A case of autoimmune hemolytic anemia with acquired functional mutation of the TLR7

Abstract

This article presents a case of recurrent autoimmune hemolytic anemia in a child with a gain-of-function (GOF) mutation of the TLR7. This patient’s condition contrasts with the six previously documented cases of GOF mutations in the TLR7, thereby expanding the phenotypic spectrum of such mutations and enhancing clinical comprehension of childhood systemic lupus erythematosus (cSLE). The article discusses the mechanisms by which TLR7 GOF mutations can result in autoimmune hemolytic anemia, explores the influence of cytomegalovirus (CMV) infection on the disease’s development and progression, and emphasizes the therapeutic potential of hematopoietic stem cell transplantation for cases of TLR7 GOF mutations.

Case presentation

An 11-month-old girl presented for evaluation of abnormal blood test results discovered one day earlier. Physical examination demonstrated hepatomegaly in the absence of splenomegaly, along with the development of a prominent generalized rash on the face and back later. Initial blood tests indicated: WBC 22.93 G/L, Hb 40 g/L, PLT 443 G/L, ANC 8.83 G/L. Further tests showed elevated total bilirubin, slightly elevated myocardial enzymes, positive results for blood type-specific antibodies, and strong positive results for direct and indirect Coombs tests. Subsequently, she developed high fever with progressive liver enzyme elevation, along with thrombocytopenia, elevated triglycerides, decreased fibrinogen, and significantly elevated levels of IL-6, IL-10, TNF-α, ferritin, and sCD25. This raised concerns about other infections, the immune system, and hematological diseases such as hemophagocytic syndrome and malignancies. Further tests showed no decrease in NK cell activity, and Next-generation sequencing (NGS) of blood samples identified the presence of cytomegalovirus (CMV). Peripheral blood smears indicated an increased ratio of atypical lymphocytes. Bone marrow cytology showed active erythroid hyperplasia at 66.5%, mainly with late-stage erythroblasts, visible nuclear-cytoplasmic budding and lobulated erythroblasts (10% of Erythroblast) Bone marrow karyotype analysis: 46, XX[20]; clinically considered diagnosis of autoimmune hemolytic anemia, hemophagocytic syndrome, cytomegalovirus infection. After treatment with ganciclovir antiviral therapy, immunoglobulin infusion, plasma exchange, high-dose methylprednisolone treatment, JAK1/2 inhibitor ruxolitinib for immune modulation, and other symptomatic supportive treatments, the child’s temperature was controlled, hemoglobin and platelet levels improved, and the child was discharged in good condition. Subsequently, the child was hospitalized multiple times due to CMV infection and autoimmune hemolytic anemia. Upon admission, the hemoglobin level fluctuated between 40 and 50 g/L. After antiviral treatment, immunoglobulin therapy, and glucocorticoid therapy, the hemoglobin level could rise to around 90 g/L, but the condition was prone to relapse.

Considering the child’s presentation of hemophagocytic syndrome after CMV infection, followed by recurrent autoimmune hemolytic anemia and transient abnormalities in autoimmune-related indicators during disease progression, it is suspected that the child has immune dysfunction. Further comprehensive genetic testing revealed that the patient carries a missense mutation in exon 3 of the TLR7, c.T1520C (p.F507S), which causes the 507th amino acid to change from phenylalanine (Phe) to serine (Ser). Parentage verification revealed that the mutation did not come from the child’s father. Due to special circumstances, we were unable to trace the genotype and clinical phenotype of the child’s biological mother, making it impossible to determine the mode of inheritance of the mutation or to verify it. Based on existing case reports, the likelihood of this gene mutation being pathogenic is considerable.

