Background

Complement C4 plays a crucial role in the activation of the classical and lectin complement pathways. It has two isotypes, C4A and C4B. While deficiency of C4A and C4B are detectable at low frequency in healthy populations, they have also been reported to be associated with various autoimmune and inflammatory diseases [1, 2]. However, a C4A or C4B deficiency has never been reported in autoantibody-associated encephalitides patient. Here we report a case of anti-N-methyl- D-aspartate (NMDA) receptor encephalitis associated with homozygous C4B deficiency.

Case presentation

Our patient is a fourteen-year-old boy with background of mild attention-deficit and hyperactive disorder. He has insignificant family history of neurological, psychological or immunological disorders. Preceded by two weeks of viral illness-like prodromal symptoms, he progressively developed hypersomnolence, confused speech with echolalia, self-muttering, dysarthria, mood fluctuation, bilateral upper limbs tremor, and headache. Physical examination was unremarkable, apart from having confusion, inappropriate speech with foul languages, urine and fecal incontinence. Investigations showed normal complete blood count, liver and renal function tests. Autoimmune markers including anti-nuclear antibodies and anti-double stranded DNA antibody were negative. His serum C3 levels were within normal range between 109 and 115 mg/dL, but his C4 levels were persistently below 8 mg/dL (Table 1). MRI brain was normal. Electroencephalogram showed a mild slowing of background activities. Cerebrospinal fluid (CSF) analysis demonstrated a total white cell count of 33 cells/mm2, with normal protein and glucose levels. Oligoclonal protein was detected in the CSF. Microbiological investigations including bacterial cultures and viral polymerase chain reactions for herpes simplex virus, varicella zoster virus and enteroviruses were all negative. Anti-NMDA receptor antibodies based on commercial assay (Euroimmune®, Lueback, Germany) were detectable (titer < 1:10) in the CSF but not the serum.

Table 1 Complement genetic profiles of the patient and his family members
Full size table

The diagnosis of anti-NMDA receptor encephalitis was made based on the diagnostic criteria published by Graus et al. [3] He was immediately given intravenous immunoglobulin (IVIG) 1 g/kg for on day 2 of admission for two consecutive days followed by pulse methylprednisolone 1 g on day 6 of admission for five days with gradual taper. However, he did not demonstrate any clinical improvement after IVIG and pulse methylprednisolone.

Plasmapheresis was therefore performed for a total of 7 sessions between day 14 and 29 of admission, with sedation required for the initial two sessions. He demonstrated significant improvement after the second session and was able to complete the remaining plasmapheresis without sedations. Nevertheless, his cognitive improvement remained slow and four doses of rituximab at 375 mg/m2/dose at weekly intervals were given between day 35 and 56 of admission. Accelerated improvement was observed after the second dose in terms his self-care abilities, behavior and cognitive functions. He was discharged after eight weeks of admission with some residual cognitive impairment requiring rehabilitation.

In view of his low C4 protein levels, an in-depth genetic analysis of the index patient, his parents and his maternal stepbrother was performed (Fig. 1; Table 1, Supplementary Fig. 1). Long-range mapping by pulsed field gel electrophoresis of PmeI-digested genomic DNA revealed heterozygosity with bimodular (LL) and monomodular (L) RP-C4-CYP21-TNX (RCCX) haplotypes (panel A). Regular Southern blots of TaqI digested genomic DNA revealed the presence of three long C4 genes in LL/L configurations (panel B), by which two C4 genes belong to the C4A isotype and one C4 gene belongs to the C4B isotype, as shown by PshAI/PvuII Southern blot (panel C). Immunofixation experiment of EDTA-plasma showed that the patient expressed C4A protein but no C4B protein (panel D) [4]. In other words, the patient contained a C4B gene that did not produce a C4B protein (panels C and D, arrows) and thus assigned a C4B mutant. Concurrent study of the patient’s parents and maternal stepbrother suggested the patient (Fig. 1, Table 1) inherited the mutant C4B gene originated from their mother with a LL haplotype consisting of one long C4A gene coding for C4A3 protein, and one long mutant C4B gene with no product.

Fig. 1
figure 1

Molecular genetic characterization of the encephalitis patient with

antibodies against NMDA-receptor and his family members. a. Pulsed field gel electrophoresis (PFGE) of PmeI digested genomic DNA to show RP-C4-CYP21-TNX (RCCX) haplotypes; b. TaqI restriction fragment polymorphism (RFLP) to show details of RCCX structures with long (L) and short (S) C4 genes; c. PshAI/PvuII RFLP to show the presence and ratios of C4B and C4A genes; d. Immunofixation of EDTA-plasma to show polymorphisms of C4A and C4B proteins. A red arrow showed the presence of a C4B gene in panel C but no C4B protein in panel D. Data interpretation is tabulated in Table 1. Abbreviations: P, patient; F, father; M, mother; S1, stepbrother.

Full size image

Discussion and conclusions

Deficiency of C4B proteins, low gene copy number or mutation of C4B gene has not been reported in any autoimmune encephalitides. Therefore, its potential role in the development of anti-NMDA receptor encephalitis remains to be established. Classical pathway complement proteins such as C1q and C4 have been implicated in the wiring of neurons, or the formation and pruning of synapses in the brain [5]. High copy number of C4A, which can also be presented as low copy number of C4B because a C4 gene either codes for C4A or C4B protein [2], are associated with schizophrenia [6].

Low levels of serum C4 correlate with reduced functions of the classical and lectin pathways, leading to the impairment of immune-complexes removal [7]. Our patient was a poor responder to IVIG and pulse glucocorticoids but achieved significant improvement after plasmapheresis and rituximab. While a study by Martinez-Hernandez et al. demonstrated that complement-mediated cytotoxicity, i.e. cellular destruction through deposition of C3b and formation of membrane attack complexes C5b-9, was not readily detectable in the pathogenesis of anti-NMDA receptor encephalitis, their study did not examine the activation of early classical pathway complement components such as C1q and C4 [8]. This would be relevant because complement activation on autologous cells are tightly controlled and mostly stop after the deposition of C4b. We postulate that C4B deficiency in our patient might have led to the defective removal of immune complexes and possibly impairment of yet to be characterized neurogenic pathways, which has a substantial role in autoimmune encephalitis [9,10,11]. Complement deficiency is ameliorated by plasmapheresis, which normally correct the complement deficiency, but not by IVIG. Plasmapheresis also effectively removes offending autoantibodies and immune-complexes [12]. The use of rituximab, an anti-CD20 monoclonal antibodies, depletes CD20+ B cells, preventing further autoantibody production and immune complex formation [8]. These strategies lead to a sustained clinical improvement. It remains to be determined if routine plasmapheresis is required to keep patient’s disease in remission.

Further studies investigating the complement profiles and circulating immune complexes in patients with anti-NMDA receptor and other autoimmune encephalitides would clarify the role of C4B deficiency. A study by Shu et al. demonstrated that southern Chinese patients with anti-NMDA receptor encephalitis had higher C4 levels among female patients (N = 19) than female healthy controls (N = 16), the sample sizes (for controls) were relatively small and required validation. Moreover, genetic deficiency of C4B was not examined [13]. While the best first-line therapeutic option (steroid, IVIG or plasmapheresis) to treat anti-NMDA receptor encephalitis remains debatable [14], inheritently low C4 levels or C4B deficiency may be a biomarker for the need of plasmapheresis upfront rather than only after non-response to steroid and IVIG.