS1 (GenBank: NP_079483.3) is shown in one-letter symbols. The MPTb domain and the FADS domain are shaded in violet and orange, respectively, and the four Met residues upstream of the FADS domain are written in bold, and their protein positions are indicated. Peptides selected as identifiers of FADS and for use in the mass-spectrometry analysis are written in superscript and further listed below with their analysis number (P1–P5) and protein domain. The signal from peptide 5 was treated as a nonsignificant finding because it was detected at the boarder of the detection limit of the SRM analysis in all analyzed gel bands. The analysis was performed on gel bands processed from two independent cell lysates, and each peptide mixture was injected and analyzed by msass spectrometry twice.