Discussion

Currently, only six cases of gain-of-function mutations in the TLR7 have been reported (as shown in the Table 1). Among them, five probands—a 1-year-old girl with the TLR7 L528I variant, a 4-year-old girl and a 3-month-old boy with the F507S variant [1], a 4-year-old girl with the Y264H variant [2], and a 13-month-old girl with the p.P267L variant [3]—have been found to have SLE or SLE-like diseases. Additionally, an eight-month-old infant girl with the G818V variant only exhibited refractory immune thrombocytopenic purpura (ITP) [4]. The mutation site F507S in this patient is located in the leucine-rich repeat (LRR) domain of TLR7 (according to UniProt). As mentioned above, this site has been previously reported, with patients carrying this mutation exhibiting SLE and neurological symptoms. Additionally, related in vitro studies have shown that the F507S variant has a gain-of-function effect on TLR7 activity [1]. However, to date, this patient has presented with hemophagocytic syndrome and recurrent autoimmune hemolytic anemia. Unlike previously reported cases, this patient only exhibits hematological system diseases, representing a novel phenotype for this mutation.

Table 1.

This data is mandatory

Age at First Manifestation	Sex	Amino Acid	Clinical Manifestations	Immunologic Readouts	Treatments and Outcomes
2Y	F	P267L	NMDA receptor encephalitis, massive pericardial effusion, severe hemolytic anemia, inflammatory vasculitis, accompanied by sudden status epilepticus	Complement C3: 0.25 g/L Complement C4: <0.05 g/L Anti-dsDNA: >189.5 IU/mL Anti-nuclear antibody: Positive Antiphospholipid antibodies: β2GPI positive ENA screen: Anti-Smith positive, Anti-RNP positive Direct Antiglobulin Test (DAT): Positive	Clinical symptoms improved after immunosuppressive therapy including JAK inhibitors, followed by HSCT.And then SARS-CoV-2 triggered symptomatic AIHA. Despite immunomodulation, DAT-positive hemolysis persists with occasional flares.
7Y	F	Y264H	Refractory autoimmune thrombocytopenia, inflammatory arthralgia, mitral valve insufficiency, and renal involvement.	Elevated antinuclear antibody (ANA) and hypocomplementemia.Lymphocyte Subset Panel: CD3 + Total T cells: 6,532 cells/uL (High) CD3 + CD4 + Helper T cells: 4,812 cells/uL (High) CD3 + CD8 + Cytotoxic T cells: 1,520 cells/uL CD19 + B cells: 2,142 cells/uL CD16 + CD56 + NK cells: 435 cells/uL CD4/CD8 Ratio: 3.2 CD3+ % (of lymphocytes): 71% CD4+ %: 52% CD8+ %: 16% CD19+ %: 23% CD16 + CD56+ %: 5% CD45+ % (Lymphocytes): 99%	Treatment with rituximab and baricitinib induced a hematologic remission, characterized by normalized platelet counts and the disappearance of antiplatelet antibodies. After disease relapse upon B-cell reconstitution, the patient was scheduled for allo-HSCT.
4Y	F	F507S	Fever, malar rash, and bicytopenia (lymphopenia and anemia). Headache, hallucinations. Generalized cerebral vasculitis, cerebral infarction, Class III/IV lupus nephritis, and intestinal ischemia.	Titers of antinuclear antibodies (ANA) and anti-dsDNA were elevated, and there was C3 and C4 hypocomplementemia.	Management consisted of glucocorticoids, hydroxychloroquine, mycophenolate mofetil, and fondaparinux sodium (for secondary thrombosis prevention). Acute myocardial infarction resulted in death at age 17, after a follow-up period of persistently low complement levels with concurrent negative autoantibody screening.
2D	M		Refractory epilepsy, recurrent fever, rash on limbs, trunk, and face, dystonic movement disorder.	Hypocomplementemia (C3/C4), autoantibody screening (ANA, anti-dsDNA, IgG/IgM anticardiolipin, IgG/IgM anti-B2-glycoprotein and lupus anticoagulant) was negative	Good response(resolution of fever, hematological and skin manifestations) to immunosuppressive therapy with glucocorticoids, hydroxychloroquine, mycophenolate mofetil, and JAK1/2 inhibitor ruxolitinib.
11 M	F		Recurrent autoimmune hemolytic anemia.	Hypocomplementemia (C3/C4), positive anti-thyroid peroxidase and thyroglobulin antibodies; positive direct antiglobulin test.	Partial response to glucocorticoids, IVIG, and JAK1/2 inhibitor ruxolitinib but prone to relapse; Following allo-HSCT, the monospecific antibody persisted, yet hemoglobin levels stabilized at approximately 100 g/L.
8 M	M	G818V	Refractory immune thrombocytopenia (ITP) with mild Coombs-negative non-hemolytic anemia.	Normal immunoglobulins (including IgG levels and lymphocyte subsets). Mildly elevated CD3 + T cells. ITP antiplatelet antibody panel showed very high anti-GP IIB/IIIA antibody titers.	Poor response to glucocorticoids, IVIG, TPO, sirolimus, baricitinib (JAK inhibitor), eltrombopag (TPO-RA), or rituximab; planned for allogeneic hematopoietic stem cell transplantation.
1Y	F	L528I	Evans-like/SLE-like syndrome, stereotyped movement disorder (e.g., head nodding).	Negative Coombs reaction, positive anti-SSA	No response to glucocorticoids, IVIG, mycophenolate mofetil, rituximab, or bortezomib; underwent allogeneic hematopoietic stem cell transplantation.Chronic graft-versus-host disease developed after hematopoietic stem cell transplantation (HSCT), with complete normalization of blood counts achieved by 22 months post-transplant.

