PMC3540165_01
Brain imaging and biopsy. a,c, Brain MRI, Axial T2, on admission for Patient A (a) and Patient B (c). b,d, Brain MRI, Axial T2, Post-discharge. e,f, White matter with multiple foci of perivascular myelin degradation, hemorrhage, edema, and scattered infiltrating neutrophils (arrowheads), foamy macrophages and hemosiderin-laden macrophages in brain biopsies from Patient A (e) and Patient B (f). (H&E, X100)
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F1
Initial CT imaging of the brain demonstrated vasogenic edema requiring a left posterior craniotomy and MRI revealed the extensive white matter signal abnormalities in the corpus callosum and left temporoparietooccipital lobe consistent with encephalitis (Figure 1c). Histopathologic examination of brain biopsy tissue revealed neutrophilic inflammation with multiple foci of myelin degradation, predominantly surrounding the vascular structures, with reactive endothelial cell changes and no distinct evidence of vasculitis (Figure 1f). Six months after the initiation of treatment, MRI revealed improvement of edema and evolving necrosis at the corpus collosum, without evidence of new white matter lesions (Figure 1d). Immunohistochemical evaluation of brain biopsy tissue from Patient B, revealed such an increase in C3, as well as increased membrane attack complex (MAC) deposition on neuronal cell surfaces compared to normal brain tissue (Figure 5a,b, Supplemental Figures S1,S2). In addition, immunohistochemical interrogation of the brain biopsy tissue of Patient B demonstrated IL-1beta positivity (Figure 5c) that was not observed in normal brain tissue (Supplemental Figure S1), supporting the use of anakinra as a specific treatment of AHLE.
FI mutations and serum levels. Pedigrees identifying mutations in CFI and their respective serum complement factor I (FI) levels (normal 29-59 microg/mL). Probands with AHLE are indicated by arrowheads. Left, Family A. Two novel mutations identified in the CFI gene. Patient and her sister are compound heterozygotes. Right, Family B. Patient and father share the G71V mutation also found in Family A. A second coding mutation was not identified, but a noncoding or regulatory variant could also explain the markedly decreased FI serum levels
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F2
A and her sister are compound heterozygotes for the glycine 71 to valine (G71V) amino acid substitution encoded by exon 2 and the cysteine 196 to serine (C196S) amino acid substitution encoded by exon 4, while her mother, father and brother are heterozygotes with single CFI mutations, consistent with their reduced serum FI levels (Figure 2, Left). Although his FI serum levels are consistent with 2 affected alleles, Patient B is only heterozygous for the exon 2 mutation and lacks any other CFI coding or splice site mutations. His father is also heterozygous for the exon 2 mutation and no mutations were identified in his brother or mother, despite the mildly reduced FI levels similar to the heterozygotes from Family A (Figure 2, Right).
PMC3540165_02
Brain imaging and biopsy. a,c, Brain MRI, Axial T2, on admission for Patient A (a) and Patient B (c). b,d, Brain MRI, Axial T2, Post-discharge. e,f, White matter with multiple foci of perivascular myelin degradation, hemorrhage, edema, and scattered infiltrating neutrophils (arrowheads), foamy macrophages and hemosiderin-laden macrophages in brain biopsies from Patient A (e) and Patient B (f). (H&E, X100)
nihms406272f1.jpg
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F1
Initial CT imaging of the brain demonstrated vasogenic edema requiring a left posterior craniotomy and MRI revealed the extensive white matter signal abnormalities in the corpus callosum and left temporoparietooccipital lobe consistent with encephalitis (Figure 1c). Histopathologic examination of brain biopsy tissue revealed neutrophilic inflammation with multiple foci of myelin degradation, predominantly surrounding the vascular structures, with reactive endothelial cell changes and no distinct evidence of vasculitis (Figure 1f). Six months after the initiation of treatment, MRI revealed improvement of edema and evolving necrosis at the corpus collosum, without evidence of new white matter lesions (Figure 1d). Immunohistochemical evaluation of brain biopsy tissue from Patient B, revealed such an increase in C3, as well as increased membrane attack complex (MAC) deposition on neuronal cell surfaces compared to normal brain tissue (Figure 5a,b, Supplemental Figures S1,S2). In addition, immunohistochemical interrogation of the brain biopsy tissue of Patient B demonstrated IL-1beta positivity (Figure 5c) that was not observed in normal brain tissue (Supplemental Figure S1), supporting the use of anakinra as a specific treatment of AHLE.
