PMC6568211_01
(A) Pedigree of Patient A. (B) Capillary zone electrophoresis trace from Patient A demonstrating absent beta-2 peak. (C) Sequencing chromatograms from Patient A demonstrating c.129C>A and c.559C>T mutations in the heterozygous states. Codon positions and wild-type amino-acids are indicated below. (D) Schematic of the domain structure of complement factor I protein demonstrating position of mutations from Patient A (black) and Patient B (blue).
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F1
Filtering of all the variants identified by WGS based on quality metrics, deleteriousness, inheritance pattern and biological function led to a short list of 5 genes that were investigated further (Supplementary Figures 1, 2). WGS revealed two heterozygous variants in CFI (c.191C>T; p.P64L and c.262C>A; p.Q88K) lying within the CFI FIMAC domain (Figure 1D).
PMC6568211_02
(A) Pedigree of Patient A. (B) Capillary zone electrophoresis trace from Patient A demonstrating absent beta-2 peak. (C) Sequencing chromatograms from Patient A demonstrating c.129C>A and c.559C>T mutations in the heterozygous states. Codon positions and wild-type amino-acids are indicated below. (D) Schematic of the domain structure of complement factor I protein demonstrating position of mutations from Patient A (black) and Patient B (blue).
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F1
Filtering of all the variants identified by WGS based on quality metrics, deleteriousness, inheritance pattern and biological function led to a short list of 5 genes that were investigated further (Supplementary Figures 1, 2). WGS revealed two heterozygous variants in CFI (c.191C>T; p.P64L and c.262C>A; p.Q88K) lying within the CFI FIMAC domain (Figure 1D).
(A) T2 weighted MRI imaging from Patient B during acute encephalomyelitis and following treatment with pulsed methylprednisolone. (B) Pedigree of Patient B showing genotypes of rare CFI variants. NA, DNA not available for deceased individuals. (C) Visualization of read-level information shows that the two CFI variants, lying 71 bp apart, are easily phased by the 150 bp reads and are in trans (boxed). The absence of the c.191C>T variant in the mother (lower panel) is insufficient to confirm an in trans orientation as the variant may have arisen de novo. (D) Free energy change calculations for known missense, disease-causing point mutations in complement factor I with mutations from Patient B shown in red.
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F2
MRI neuroimaging showed diffuse, confluent cerebral and cerebellar white matter high signal changes, oedema, and mass effect without DWI change (Figure 2A). Her sister had died of fulminant haemorrhagic leukencephalopathy at the age of 16 (Figure 2B). Filtering of all the variants identified by WGS based on quality metrics, deleteriousness, inheritance pattern and biological function led to a short list of 5 genes that were investigated further (Supplementary Figures 1, 2). Although paternal DNA was not available, Illumina read-level information was used to confirm the variants lay in trans (Figure 2C). To further assess the pathogenicity of these variants, mutation Cutoff Scanning Matrix (mCSM) analysis was performed (Figure 2D), an approach that predicts the effects of amino acid variation on protein stability by estimating free energy changes. Heterozygous variants in three other immunologically relevant genes were identified by WGS in Patient B: C6, PTPRC and CD74 (Supplementary Figure 2).
Three-dimensional topology of selected missense mutations in complement factor I (light gray) and their relationship with C3b (dark gray). (A) P64 residue of complement factor I forms a contact point with V1658 of C3b (blue). Q88 lies in close apposition to this contact site. (B) G71 (previously reported by Broderick et al and associated with a similar clinical phenotype) lies on a side chain between P64 and Q88. Figures produced using PyMOL v2.2 using a crystal structure of CFI and C3b solved to a resolution of 4.2 A (Protein Data Bank Reference - 5O32).
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F3
P64 is highly conserved across taxa (Supplementary Figure 3). Previous reports have associated the p.P64L variant with atypical haemolytic uraemic syndrome and age related macular degeneration. Q88 is also highly conserved across taxa (Supplementary Figure 3). Three-dimensional modeling of the protein structure of CFI in complex with C3b shows the close topological relationship between the P64 and Q88 residues of CFI and the V1658 residue of C3b (Figure 3A). Furthermore, the G71 residue, mutations of which have also been associated with neurological presentations of CFI deficiency, lies on a side chain between these two mutations Figure 3B.