The TLR7 is located on the short arm of the X chromosome (Xp22) [5]. Its translation product, TLR7, as a pattern recognition receptor, is an important component of innate immunity. It recognizes single-stranded RNA (ssRNA) rich in uridine and guanosine in the cytoplasm, transmits signals through the MyD88-dependent pathway, activates the NF-κB and MAPK pathways, stimulates pro-inflammatory cytokines (such as IL-1, IL-6, TNFα, etc.) and chemokines (such as MCP-1, CCL, and CXCL), and induces the upregulation of type I and type II interferons [6].

Recent studies have increasingly shown that TLR7 promotes the phenotype of systemic lupus erythematosus (SLE), such as in mouse strains with the yaa translocation (a chromosomal translocation containing the mouse TLR7) and the additional copies of TLR7 it causes, which accelerate lupus pathology and symptoms of anti-DNA autoantibodies [7]. Consistent with this, genetic ablation of TLR7 eliminates the development of spontaneous germinal centers in mouse lupus models, which aligns with the improvement in the pathogenesis of these models [8].

The specific mechanism by which gain-of-function mutations in TLR7 lead to SLE is still unclear. Brown and others found that gain-of-function mutations in the TLR7 can cause B-cell-driven autoimmune diseases, including systemic lupus erythematosus (SLE), because the TLR7 Y264H variant increases affinity for guanosine, leading to a lower threshold for TLR7 activation. TLR7 gain-of-function mutations promote the survival of immature B cells activated by BCR, which are rich in auto-reactivity [2]. In the presence of constitutive signal 2, the activation of autoreactive B cells by self-antigens may promote B cell differentiation and the production of autoantibodies [4]. Additionally, David et al. found that compared to the wild type, HEK Blue™ cells transfected with the G818V mutation had a threefold increase in NF-κB/AP-1 activation, and this enhanced stimulation also occurred in the absence of exogenous ligands [10]. Therefore, it can be inferred that under such conditions, the low or absent TLR7 activation threshold may activate self-cells expressing TLR7, including platelets, leading to an early and specific breakthrough in tolerance to self-cell antigens, making them appear as new antigens. This results in the production of high titers of antibodies against self (including red blood cells), leading to the SLE and SLE-like manifestations in the aforementioned cases.

In this case, the child’s prominent symptom is recurrent autoimmune hemolytic anemia, which usually sees a rise in hemoglobin levels after treatment with hormones or immunoglobulins, but the condition is prone to relapse. Plotting the child’s hemoglobin levels against intracellular, extracellular, and urinary CMV-DNA curves (as shown in the Fig. 1) revealed that the child experienced severe onset of hemolytic anemia, accompanied by CMV infection. After controlling the CMV infection, the child’s hemoglobin levels were generally higher than when the CMV infection was uncontrolled. It seems that the control of autoimmune hemolytic anemia (AIHA) symptoms is closely related to the clearance of CMV infection.

Fig. 1.