FI mutations and serum levels. Pedigrees identifying mutations in CFI and their respective serum complement factor I (FI) levels (normal 29-59 microg/mL). Probands with AHLE are indicated by arrowheads. Left, Family A. Two novel mutations identified in the CFI gene. Patient and her sister are compound heterozygotes. Right, Family B. Patient and father share the G71V mutation also found in Family A. A second coding mutation was not identified, but a noncoding or regulatory variant could also explain the markedly decreased FI serum levels
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F2
A and her sister are compound heterozygotes for the glycine 71 to valine (G71V) amino acid substitution encoded by exon 2 and the cysteine 196 to serine (C196S) amino acid substitution encoded by exon 4, while her mother, father and brother are heterozygotes with single CFI mutations, consistent with their reduced serum FI levels (Figure 2, Left). Although his FI serum levels are consistent with 2 affected alleles, Patient B is only heterozygous for the exon 2 mutation and lacks any other CFI coding or splice site mutations. His father is also heterozygous for the exon 2 mutation and no mutations were identified in his brother or mother, despite the mildly reduced FI levels similar to the heterozygotes from Family A (Figure 2, Right).
Molecular modeling of CFI mutations. a, Domain structure with the positions of mutations associated with recurrent infections, aHUS, and AHLE. Mutations are numbered inclusive of the signal peptide (+18 relative to position numbers when the signal peptide is excluded). b, Structure of human FI with AHLE mutations (red spheres) c. Structure with aHUS mutations (red spheres). Molecular images generated by PyMol. FIMAC, Factor I/membrane attack complex; SRCR, scavenger receptor cysteine-rich
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F3
We modeled these mutations and more than 40 identified mutations in CFI on the FI structure (Figure 3). Remarkably, both substitutions could change the packing of the helical linker, thereby disrupting the disulfide bond between the helical linker and the light chain (Figure 3, violet).
Recombinant FI protein expression. a, Flow cytometry demonstrates similar intracellular expression of wild-type FI and mutants in transiently transfected HEK293 cells following treatment with Brefeldin A. b, Western blot of purified cell culture supernatants following transient transfection with wild-type (wt) or mutant CFI His-tagged plasmids. Images are representative of five independent experiments
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F4
Intracellular FI was detected by flow cytometry similarly in the wild-type and mutant CFI transfected cells suggesting no effect of the mutation on the rate of protein translation (Figure 4a). However, subsequent analyses of the cell culture supernatants demonstrated that no FI protein could be detected from cells transfected with constructs containing either of our patients' mutations (Figure 4b).
Histologic analysis of brain tissue. a-c, Immunohistochemistry of brain biopsy sections from Patient B demonstrating positive staining for C3 (a, X100), C5b-9 (MAC, b, X400) and IL-1 (c, X100). Arrowheads indicate positively staining cells.
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F5
Immunohistochemical evaluation of brain biopsy tissue from Patient B, revealed such an increase in C3, as well as increased membrane attack complex (MAC) deposition on neuronal cell surfaces compared to normal brain tissue (Figure 5a,b, Supplemental Figures S1,S2). In addition, immunohistochemical interrogation of the brain biopsy tissue of Patient B demonstrated IL-1beta positivity (Figure 5c) that was not observed in normal brain tissue (Supplemental Figure S1), supporting the use of anakinra as a specific treatment of AHLE.