Hemoglobin level and intracellular titer of CMV-DNA

CMV infection is closely related to autoimmunity. After CMV infection, it can trigger or maintain autoimmunity through three mechanisms: the production of autoantibodies, enhanced inflammation, and vascular damage. Autoimmunity can be induced or sustained through antigen-specific (i.e., molecular mimicry) and non-antigen-specific (i.e., bystander activation) processes [10]. CMV infection may enhance B cell proliferation and autoantibody production by engaging TLR7/TLR9 signaling in plasmacytoid dendritic cells [11]. In monocytes, CMV downregulates complement receptor 3 (CR3, ITGAM/ITGB2), a negative regulator of TLR pathways [12], potentially leading to hyper responsiveness to TLR stimuli and increased production of type I interferon and pro-inflammatory cytokines [13]. It seems that perhaps it is under the influence of CMV infection, through the downregulation of CR3 and other mechanisms, that the signaling of TLRs, including TLR7, is enhanced, leading to sustained activation of autoimmunity, the production of erythrocyte antibodies, and thus the manifestation of autoimmune hemolytic anemia, which may even result in the production of antibodies against other self-antigens. In refractory or severe AIHA, especially in infants, CMV can be a significant trigger, and its viral load correlates with hemolysis severity [14]. CMV DNAemia in active SLE is also linked to poorer outcomes [15]. Thus, monitoring and early treatment of CMV may improve prognosis in these patients.

Regarding treatment, the aforementioned cases emphasized the importance of B-cell depletion and the use of JAK inhibitors or mTOR inhibitors. The patient in this case, as well as two patients from the aforementioned cases, underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). One patient with hematological manifestations (refractory autoimmune thrombocytopenic purpura and hemolytic anemia) had normal blood counts 22 months post-transplant [9]. Another patient still experienced chronic hemolysis post-transplant, but their overall condition remained good, and they made excellent progress in immune reconstitution, growth, and neurodevelopment [12]. The patient in this case received an allogeneic hematopoietic stem cell transplantation, and after that, the monospecific antibody persisted, yet hemoglobin levels stabilized at approximately 100 g/L, and other abnormal measured parameters also showed improvement (as shown in the Table 2),which highlighting the therapeutic potential of this approach for treating TLR7 gain-of-function mutations. Moreover, some studies suggest that TLR7, as a key upstream driver of human SLE, drives autoimmunity in a GC (germinal center)-independent manner [16]. This implies that when treating SLE with TLR7 gain-of-function mutations, therapies targeting TLR7 itself or My88 may be more effective than therapies targeting GC.

Table 2.

This data is mandatory

Measured Relevant Parameters	Normal Reference Range	Initial Visit	3-Month after HSCT	6-Month after HSCT
Hemoglobin (g/L)	112–149	37↓	95 ↓	101 ↓
Total Bilirubin (µmol/L)	5.1–19.0	27.9 ↑	9.6	4.2 ↓
Alanine Aminotransferase (U/L)	8.0–30.0	477 ↑	13	25
Aspartate Aminotransferase (U/L)	14–44	255 ↑	24	36
Ferritin (µg/L)	4.6–204	1695.2 ↑	1175.9 ↑	469.6 ↑
sCD25 (U/ml)	228–724	7114.22 ↑	888.10 ↑	NT
CMV-DNA (copies/ml)	/	251	ND	ND
CMV-DNA (Intracellular) (copies/ml)	/	3330	ND	ND
Complement C3 (g/L)	0.790–1.520	0.734 ↓	0.955	0.881
Complement C4 (g/L)	0.160–0.380	0.734 ↑	0.235	0.253
IL-6 (pg/ml)	1.70–16.60	25.99 ↑	9.31	4.88
IL-10 (pg/ml)	2.60–4.90	45.87 ↑	3.93	3.6
IL-17 A (pg/ml)	0.00–20.60	ND	0.98	2.35
TNF-α (pg/ml)	0.10–5.20	0.78	0.98	3.37

Considering the clear diagnosis of hereditary anemia in this case, we will continue to monitor the specific condition of the child post-transplant, closely observing for any signs of other autoimmune disorders, lupus, or neuro-inflammation.

Author contributions

Y.H. and H.C. wrote the main manuscript text and Y.H. prepared figure1; Table 1、2. All authors reviewed the manuscript and edit.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.